Guillemette_Supplemental Table 1 Gene Mature Sequence Gene Mature Sequence ATTCATAGGATGTCAGCAG LOC157193 TATGCGTAAGTAATAAATTGCT TTAGTTCTGTCTTGGAGG LOC152225 CGCATATATGTTCTGACTT ALDH2 CACTTCAGTGTATGCCT LRRC1B ATCCCTGAATCTGGATGCAA ALG6 AACAGTGTCTCTTAATTCTT LRRC8B ACTCAAAGACATTGTCTGGG ANKRD62 ACGTCGATTGGAGGACATTG MKI67 AGGCTACAAACTCCTAAGGAAA ARMCX5 CGGTCAATAATCCCAATGTTA MYO15B TGTCCAGGGCACGCTGAGGT ASXL2 CGCCTTGAAGATTCTAAG NCRNA294 CGCTATTAATAAGTTTGACTTA ATP11B CTATAAATTTCTGAGGG NOL4 TTAGACCTCAGTGAATATGG BICD1 TAACAGGAGAGATTGTGGT NOL4 TTCATCTACAAACAGCCTGAC BMS1 ACCTCATCTTCTGCTAAG NR4A2 AGCACATGATCGAGCAGAGGAA C1orf81 AACAAAGGAAATATGCCT PDLIM5 CTGCCAGTAAGAGATGTTA C9orf23 CGCCAAGTTCTGCATTGC PLCL1 AACTTTAACCTGATCTTGGT CCDC136 CGGCTAAGTCCTCCAAATG PMS2L2 CCCGGTGCCCATTAGATTTCT CDC6 AGCTTTGTTCAATGCCCGG POLK CGGACATTCAGTGAGATAAATA CDO1 AGAGACCAAAGTATTTCCG PPP1CC TTCATTCTCCTGAAGCTGGG COL28A1 TGATCTGAAAGTTCTCTGGT PPP2CB CGGGAATGCCAACGTTTGG COMMD1 CGGAGTTGTTTGATTTCTA PRMT5 CATTGTCAGCAAATGAGCT CPEB1 AAGTATCCCATTTGTTCT PSMC1 CAATAAACACGATGGACGGG CPNE3 TATAGTCAAGGAATGTG PTGES CTTAAATAGAGTCTCCCTTT CRLS1 CCCACTTACTTACATGATC RASSF9 CAGCTCTAATTTAGAGAGTG CXorf23 CCCTAAAGACGTGGAGA REV3L ATCTTAGCAGAAGATGAGGT CYP8B1 ACCTCTTTCGCTTCTGCTA RNF2 TTCTGGAAATTTGATACGG DDX1 TATAACATAAGGAACACCGG SCIN CGCTCATTATTTACAAG DENND2C ACCCAGTGGGATGAATAGA SETDB1 ACCACAAGGTGTCTTACC DGKG CGCAGCCATGTTGCACGA SFPQ ACCATTCATGCTTCTATGC EFCAB2 GACAGGCTCCTTTATCAGGT SHCBP1L CGCTGATGAAGTTATGGG EIF4B TAATCCTCGAAAGAGTTCCG SLC15A4 ACATGTTGCCTGGATTGATTTC EIF4H AAGCTATATGACAGAAAGT SLC7A11 CGATGAAACTGTCCAGATT ELL AATAAATCCTCTCTCACCT SLCO4A1 AGGAGGAACTTGCATAAATATA ETV3 CGCAGTGTCTTTGATCTGCAA SPA17 CAGATTTGATGTTGTGAA ETV3L CCCTGCATAAGACCAAAGGC SUMF1 AAACCGCCTTGTGAGACC FAM48B1 ATTTCAATGATATTCTCTGCT TNFRSF1A CCCTCTTCATTGGTTTAATG FECH TCAACTCCACACTCCTTGGT TTC38 ACCAAAGAGGCTTTATC FMO9P ATGGAAGTCTGTAGTAGT UBE2D4 GGGTACCAGAGACAGACCT FUT9 AATTTATGGATTACTGACCT UBE2G2 AGGTATTATACCTATGTTG HECTD1 ACCGAACAGTGTGTAAAGGTAA USP44 TTCTCAATCCTGTTACACC HSD17B12 ATATTTCATCTGGCAGTGGG VDBP GAGAATTACTTTCACAGG HSPA5 AGTATCTCCATTAGTGGCT WDHD1 GCTCTCATATCTGTG HTR3A TTTCTGCATAGTAGTTACTGCG WDR52 AGTGAGAGGAGAAACAGCT IL2RB AAGATACAGCAGCCAAG ZBTB38 TGTAGTTGTAGTAAGGGCGT KCNH6 TATCTTGGAGTTCAGTGGCT ZNF154 ACCTGAATTGAACATCCTA KCNJ5 ACCATGGTGAATGTTACC ZNF56 ACATTCAGACGTGTTGCCTT KIAA195 CTGAAGGAGGAGCACCTGGG ZNF673 TAATGGAAGCATACTTTCCG KIAA355 CGCCGCAGGGACCTAGAAA KIAA494 TTGTCAAGTTCTGATGTGGT KIAA556 TAATGGAAGCATACTTTCCG Supplemental Table 1. List of genes and mature shrna sequences identified from the shrna screen in BRCA2mutant PEO1 cells.
Guillemette_Supplemental Fig 1. A B PEO1 cells βactin PEO1 cells % Surviving Colonies 12 sh3 1 sh4 8 6 4 2 C D.25.5.75 1 Cisplatin [μm] PEO1 cells +GFP sh+gfp sh+gfpha 12 % Survival 1 HA GAPDH 2 8 6 4 2 sh 3 UTR PEO1 cells.5.75 1 µm Cisplatin F MCM7 PEO1 Cells G H PEO1 cells 12 shmbd21 1 shmbd22 8 CHD3 MCM7 6 4 PEO1 Cells 2 PEO1 cells 12 % Surviving Colonies MBD2 % Surviving Colonies E shchd31 1 shchd32 8 6 4 2.5 Cisplatin [μm].75.5.75 Cisplatin [μm] Supplemental Figure 1. The toxicity of cisplatin in PEO1 cells is not altered by depletion of other NuRD complex members (A) PEO1 cells containing two unique plko.1 shrna vectors (3 or 4) targeting or nonsilencing control (NSC) were analyzed by immunoblot and (B) were left untreated or cisplatin treated and analyzed for colony survival. (C) PEO1 cells containing a plko.1 shrna vector targeting the 3 UTR of or NSC were transfected with GFP or GFPHA tagged and analyzed by immunoblot. (D) Cells were left untreated or cisplatin treated and analyzed for survival using a Coulter Counter 5 days after treatment. (E) PEO1 cells containing two unique plko.1 shrna vectors targeting MBD2 or NSC were analyzed by immunoblot and (F) were treated as in B. (G) PEO1 cells containing two unique plko.1 shrna vectors targeting CHD3 or NSC were analyzed by immunoblot and (H) were treated as in B. Where shown, error bars represent the standard deviation of the mean of three independent experiments.
Guillemette_Supplemental Fig 2. A.5 μm Cisplatin Untreated 24 h 36 h BRCA2mutant CAPAN1 + S/G2: 43% 63% 71% Sub G1 5 1 15 2 25 5 1 15 2 25 5 1 15 2 25 BRCA2revertant BRCA2mutant CAPAN1 + sh C25 + 35% 5 1 15 2 25 29% 53% 53% 5 1 15 2 25 5 1 15 2 25 57% 55% 5 1 15 2 25 5 1 15 2 25 5 1 15 2 25 B.5μM Cisplatin Untreated 24 h FAD1 + S/G2: 72% 82% 5 1 15 2 25 5 1 15 2 25 FAD1 + sh 64% 69% 5 1 15 2 25 5 1 15 2 25 Supplemental Figure 2. depletion alleviates cisplatin induced cell cycle progression defects in BRCA2mutant cells (A) CAPAN1 and (B) FAD1 cells were left untreated or cisplatin treated and analyzed for cell cycle with propidium iodide.
Guillemette_Supplemental Fig 3. % Surviving Colonies % Surviving Colonies % Surviving Colonies A C42 cells B 12 12 1 8 6 4 2 D 12 G 1 2 1 8 6 4 2 Olaparib [μm] sh1 sh2 1 2 12 1 8 6 4 2 PEO1 cells sh1 sh2 Olaparib [μm] PEO1 cells sh % Surviving Colonies % Surviving Colonies 1 8 6 4 2 E 12 H % Surviving Colonies C42 cells sh1 sh2 25 5 1 2 5 1 8 6 4 2 12 1 Zeocin [µg/ml] PEO1 cells sh1 sh2 25 5 1 2 5 8 6 4 2 Zeocin [µg/ml] PEO1 cells sh C F % Surviving Colonies % Surviving Colonies 12 1 8 6 4 2 C42 cells sh.25.5 12 1 8 6 4 2 Aphidicolin [μg/ml] PEO1 cells sh.25.5 Aphidicolin [μg/ml].6251.25 2.5 5 1 6Thioguanine [μm] 32.5 62.5 125 25 5 Melphalan [μg/ml] Supplemental Figure 3. depletion has differential effects in BRCA2 proficient verses BRCA2mutant cells (A) BRCA2 revertant C42 cells containing plko.1 shrna vectors targeting or NSC were left untreated or treated and analyzed for colony survival in response to the PARP inhibitor olaparib and (B) zeocin, (C) aphidicolin. (D) PEO1 cells were treated as in A and analyzed for colony survival after treatment with olaparib, (E) zeocin, (F) aphidicolin, (G) 6thioguanine, and (H) melphalan. Where shown, error bars represent the standard deviation of the mean of three independent experiments.
Guillemette_Supplemental Fig 4. A 1 C42 cells C C42 cells D PEO1 Cells E sh sh HCC1937 Cells sh % Colony Forming Efficiency B % Colony Forming Efficiency 8 6 4 2 1 8 6 4 2 *** sh PEO1 cells DAPI γh2ax % Cells with 5 γh2ax foci 1 5 C42 cells γh2ax ** 1 5 PEO1 cells γh2ax 1 5 HCC1937 cells γh2ax ** sh sh sh sh F G H I J γh2ax MCM7 CASPASE3 (Asp175) βactin Nuclear Extract Cell Growth (x1 3 ) Cytoplasmic Extract C42 Cells 6 5 4 3 2 1 C42 Cells sh 3 5 8 Days γh2ax MCM7 CASPASE3 (Asp175) GAPDH Nuclear Extract Cytoplasmic Extract C42 Cells Cell Growth (x1 3 ) 6 5 4 3 2 1 C42 Cells shmbd21 shmbd22 3 5 8 Days γh2ax MCM7 CASPASE3 (Asp175) βactin Nuclear Extract Cytoplasmic Extract PEO1 Cells Supplemental Figure 4. depletion has toxic effects in BRCA2proficient, but not BRCA2mutant cells (A) C42 and (B) PEO1 cells containing plko.1 shrna vectors targeting or NSC were plated at low density and quantified for colony formation efficiency. (C) C42, (D) PEO1, or (E) HCC1937 cells containing plko.1 shrna vectors targeting or NSC were left untreated and analyzed for the induction of γh2ax foci by immunostaining and quantified. (F) C42 cells were treated as in A and analyzed by immunoblot with the indicated abs or (G) analyzed for overall growth using a Coulter counter. (H) C42 cells containing plko.1 shrna vectors targeting MBD2 or NSC were analyzed by immunoblot with the indicated abs or (I) analyzed for overall growth as in G. (J) PEO1 cells containing plko.1 shrna vectors targeting or NSC were analyzed by immunoblot with the indicated abs. Where shown, error bars represent the standard deviation of the mean of three independent experiments. Asterisks denote significance from student s twotailed, unpaired ttest; *p.5, **p.1, *** p.5.
Guillemette_Supplemental Fig 5. A B C D sh1 βactin βactin HCC1937 cells BRCA1mutant SUM1315MO2 cells BRCA1mutant HCC1937 cells Bright field E sh1 SUM1315MO2 cells Bright field F 6 5 %Survival BRCA1 4 ** ** 3 2 1 GAPDH sh4 PEO1 cells sh4.5 µm Cisplatin G I K βactin βactin FAJ cells FANCJmutant βactin PD2 cells FANCD2mutant XPF cells XPFmutant J L sh 5.25.5.75 1 Cisplatin [μm] 2 1 % Surviving Colonies 1 XPF cells PD2 cells FAJ cells sh 5 1 % Surviving Colonies H % Surviving Colonies sh 5.25.5.75 1 Cisplatin [μm] 2.25.5.75 1 2 Cisplatin [μm] Supplemental Figure 5. depletion does not rescue HR defective cells (A) BRCA1 mutant HCC1937 cells containing plko.1 shrna vectors targeting or NSC were analyzed by immunoblot and (B) representative bright field images are shown 4 days after lentiviral infection. (C) BRCA1 mutant SUM1315MO2 cells were treated as in A, analyzed by immunoblot and (D) imaged as in B. (E) PEO1 cells expressing plko.1 shrnas targeting NSC or were codepleted of BRCA1 with two unique shrnas vs NSC and analyzed by immunoblot blot with the indicated abs. (F) Cells were left untreated or cisplatin treated and quantified for survival 5 days later using a Coulter counter. (G,H) FAJ cells, (I,J) PD2 cells and (K,L) XPF cells were examined by immunoblot, left untreated or cisplatin treated and quantified for colony survival.
Guillemette_Supplemental Fig 6. A 12 36 sh4 48 12 36 48 (h).5 μm Cisplatin RAD51 MCM7 Nuclear Extracts PEO1 Cells B 12 24 sh4 12 24 (h).5 μm Cisplatin C MCM7 MCM7 Ubiquitin Ubiquitin sh4 24 24 (h).5 μm Cisplatin Nuclear Extracts FAD1 Cells Nuclear Extracts PEO1 Cells D + +.5 µm Cisplatin MBD2 MCM7 PCNA Supplemental Figure 6. depletion in BRCA2mutant cells does not increase nuclear RAD51, but does increase ubiquitination. (A,B) BRCA2mutant PEO1 cells and (C) BRCA2mutant FAD1 cells expressing shrnas targeting NSC or were left untreated or cisplatin treated and nuclear extracts were analyzed with the indicated Abs. Arrows indicate molecular weight of ~37 kilodaltons. (D) BRCA2mutant PEO1 cells expressing shrnas targeting NSC or MBD2 were left untreated or cisplatin treated and nuclear extracts were analyzed with the indicated Abs.
Guillemette_Supplemental Fig 7. A B Survival fraction Survival fraction 1..9.8.7.6.5.4.3.2.1 1..9.8.7.6.5.4.3 Overall Survival All cancers p =.485 overexpressed (n=262) underexpressed (n=263) 2 4 6 8 1 12 14 16 18 2 22 24 time.2.1 BRCA2mutant p =.127 overexpressed (n=11) underexpressed (n=11) 1 2 3 4 5 6 7 8 9 1 11 time Supplemental Figure 7. In BRCA2mutant breast cancers, reduced expression correlates with poor patient response to chemotherapy. (A) Above median or below median mrna levels were correlated with Overall Survival (OS) in sporadic breast cancers and (B) BRCA2mutant breast cancers, pvalues were determined by twotailed log rank tests.