population. TKC. T1. T1 cells bearing a scrambled shRNA knockdown control. ... Klf2. AAGAGCTCGCACCTAAAGGC. CTTTCGGTAGTGGCGGGTAA. 123. Lhx5.
Supplemental Data The Aryl Hydrocarbon Receptor Represses Mid1 and Delays Mitotic Progression in Undifferentiated Embryonic Stem Cells Causing Loss of Pluripotency Chia-I Ko, Yunxia Fan, Matthew De Gannes, Qin Wang, Ying Xia and Alvaro Puga¶
Supplemental Table ST1. Mouse embryonic stem cell lines. Cell line C2
Background C57Bl/6J Ahr+/+
T1
C57Bl/6J Ahr-/-
OCT4-GFP
R1 (129/SV x 129/SVJ)
AH
C2
TAH
T1
AE
AH
BA
C2
TBA
T1
PB
BA
TKC TMK
T1 T1
Description of genetic modification C57Bl/6J ES cells bearing the high-affinity Ahrb1 allele. ES cells from Ahr-/- mice bred for 14 generation into a C57Bl/6 genetic background. R1 ES cells expressing GFP under the control of Oct4 promoter. C2 cells contain a randomly integrated puromycinresistance-IRES2-eGFP cassette under control of the Cyp1a1 promoter. T1 cells contain a randomly integrated puromycinresistance-IRES2-eGFP cassette under control of the Cyp1a1 promoter. AHR-expressing ES cells obtained from AH cells after 3 days of puromycin treatment. C2 cells contain a randomly integrated puromycinresistance-IRES2-eGFP cassette under control of actin promoter. T1 cells contain a randomly integrated puromycinresistance-IRES2-eGFP cassette under control of actin promoter. ES cell population obtained from BA cells after 3 days of puromycin treatment. Control ES cells for AE cell population. T1 cells bearing a scrambled shRNA knockdown control. Mid1 knockdown by shRNA in T1 cells.
Supplemental Table ST2. List of primer sequences. Target genes Ahr Atbf1 Bmp2 Bmp4 Brachyury (T) Cd31 Creb3l2 Dlx5 Esrrb Fgf4 Fn1 Foxa2 Gapdh Gata4 Gata6 Gdf3 Gfap Hand1 Hoxb1 Isl1 Klf2 Lhx5 Meis1 Mid1 Myf5 Myh7 Nanog Nes Neurog Nkx2.5 Notch1 Oct4 Onecut1 Otx1 Pax6 Pdgfra Shox2 Snai1 Sox1 Sox2 Sox17 Tcf23 Tubb3
Primer sequence Forwards
Reverse
GGCCAAGAGCTTCTTTGAATG
TGCCAGTCTCTGATTGTGC
CCAGATCTTCACCATCCGCA
GGGTCCTTCGGCATCTTCAA
CCAGCACCAGCACCAGCCAACT
TGGGGCGTTAACTGCATCTGGC
AGGAGGAGGAGGAAGAGCAG
CACTGGTCCCTGGGATGTTC
ACCATTGCTCACAGACCAGAGACT
TGCTGCAGTCCCATGATAACTGGT
GAAAGCCAAGGCCAAACA
TCCAGAAACATCATCATAACCG
TCACCATTTACCCATGCGGC
TGGCTCTGTCTTGATGGTAGC
CCAGTACCACGGCGTGAA
CTCCGCCACTTCTTTCTCTGG
GGGATGCTGAAGGAAGGTGT
TTAGCAGGTGGGGAAATCGG
TGCCCTGTTCTGATGGGATTCTCT
AAGGGAGCTAGCTGGCTGAAGAAA
AGGATTGGCTTCAAGCTGGGTGTA
TCGCAGGACTTGGATGGTGTAAGT
CATGAACTCGATGAGCCCCA
TGTAGCTGCGTCGGTATGTC
AACTTTGGCATTGTGGAAGG
GGATGCAGGGATGATGTTCT
AGTGGTGCCTCCAGCGGTAACT
CCGGACACAGTACTGAATGTCTGGGA
AGTGGCTCTGTCCCTATGACT
CGTTTTCTCCCACTGCAGAC
TATGCTACGTGAAGGAGCTGGGT
AGACAGCAGCCATCTTGGAAAGGT
ACGCTTCTCCTTGTCTCGAA
AAGCGGACCTTCTCGATGTA
GGCTACCAGTTACATCGCCT
CTGCTGAGGCAACTCCCTTT
AATCGCCTTGCTCGTCAGAA
CGGACACTTCGCTGTCTTA
GCAAGGACAAGAAACGCAGCATCA
ACTGGGTTAGCCTGTAAACCACCA
AAGAGCTCGCACCTAAAGGC
CTTTCGGTAGTGGCGGGTAA
GTCTCAACTGGGTGTCGTCC
TTTGGGGTCGTCCTGTAACG
GGAAAATGCCTATCGATTTGG
AGTGGATGCCGTGTCAT
GGCAACGTGTTCATCGACAG
CTTCCCGATCCACTCGTGTT
TGACGGCATGCCTGAATGTA
CAATCCAAGCTGGACACGGA
TGCAAAGGCTCCAGGTCTGAGGGC
GCCAACACCAACCTGTCCAAGTTC
AGGGTCTGCTACTGAGATGCTCTG
CAACCACTGGTTTTTCTGCCACCG
GCCAGCACTCCCATCCCACCT
AGCCTGTTGCCTCCCACCCTG
CAGGACGAAGAGCAGGAACG
GAGGTTGTGCATGCGGTTG
CGACGGAAGCCACGCGTGCT
CCGCTGTCGCTTGCACTTG
TCTGCTTATGCCTCAAGGGAACCA
AATCTTGTCCAGACAGGTGCCAGA
GGCGTTCTCTTTGGAAAGGTGTTC
CTCGAACCACATCCTTCTCT
AGCAAACTCAAGTCGGGTCG
CTTTTTGGGGGTGTTGCCTC
CAGACATCTTCATGCGCGAG
TTCGTTCCATTCCCGCTCTG
TTCTGCAGGTATCCAACGGT
TCTTGGCTTACTCCCTCCGA
CACCGGATGGTACACTTGCT
GTCATCCCGAGAGGCACAAA
GGTGTCCTTATAGGAGCCGC
CTGAAAGGACAAGGGCGTCA
GTCTGCACGACCTGTGGAAA
GGTCAGCAAAAGCACGGTTG
CCCATGCACCGCTACGACAT
CGCTCATGTAGCCCTGAGAGT
ACAGATGCAACCGATGCACC
TGGAGTTGTACTGCAGGGCG
CTGCACAACGCAGAGCTAAG
GCTTCATGCGCTTCACCT
GAGGGACCAGAGCTAGGGGA
AGAAAGGCCTGACGAAGCGT
TTCTGGTGGACTTGGAACCTGGAA
TCACACTCTTTCCGCACGACATCT
Amplicon size (bp) 93 70 149 126 131 133 111 148 119 100 138 80 132 146 133 106 162 139 95 145 123 77 134 117 125 203 364 108 132 180 101 313 150 124 98 120 126 118 70 53 172 103 184
Supplemental Table ST3. List of primary antibodies Target protein 14-3-3 AHR α4 βactin CDC25B CDK1 CDK2 Cyclin A Cyclin B EF1α Histone H3 MID1 NANOG OCT4 phS10H3 pS323CDC25B pY15CDK1 PP2A, B55α subunit PP2A, C subunit SOX2 WEE1
Manufacturer Sigma-Aldrich Abcam Proteintech Sigma-Aldrich BD Biosciences Abcam Santa Cruz Biotechnology Santa Cruz Biotechnology Abcam Millipore Abcam Sigma-Aldrich Millipore Santa Cruz Biotechnology Cell signaling LifeSpan BioSciences Cell Signaling Technology Santa Cruz Biotechnology Millipore Cell Signaling Technology Santa Cruz Biotechnology
Reference HPA007925 ab84833 14952-1-AP A1978 610528 ab32384 sc-163 sc-751 ab2949 05-235 Ab1791 AV34680 AB5731 sc-9081 9701 LS-C380653 9111 sc-81606 05-421 2748 sc-325
Supplemental Figure S1
Supplemental Figure S1. AHR expression in ES cells. (A) Expression of AHR in T1 (Ahr-/-) and C2 (Ahr+/+) cells was detected by immunostaining and quantified in the bar graph next to the images. (B) Map of the pAHRPuroIRESeGFP vector, showing Cyp1a1-promoter driven expression of eGFP and the Pac gene, encoding puromycin resistance. (C) eGFP expression in TAH and TBA cells driven by the Cyp1a1 and beta actin promoters, respectively. Scale bars indicate 10 μm. Data represent the means ± SD of at least two experiments. * indicates statistical significance, relative to T1 cells, at p-value ≤ 0.05.
Supplemental Figure S2
Supplemental Figure S2. Correlation of eGFP and AHR expression in C2-derived ES cells. (A), AHR expression in AH, AE, BA, and PB cells detected by immunostaining compared to the expression of eGFP. (B), Ahr transcript levels measured by real-time PCR. Scale bars indicate 10 μm. Data represent the means ± SD of at least two experiments. * indicates statistical significance, relative to AH cells, at p-value ≤ 0.05.
Supplemental Figure S3.
Supplemental Figure S3. Mutual exclusion of gene expression between OCT4 and AHR. (A, D): Expression of AHR detected by immunostaining in (A), ES cells and (D), cells that have differentiated for 9 days, where GFP denotes OCT4 expression. (B, E): Bar graphs represent the percent of OCT4-negative (OCT4-), OCT4-positive (OCT4+), AHR-negative (AHR-), and AHRpositive (AHR+) cells in (B), ES cells and (E), differentiated cell population. (C, F): Stacked bar graphs represent the percent of AHR- and AHR+ cells in both OCT4- and OCT4+ (C), ES cells and (F), differentiated cell population. (G): Ahr mRNA levels were determined by real-time RTPCR in Ahr-/- cells with ectopic Ahr expression (indicated as AHR) relative to cells transfected with a control vector (Script). (H): OCT4 expression detected by immunoblotting in Ahr-/- cells with ectopic Ahr expression and cells transfected with a control vector. All experiments were
done in biological duplicates or triplicates; data are presented as means ± SD. Comparisons were made by unpaired t-test followed by Bonferroni test to compare variances. A p-value equal to or less than 0.05 was considered statistically significant; *, **, and *** indicate the statistical significance at p-value ≤ 0.05, ≤ 0.01, and ≤ 0.001, respectively.
Supplemental Figure S4.
Supplemental Figure S4. AHR expression disrupts ES mitotic progression. (A) Cell proliferation profile of Ahr-knocked down (shAhr) C2 cells and cells bearing a scrambled control shRNA. (B, F) pS10H3 expression detected by immunostaining in (B): AH and AE and (F): C2 and T1 cells. Arrowheads indicate cells that cycle at metaphase. Scale bars indicate 10 μm. (C, G) pS10H3
expression as measured by immunoblotting in (C) AH and AE and (G) C2 and T1 cells. (D)
Expression of mitotic regulators detected by immunoblotting in AH and AE cells arrested at and released from M-phase. (E) B55α-subunit specific PP2A phosphatase activity in AH and AE cells.
All experiments were done in biological duplicates or triplicates; data are presented as
means ± SD. Comparisons were made by unpaired t-test followed by Bonferroni test to compare variances. A p-value equal to or less than 0.05 was considered statistically significant; *, **, and *** indicate the statistical significance at p-value ≤ 0.05, ≤ 0.01, and ≤ 0.001, respectively.
Supplemental Figure S5.
Supplemental Figure S5. Regulation of MID1 subsequent to changes of AHR expression. MID1 expression as measured by immunoblotting in (A): T1 cells with ectopic Ahr expression (indicated as AHR) and cells transfected with a control vector (Script); and in (B) Ahr-knocked down (shAhr) in C2 cells and cells bearing a scrambled control shRNA (Scb.). All experiments
were done in biological duplicates or triplicates; data are presented as means ± SD. Comparisons were made by unpaired t-test followed by Bonferroni test to compare variances. A p-value equal to or less than 0.05 was considered statistically significant; *, **, and *** indicate the statistical significance at p-value ≤ 0.05, ≤ 0.01, and ≤ 0.001, respectively.
Supplemental Figure S6.
Supplemental Summary Figure S6. Schematic illustration of pluripotency and mitotic
progression regulation in AHR-positive and AHR-negative mouse ES cells. In pluripotent cells where Ahr is repressed, MID1 interactions with the elongation factor EF1α and the PP2A phosphatase complex maintain pluripotency and cell cycle progression. Untimely Ahr upregulation represses Mid1 and decreases the expression of PP2A-B55α and 14-3-3, blocking the inhibitory CDC25B degradation. Decreased PP2A-B55α and increased CDC25B expression levels dephosphorylate pY15-CDK1, accelerating mitotic entry. Downregulation of MID1 expression may also lead to failure of MID1-EF1α interactions and directly regulate expression of pluripotency factors and the pluripotency of AHR-
expressing cells. In contrast, AHR-negative cells upregulate MID1, PP2A-B55α, 14-3-3, and OCT4 expression and have elevated rates of mitotic completion.