Supplemental Data The Aryl Hydrocarbon Receptor

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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.