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Measuring p66Shc Signaling Pathway Activation and Mitochondrial Translocation in Cultured Cells

UNIT 25.6

Mariusz R. Wieckowski,2 Cláudia M. Deus,1 Renata Couto,1 Monika Oparka,2 Magdalena Lebiedzińska-Arciszewska,2 Jerzy Duszyński,2 and Paulo J. Oliveira1 1

CNC—Center for Neuroscience and Cell Biology, University of Coimbra, Cantanhede, Portugal 2 Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland

The adaptor protein p66Shc links membrane receptors to intracellular signaling pathways, with downstream consequences on mitochondrial metabolism and reactive oxygen species production. Moreover, p66Shc has also been implicated in cancer development, progression, and metastasis. Increased phosphorylation of serine 36 residue of p66Shc very often correlates with oxidative stress– associated pathologies. The pro-oxidative role of p66Shc also appears to be involved in chemical toxicity, being an important component of stress responses triggered by xenobiotics. Here, we present a protocol that can be used: (a) for isolation of mitochondrial, cytosolic, and mitochondrial-associated membrane fractions from adherent cells lines; (b) to perform p66Shc detection with specific antibodies in order to monitor its translocation between different cellular compartments in response to the oxidative stress; and (c) to modulate the p66Shc pathway with the use of pharmacological approaches or gene-silencing C 2015 by John Wiley & Sons, Inc. methods. Keywords: cell signaling pathways r mitochondria r oxidative stress r protein phosphorylation and p66Shc protein

How to cite this article: Wieckowski, M.R., Deus, C.M., Couto, R., Oparka, M., Lebiedzińska-Arciszewska, M., Duszyński, J., and Oliveira, P.J. 2015. Measuring p66Shc signaling pathway activation and mitochondrial translocation in cultured cells. Curr. Protoc. Toxicol. 66:25.6.1-25.6.21. doi: 10.1002/0471140856.tx2506s66

INTRODUCTION The adaptor protein p66Shc belongs to the Src homology and collagen protein (ShcA) family. Initially characterized in the 90s, the p66Shc protein can be involved in several crucial processes including cell proliferation, differentiation, and pro-apoptotic signaling. Interestingly, the role of p66Shc protein seems to be determined by the presence of numerous critical phosphorylation residues in its sequence (Wills and Jones, 2012). The basic role of ShcA family proteins is their implication in the signal transduction pathway through the Ras protein. Under physiological conditions, the involvement of ShcA proteins (p46Shc, p52Shc and p66Shc) in signal transduction pathways requires the phosphorylation of tyrosine residues located in their central CH1 domains by receptor tyrosine kinases (TRKs). Activated TRKs are dimerized and autophosphorylated in their cytosolic domains, which then bind the PTB domain of ShcA proteins (Ravichandran, 2001). Phosphorylation of tyrosine residues in ShcA proteins allows the interaction and Current Protocols in Toxicology 25.6.1-25.6.21, November 2015 Published online November 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471140856.tx2506s66 C 2015 John Wiley & Sons, Inc. Copyright

Mitochondrial Toxicity

25.6.1 Supplement 66

Figure 25.6.1 Activation of protein kinase C-β isoform by oxidative stress leads to Ser36 phosphorylation of p66Shc. Several oxidative stress stimuli lead to activation of PKC-β, which induces p66Shc phosphorylation at Ser36 and translocation to mitochondria. It has been proposed that p66Shc binds to a member of the TOM/TIM import system, with linkage destabilized by pro-apoptotic stimuli, resulting in the release of p66Shc in its monomeric form. In the mitochondrial intermembrane space, p66Shc oxidizes cytochrome c and catalyzes the reduction of oxygen to hydrogen peroxide, which may culminate in the opening of the mitochondrial permeability transition pore (PTP). This process increases mitochondrial membrane permeability to ions, leading to swelling and disruption of the outer mitochondrial membrane with consequent release of pro-apoptotic factors to the cytosol, triggering cell death. Abbreviations: ADP, adenosine diphosphate; ANT, adenine nucleotide translocator; ATP, adenosine triphosphate; CK, creatine kinase; CyD, cyclophilin D; Cyt C, cytochrome c; FAD, flavin adenine dinucleotide; HK, hexokinase; H2 O2 , hydrogen peroxide; IMM, inner mitochondrial membrane; MTHSP70, mitochondrial heat shock proteins, 70 kDa; NADH, nicotinamide adenine dinucleotide; OMM, outer mitochondrial membrane; PBR, peripheral benzodiazepine receptor; PKC-β, protein kinase C–β isoform; Prx1, paired related homeobox 1; Q, coenzyme Q; ROS, reactive oxidative species; TIM, mitochondrial translocase of the inner membrane; TOM, mitochondrial translocase of the outer membrane; VDAC, voltage-dependent anion channel.

p66Shc Signaling in Toxicology

formation of a complex with growth factor receptor–bound protein 2 (Grb2) and son of sevenless homolog 1 protein (Sos), which are then able to exchange guanosine nucleotides in Ras protein (Ravichandran, 2001). In contrast to p52Shc and p46Shc tyrosine residues (Y239/240, Y317), phosphorylation in the CH1 domain of p66Shc converts this protein into a negative regulator of proliferation (Migliaccio et al., 1997). Interestingly, p66Shc differs from p46Shc and p52Shc by possessing an additional N-terminal CH2 (collagen homology domain 2) domain (Luzi et al., 2000). This domain contains an unique serine phosphorylation residue (Ser36) which is particularly important for the role of p66Shc for cellular responses to oxidative stress. Under oxidative stress conditions, activation of several kinases including protein kinase Cβ (PKCβ), protein kinase Cδ (PKCδ), and c-Jun N-terminal kinase (JNK) has been described (Le et al., 2001; Pinton et al., 2007; Song et al., 2014; La Colla et al., 2015). The most well-characterized kinase involved in the pathway, PKCβ, phosphorylates the Ser36 residue in the p66Shc protein as a response to hydrogen peroxide–evoked oxidative stress (Almeida et al., 2010) or phorbol 12-myristate 13-acetate (PMA) treatment (Lee et al., 2011) (Fig. 25.6.1). Serine 36 phosphorylation is followed by Pin1-dependent cis-trans isomerization of p66Shc, which is


Current Protocols in Toxicology

then dephosphorylated by protein phosphatase 2 A (PP2A). One hypothesis states that such modified p66Shc is "translocated" to the mitochondrial fraction, where it interacts with cytochrome c (cyt c), playing an important role in an increased formation of reactive oxygen species (ROS) in mitochondria (Fig. 25.6.1). However, translocation of p66Shc in response to the oxidative stress has been found to occur also to mitochondria-associated membranes (MAM), a fraction of the endoplasmatic reticulum interacting with the outer mitochondrial membrane (Lebiedzinska et al., 2009). Interestingly, a tetramerization of p66Shc due to the oxidation of a cysteine 59 residue has been described, which is another oxidative-stress-related modification of this protein (Gertz et al., 2008). Independently of ROS production, p66Shc also negatively regulates cellular antioxidant responses by interaction with forkhead transcription factor 3a (FOXO3a), leading to lower expression of SOD2 and catalase (Nemoto and Finkel, 2002) and to accumulation of oxidatively damaged cellular components such as proteins, lipids, and nucleic acids (Pani et al., 2009). Numerous studies confirmed the involvement of p66Shc in the accumulation of oxidative stress–related damage observed in multiple pathologies, such as neurodegenerative diseases, diabetes, and cancer (Almeida et al., 2010; Zhou et al., 2011; Savino et al., 2013). Increased Ser36 phosphorylation of p66Shc is very often observed in oxidative stress–associated diabetic complications including renal failure (Sun et al., 2010). Additionally, increased Ser36 phosphorylation of p66Shc has also been correlated with the toxicity of ethanol (Koch et al., 2008), taxol (Yang and Horwitz, 2000), and doxorubicin (Marques-Aleixo et al., 2015). Moreover, higher Ser36 phosphorylation status of p66Shc has been found in fibroblasts derived from patients with diagnosed mitochondrial disorders. This indicates that oxidative stress caused by abnormalities in the mitochondrial respiratory chain is also able to promote pro-oxidative activity of the p66Shc protein (Lebiedzinska et al., 2010). On the other hand, increased tyrosine phosphorylation of p66Shc has been found to be predominantly altered in breast (Xie and Hung, 1996; Lee et al., 2004), prostate (Lee et al., 2004), and lung cancer (Li et al., 2014). Based on a growing body of evidence, there is no doubt that p66Shc is a multifunctional protein, and its final role can be determined by several of intracellular as well as external stimuli. It is reasonable to believe that p66Shc-related pathways, especially those involved in cellular responses to oxidative stress, should be investigated in the context of xenobiotic-induced cell and tissue toxicity. The protocols here described will help the researcher to investigate the status of p66Shc in the context of cell toxicity by multiple agents.

ISOLATION OF MITOCHONDRIAL AND CYTOSOLIC FRACTIONS The isolation of mitochondria from different organs has been described since the late 1940s (Hogeboom et al., 1948). In general, all isolation methods are based on differential centrifugation after tissue or cellular homogenization, followed by density-gradient centrifugations to further purify the fractions (Clayton and Shadel, 2014). The present protocol describes a method to obtain crude mitochondrial fractions from cell cultures (Fig. 25.6.2).

BASIC PROTOCOL 1

Materials Cells of interest Appropriate growth medium for cells, containing 10% fetal bovine serum (FBS) Phosphate buffered saline (PBS; APPENDIX 2A) 0.05% (w/v) trypsin-EDTA (Invitrogen, cat. no. 25300-062) Homogenization medium (see recipe) Mitochondria isolation medium (see recipe) Mitochondria resuspension buffer (see recipe)


25.6.3 Current Protocols in Toxicology

Supplement 66

Figure 25.6.2

Timing and schematic steps of crude mitochondria fraction isolation from cells.

10-cm2 cell culture dishes 15-ml conical polypropylene tubes (e.g., Corning Falcon) Refrigerated low-speed tabletop centrifuge (e.g., Sigma Model 2K15) Stirrer motor with electronic speed controller Motor-driven tightly fitting glass/Teflon Potter- Elvehjem homogenizer 30-ml polypropylene centrifugation tubes Ultracentrifuge (e.g., Beckman Coulter Optima L-100 XP) Additional reagents and equipment for pharmacological inhibition (Support Protocol 1) or knockdown (Support Protocol 2) of p66She pathway 1. Grow cells on cell culture dishes using an appropriate cell culture medium and perform the desired treatment with the xenobiotic being tested (See Support Protocols 1 and 2). 2. Remove the medium and wash cells twice with PBS. Afterwards, cover the cell layer with proper amount (3 to 5 ml) of 0.05% trypsin-EDTA and keep the plate in the incubator for 3 to 5 min at 37°C in order to harvest adherent cells. Add 3 to 5 ml of growth medium containing 10% fetal bovine serum (FBS) to inhibit trypsin. Transfer the cell suspension to 15-ml polypropylene tubes. 3. Centrifuge cell suspension for 5 min at 600 × g, 4°C, using the “low-speed” centrifuge. p66Shc Signaling in Toxicology

4. Discard the supernatant and wash the cell pellet with 2 ml PBS. Centrifuge again as described in step 3.



5. Discard the supernatant, resuspend the pellet in 3 ml of homogenization medium, and incubate for 15 min on ice. IMPORTANT NOTE: Homogenization and the following remaining steps should always be carried out at 4°C in order to avoid the activation of proteases and phospholipases. Moreover, the researcher should use pre-cooled glassware, including the homogenizer and pestle, by using an ice bath for 5 min before starting the homogenization.

6. Homogenize cells using Potter-Elvehjem homogenizer with a Teflon pestle (30 to 40 strokes). As an alternative, it is also possible to force the cell suspension 10 times through a 25-G needle using a 1-ml syringe. This process should always be performed on ice. It is important to control the cell integrity after a certain number of strokes, using a light microscope. The homogenization process is completed when 80% to 90% of cell disintegration is observed. IMPORTANT NOTE: The homogenization process must be slow and done smoothly, since when higher force and speed are used, the mitochondrial integrity can be affected.

7. Transfer the homogenate to a 30-ml polypropylene centrifuge tube and centrifuge 5 min at 600 × g, 4°C, using the “low-speed” centrifuge. 8. Discard the pellet, which contains unbroken cells and nuclei, and collect the supernatant (containing mitochondrial and cytosolic fractions), which should be kept on ice. 9. Centrifuge the supernatant collected in step 8 for 5 min at 600 × g, 4°C, using the “low-speed” centrifuge. 10. Collect the supernatant and discard the pellet (which includes unbroken cells and nuclei), and centrifuge 10 min at 7000 × g, 4°C, using the “low-speed” centrifuge. 11. Remove the supernatant, which contains the cytosolic fraction and place it on ice for further separation. Resuspend the pellet containing the mitochondrial fraction in 5 to 10 ml of mitochondrial isolation medium. The supernatant can be stored at 4°C up to 1.5 hr for further separation of the cytosolic fraction.

12. Centrifuge the mitochondrial suspension 10 min at 7000 × g, 4°C, using the “lowspeed” centrifuge. After that, discard the supernatant, and resuspend the pellet in 5 to 10 ml (depending on the pellet volume) of mitochondrial isolation medium. 13. Centrifuge the mitochondrial suspension again 10 min at 10,000 × g, 4°C, using the “low-speed” centrifuge. 14. Discard the supernatant and gently resuspend the mitochondrial pellet in approximately 2 ml of mitochondria resuspension buffer and preserve a desired volume of the sample at −80°C for further analysis (Fig. 25.6.3). Separate a part of the resuspended mitochondrial fraction for further isolation of mitochondrial-associated membranes (Basic Protocol 2). 15. To perform subfractionation of cytosol, lysosomal, and endoplasmic reticulum fractions, centrifuge the supernatant collected in step 11 for 30 min at 20,000 × g, 4°C, in an ultracentrifuge, using ultracentrifuge tubes. 16. Discard the pellet containing lysosomal and plasma membrane fractions and centrifuge the supernatant 30 min at 100,000 × g, 4°C. Afterwards, remove the pellet (endoplasmic reticulum fraction) and collect the supernatant, which contains the cytosolic fraction. The supernatant can be concentrated by lyophilization or by tangential flow filtration and stored at −80°C until further use (Fig. 25.6.3).



Supplement 66

Figure 25.6.3 Schematic representation of different types of pellet and fractions obtained in the mitochondrial isolation process. The diagram represents a typical result of cell fractionation. The pellets obtained can have different sizes, depending on the amount of starting material and cell type used.

BASIC PROTOCOL 2

ISOLATION OF MITOCHONDRIAL-ASSOCIATED MEMBRANES It has been demonstrated that p66Shc can also be found in mitochondria-associated membranes (MAM), a fraction of the endoplasmic reticulum that interacts with the mitochondrial outer membrane (Lebiedzinska et al., 2009). Basic Protocol 2 outlines the procedure for the mitochondrial-associated membranes (MAM) isolation from HeLa cells (Wieckowski et al., 2009). Other cell lines may also be used, but must be optimized to provide sufficient crude mitochondrial fraction quantity for further MAM isolation procedure.

Materials Crude mitochondrial fraction (Basic Protocol 1) MAM isolation buffer I (see recipe) Mitochondria resuspension buffer (see recipe)


Ultra-Clear 14-ml polyallomer ultracentrifuge tubes Ultracentrifuge: e.g., Beckman Coulter Optima L-100 XP with, e.g., SW 40 Ti rotor, swinging-bucket, 6 × 14 ml, and Beckman Type 70 Ti rotor, fixed-angle, 8 × 39 ml Refrigerated low-speed centrifuge 30-ml tubes for refrigerated low-speed centrifuge Polycarbonate tubes with cap assembly (Beckman, cat. no. 355618, for use with 70Ti rotor)



1. Isolate the crude mitochondria fraction as described in Basic Protocol 1. Cell culture should be handled as indicated by the supplier or by the protocol established in the laboratory.

2. Add 8 ml of MAM isolation buffer to a 14-ml thinwall, polyallomer ultracentrifuge tube (for SW40 rotor or similar). 3. Layer crude mitochondria suspension on top of 8 ml of MAM isolation buffer in the ultracentrifuge tube. Afterward, gently layer mitochondria resuspension buffer (about 3.5 to 4 ml) on top of the mitochondrial suspension to fill up the ultracentrifuge tube (the surface of the liquid should be 4 to 5 mm from the rim of the tube). 4. Centrifuge 30 min at 95,000 × g, 4°C (e.g., in a Beckman Coulter Optima L-100 XP ultracentrifuge). A dense band containing purified mitochondria will be localized near the bottom of the ultracentrifuge tube. The MAM fraction, visible as the diffused white band, is located above the mitochondrial fraction.

5. Collect the MAM fraction from the Percoll gradient with a Pasteur pipet and dilute 10× mitochondria resuspension buffer. 6. Collect pure mitochondria band in a 30-ml tube and dilute 10× with mitochondria resuspension buffer. 7. Centrifuge MAM and purified mitochondria fractions 10 min at 6,300 × g, 4°C, refrigerated low-speed centrifuge. 8. After the centrifugation of MAM fraction, discard the pellet containing mitochondrial contamination, collect and transfer the MAM-containing supernatant to a polycarbonate tubes with a cap assembly, and ultracentrifuge 1 hr at 100,000 × g, 4°C (e.g., in an ultracentrifuge using a Beckman 70 Ti rotor). 9. Discard the supernatant and resuspend MAM pellet in a small volume of mitochondrial resuspension buffer (50 to 100 μl) 10. Discard the supernatant contaminated with MAM from the tube with the mitochondrial fraction. 11. Gently resuspend the mitochondrial pellet in 20 ml of mitochondria resuspension buffer and centrifuge 10 min at 6,300 × g, 4°C, in a refrigerated low-speed centrifuge. 12. Discard the supernatant and resuspend the pellet of pure mitochondria in a small volume of mitochondrial resuspension buffer (500 μl) (Fig. 25.6.3). 13. Store the pure mitochondrial suspension at −80°C.

IMMUNOBLOTTING OF p66Shc AND pSer36-p66Shc IN DIFFERENT FRACTIONS

BASIC PROTOCOL 3

The immunoblotting technique or western blotting is used to detect proteins of interest in a complex mixture of proteins in different cellular or tissue samples using specific antibodies (Magi and Liberatori, 2005). This protocol outlines the procedure for evaluating the protein content of p66Shc and pSer36-p66Shc in mitochondrial, cytosolic, and MAM fractions prepared as previously described (Basic Protocols 1 and 2).

Materials Mitochondrial, cytosolic, and MAM fractions (see Basic Protocols 1 and 2, respectively) 10× Cell Lysis Buffer (Cell Signaling, cat. no. 1679803S)



Supplement 66

Phenylmethylsulfonyl fluoride (PMSF) Protease and phosphatase inhibitors (Sigma-Aldrich, cat. no. P8340 and P5726, respectively) Bradford reagent (see recipe) 6× Laemmli buffer (see recipe) 10× SDS-PAGE running buffer (see recipe) Standard protein marker (e.g., Precision Plus Protein Dual Color Standard; BioRad, cat. no. 161-0374) Methanol 10× transfer buffer (see recipe) Bovine serum albumin (BSA; Sigma-Aldrich, cat. no. A6003) 10× Tris-buffered saline/Tween 20 (TBST; see recipe) Mouse monoclonal anti-SHC/p66 – pSER36 (Calbiochem, cat. no. 566807) Goat anti-mouse IgG-AP (Santa Cruz Biotechnology, cat. no. sc-2008) ECF substrate (Thermo Fisher Scientific, cat. no. RPN3685) Mouse monoclonal anti-SHC (BD Bioscience, cat. no. 610879) Anti-actin antibody, clone C4 (Millipore, cat. no. MAB1501) 15- and 50-ml disposable polystyrene tubes (e.g., BD Falcon) 0.5- and 1.5 ml microcentrifuge tubes (e.g., VWR) Accublock digital dry bath (LabNet international, Inc.) Polyvinylidene difluoride (PVDF) membrane (Millipore, cat. no. IPVH00010) or nitrocellulose membrane (also see Gallagher et al., 2008) Mini Trans-Blot Cell (BioRad, cat. no. 170-3930) Shaker plate BioSpectrum multispectral imaging system (UVP) Absorbance plate reader (e.g., Victor X3 Multilabel Reader, PerkinElmer) Computer running Microsoft Excel, Quantity One 4.6.6 (BioRad), NIH ImageJ, and statistical software analysis Additional reagents and equipment for protein assay (APPENDIX 3I; Krohn, 2005), SDS–polyacrylamide gel electrophoresis (APPENDIX 3F; Gallagher, 2007), immunoblotting (Gallagher et al., 2008) Prepare samples 1. Isolate mitochondrial, cytosolic, and MAM fractions (See Basic Protocols 1 and 2, respectively). 2. Keep all fractions and reagents used on ice at all times. 3. Dilute 10× Cell Lysis Buffer to 1×. Divide into 1-ml aliquots. The 1× aliquots can be stored at −20°C.

4. Add 1 mM phenylmethylsulfonylfluoride (PMSF), as well as a 1× protease and phosphatase inhibitors . Dilute the protease and phosphatase inhibitors 1:100 in the Cell Lysis Buffer.

5. Dilute all fractions 50 to 100 μl of 1× Cell Lysis Buffer with PMSF and protease/phosphatase inhibitors. 6. Determine the protein concentration of mitochondrial, cytosolic, and MAM fractions using the Bradford assay (Bradford, 1976) or other similar technique (see APPENDIX 3I; Krohn, 2005) using a spectrophotometer or UV/visible plate reader. p66Shc Signaling in Toxicology

Use bovine serum albumin (BSA) as a standard. Make stock solution of 1 mg/ml BSA and store at −20°C.



Table 25.6.1 10% SDS-Polyacrylamide Gel Recipea

Running gel (10%)

Stacking gel (4%)

1.5 M Tris·Cl, pH 8.8

5 (ml)

—

0.5 M Tris·Cl, pH 6.8

—

2.5 (ml)

40% acrylamide/bisacrylamide

5 (ml)

1 (ml)

Milli-Q H2 O

9.8 (ml)

6.4 (ml)

10% SDS

200 (μl)

100 (μl)

10% APS

100 (μl)

50 (μl)

TEMED

10 (μl)

10 (μl)

a Abbreviations:

APS, ammonium persulfate; SDS, sodium dodecyl sulfate; TEMED, tetramethylenediamine.

7. Make sure all samples are at the same concentration by accounting for the amount of protein (determined previously) and add the same amount of Laemmli buffer to each sample using 6× Laemmli stock buffer. Prepare all samples at the same concentration. Denature samples at 95°C during 5 min in an Accublock digital dry bath or equivalent.

Perform gel electrophoresis 8. Separate proteins electrophoretically in 10% SDS-polyacrylamide gel (also see APPENDIX 3F; Gallagher, 2007) as described in steps 9 to 11). In the day prior to immunoblotting, prepare the 10% SDS-polyacrylamide gel (Table 25.6.1) and store it overnight in running buffer at 4°C. Alternatively, prepare it the same day.

9. Dilute the 10× running buffer 1×. Assemble the SDS-PAGE system (Gallagher, 2007). Put the previously prepared gels in the system and fill it to the top with 1× running buffer. Different protocols for immunoblotting procedures can be found online in different vendors (e.g., BioRad).

10. Load samples and protein weight marker e.g., Precision Plus Protein Prestained Standards (10 and 250 kD) in the desired sequence. Add 15 to 50 μg of protein in each well. Add 5 μl of standard protein marker in one well. Sample protein amount may require optimization.

11. Run protein separation at 120 to 200 V.

Perform immunoblotting Also see Gallagher et al. (2008). Figure 25.6.4 shows a schematic representation of the immunoblotting procedure. 12. Pre-soak the membrane in methanol, for polyvinylidene fluoride (PVDF) membranes, or in 1× transfer buffer, for nitrocellulose membranes. Prepare the transfer sandwich (Gallagher et al., 2008), avoiding air bubbles between gel and membrane, as this can affect proper transfer of proteins from the gel to the membrane. Note that the transfer to membrane must be performed at 4°C, with subsequent storage at that temperature.

13. Block the membrane during 1 to 2 hr at room temperature with 5% BSA in 1× TBST, or as described by the antibody vendor manufacturer.



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Figure 25.6.4 General schematic representation of the immunoblotting (western blotting) procedure (western blotting).

14. Add the primary antibody against phosphorylated p66Shc (mouse monoclonal antiSHC/p66–pSER36 ) prepared in 2% BSA in 1× TBST. Incubate in antibody overnight at 4°C with constant gentle agitation on a shaker plate. We usually use 1:1,000 dilution, although this may require optimization.

15. Wash membrane three times, each time for 5 min, with 1× TBST at room temperature under constant gentle agitation. 16. After discarding the wash solution, add the secondary antibody (goat antimouse IgG-AP) prepared in 2% BSA in 1× TBST, for 2 to 3 hr at 4°C. Use a secondary antibody dilution of 1:2500 or higher, although this requires optimization to guarantee an optimal antibody/protein ratio.

17. Wash the membrane three times, each time for 5 min, with 1× TBST at room temperature with constant gentle agitation. For the detection of immunolabeled proteins we normally use the ECF reagent (Thermo Fisher Scientific), according to the manufacturer instructions.

18. Incubate the membrane with ECF reagent and detect the luminescent signal using an imaging system (e.g., UVP BioSpectrum). 19. Rinse the membrane with methanol after using ECF, then add a primary antibody against total p66Shc (mouse monoclonal anti-SHC) prepared in 2% BSA in 1× TBST overnight at 4°C with constant gentle agitation. p66Shc Signaling in Toxicology

We normally use a 1:1,000 dilution of the total anti-p66Shc antibody, although this requires optimization.

20. Repeat steps 14 to 17 to evaluate total p66Shc protein.



21. Repeat steps 18 to 19 using a protein-loading control antibody instead of antibody against p66Shc (e.g., anti-actin). The researcher can also use different antibodies to evaluate the content of other proteins of interest. The researcher can also use different gel loading protein controls depending on the sample origin.

Data analysis 22. Save obtained images in a file format supported by the software enabling banddensity quantification (e.g., JPEG or TIFF for NIH ImageJ). 23. Calculate the ratio between the total p66Shc and pSer36-p66Shc band density value, as well as the total p66Shc band and the protein being the gel loading control. The ratio between the phosphorylated/total forms will be an indication of activation of the p66Shc pathway.

IMMUNOBLOTTING FOR ACTIVITY OF p66Shc PATHWAY-ASSOCIATED PROTEINS

ALTERNATE PROTOCOL

This protocol summarizes the procedure for evaluating levels and activity related modifications (e.g., phosphorylation, cleavage) of p66Shc-associated proteins from crude mitochondria, cytosolic, and MAM fractions.

Additional Materials (also see Basic Protocol 3) Antibodies against p66Shc-associated proteins of interest (Table 25.6.2) 1. Repeat steps 1 to 17 of Basic Protocol 3 to analyze samples with the use of proper antibodies against the proteins described in Table 25.6.2.

PHARMACOLOGICAL INHIBITION OF p66Shc PATHWAY As described above, p66Shc gains a pro-oxidant function under certain conditions, promoting cellular death and resulting in tissue damage associated with numerous pathologies and chemical toxicity (Diogo et al., 2013). Therefore, inhibition of Ser36 p66Shc phosphorylation is a viable strategy to attenuate the harmful activity of p66Shc.

SUPPORT PROTOCOL 1

Table 25.6.2 Examples of Antibodies to Use in the Alternate Protocol

Protein/antibody against

Vendor

Catalog number

Bim

Cell Signaling

2819

Forkhead box O3 (FoxO3a)

Abcam

ab4709

Forkhead box O3 (FoxO3a, phosphoSer253)

Abcam

ab47285

Pin1

Millipore

07-091

Protein kinase B (PKB) or AKT (phosphoSer473)

Cell Signaling

9271

Protein kinase B (PKB) or AKT

Cell Signaling

9272

Protein kinase Cβ (PKCβ)

Santa Cruz Biotechnology

sc-210

Protein phosphatase 2 (PP2A)

Millipore

05-421

Ras

Cell Signaling

3965

Superoxide dismutase 1 (SOD 1)

Santa Cruz Biotechnology

sc-11407

Superoxide dismutase 2 (SOD 2)

Abcam

ab13533



Supplement 66

Figure 25.6.5 p66Shc pharmacological inhibition (A) or silencing (B). p66Shc has a relevant role in oxidative signaling, contributing to an increase in cell oxidative stress. Thus, pharmacological inhibition of p66Shc phosphorylation at Ser36 is a valid strategy to block p66Shc pathway decreasing ROS production. Use of hispidin, a PKCβ inhibitor, or gene silencing by RNA-based approaches, will increase the resistance to oxidative stress-induced cell death, which is relevant in the context of drug-induced toxicity. During pharmacological inhibition or silencing, p66Shc is not activated, remaining in inactive form. Inhibition of p66Shc signaling decreases the amount of H2 O2 produced by the mitochondrial respiratory chain. Abbreviations: ADP, adenosine diphosphate; ANT, adenine nucleotide translocator; ATP, adenosine triphosphate; CK, creatine kinase; CyD, cyclophilin D; Cyt C, cytochrome c; FAD, flavin adenine dinucleotide; HK, hexokinase; H2 O2 , hydrogen peroxide; IMM, inner mitochondrial membrane; MTHSP70, mitochondrial heat shock proteins, 70 kDa; NADH, nicotinamide adenine dinucleotide; OMM, outer mitochondrial membrane; PBR, peripheral benzodiazepine receptor; PKC-β, protein kinase C–β isoform; Prx1, paired related homeobox 1; Q, coenzyme Q; ROS, reactive oxidative species; TIM, mitochondrial translocase of the inner membrane; TOM, mitochondrial translocase of the outer membrane; VDAC, voltage-dependent anion channel.

Several studies have shown that hispidin-specific PKCβ inhibitor prevents Ser36 p66Shc phosphorylation and thus reduces oxidative stress in the cell (Fig. 25.6.5); Lebiedzinska et al., 2010; Suski et al., 2011; Lebiedzinska et al., 2013). The following protocol outlines a simple procedure to inhibit p66Shc pro-oxidant activity in the cells. This protocol describes the procedure for serine 36 p66Shc phosphorylation inhibition using hispidin, performed using the H9c2 cardiomyoblast cell line as an example. Normally, we culture this cell line in DMEM supplemented with 10% FBS, 1× penicillinstreptomycin, and 1.5 g/liter sodium bicarbonate, and maintain in the atmosphere of 95% air and 5% CO2 at 37°C. Other cell lines likely can be used after culturing as specified by the supplier.

Materials


Cells of interest (e.g., H9c2 cardiomyoblasts, ATCC-CRL-1446) High-glucose DMEM (Sigma-Aldrich, cat. no. D-5648) containing 10% FBS (Invitrogen, cat. no. 16000-044), 1× penicillin-streptomycin (Invitrogen, cat. no. 15140-122) and 1.5 g/liter sodium bicarbonate (Sigma-Aldrich, cat. no. S5761) Xenobiotic(s) of interest 10-cm2 cell culture dishes



10 mM hispidin stock solution (see recipe) Dimethyl sulfoxide (DMSO; Sigma-Aldrich, cat. no. W387509) 1. One day before treatment with the desired xenobiotic, seed the cells in 10-cm2 cell culture dishes with the necessary cell density (normally aiming to achieve 80% confluence upon cell treatment). 2. Perform the treatment with the desired xenobiotic. 3. Afterwards, treat cells with 5 μM of 10 mM hispidin or 0.05% (v/v) DMSO as the vehicle control.

KNOCKDOWN OF p66Shc IN CELL LINES This protocol outlines the procedure for p66Shc knockdown. The most commonly used applications of RNA interference (RNAi) are mediated by two types of molecules: smallinterfering RNA (siRNA) (Fig. 25.6.5) and short hairpin RNA (shRNA) (Rao et al., 2009). The choice of the method depends on the factors such as cell type, integration type and delivery to the cell. Moreover, the efficiency of knockdown differs between siRNA and shRNA methods.

SUPPORT PROTOCOL 2

The choice of appropriate p66Shc gene construct is the first crucial step of the knockdown procedure. Secondly, it should be noted that the outcome of the RNAi technique is limited by the transfection capacity of the cells (Moore et al., 2010). Here we present a protocol suitable for adherent immortalized cell lines e.g., HeLa, 3T3, which uses Lipofectamine as the delivering agent for the p66Shc silencing construct.

Materials Cell line of interest Appropriate medium for cell line of interest p66Shc-constructs (siRNA, shRNA) Constructs with random sequence Opti-MEM medium (Invitrogen, cat. no. 11058-021) Lipofectamine 2000 (Invitrogen, cat. no. 11668-019) 24-well cell culture plates 0.5 ml Eppendorf microcentrifuge test tubes (e.g., VWR) Prepare cells 1. Seed the cells in to two independent 24-well plates (0.5–2 × 105 cell/well) the day before transfection. 2. Use one of the 24-well plates for the p66Shc transfection procedure while the second plate will be used for the transfection-negative control. The negative control can be a random sequence in vector/plasmid.

3. Incubate cells overnight under standard conditions characteristic for the cell type used to achieve 50% to 80% of confluence. Use medium without antibiotics.

Prepare Lipofectamine and DNA 4. Prepare dilutions of the following Lipofectamine concentrations: 2 μl, 3 μl, 4 μl, 5 μl in 50 μl of Opti-MEM medium. This will indicate the optimum amount for the cells of interest. Mix gently. 5. Prepare dilution of p66Shc-construct (p66-siRNA or p66-shRNA) at a concentration of 0.5 to 5 μg/μl in 250 μl of Opti-MEM medium. Mix/vortex gently.



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6. Mix both diluted solutions in 1:1 ratio. Vortex gently. 7. Incubate for 5 min at room temperature.

Perform transfection 8. Remove old medium from the cells and replace it with 250 μl Opti-MEM medium. 9. Add p66Shc construct-lipid complex to the cells (500 ng per well). 10. Incubate the cells for 4 to 6 hr (optimizing for the used cell lines) at 37°C in a 5% CO2 incubator. If low transfection efficiency is obtained, an antibiotic selection of transfected cells can be performed.

11. Add 0.5 ml of growth medium with serum without removing the transfection mixture (serum level is characteristic for the cells of interest). 12. Confirm p66Shc silencing by immunoblotting (see Basic Protocol 3).

REAGENTS AND SOLUTIONS For the preparation of each solutions, ultrapure distilled water (conductivity