Supplementary Figure 1: MYCER protein expressed from the transgene can enhance

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Allen Parrish
6 years ago
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1 Relative luciferase activity Relative luciferase activity MYC is a critical target FBXW7 MYC Supplementary is a critical Figures target 1-7. FBXW7 Supplementary Material A E-box sequences HSV-TK Luc+ B Relative MYC reporter activity in uninfected MCF10A cells 4 Relative MYC reporter activity in MYCER-infected MCF10A cells NT + 4OHT 0 NT + 4OHT Supplementary Figure 1: MYCER protein expressed from the transgene can enhance transcriptional activity. (A) A reporter plasmid containing 6 E-box sequences (CACGTG) followed Supplementary by a luciferase Figure 1. reporter MYCER was protein transfected expressed into from uninfected the transgene parental can MCF10A enhance or transcriptional activity. (A) A reporter plasmid containing 6 E-box sequences (CACGTG) MCF10A-MYCER cells and luminescence was measured 48 hr after transfection. (B) followed by a luciferase reporter was transfected into uninfected parental MCF10A or Luciferase activity from the reporter was normalized to Renilla luciferase activity. Each MCF10A-MYCER cells and luminescence was measured 48 hr after transfection. (B) experiment was done in triplicate and graphs show the average 3 biological replicates. Luciferase activity from the reporter was normalized to Renilla luciferase activity. Each Error experiment bars represent was done the in SEM. triplicate and graphs show the average 3 biological replicates. Error bars represent the SEM. 1

2 Asynchronous cells After double thymidine block 24h post release from DT block Supplementary Supplementary Figure Figure 2: 2. MCF10A-MYCER cells cells arrest arrest at the at G1/S the transition G1/S transition following following double double thymidine block. MCF10A-MYCER cells cells were were subjected subjected to two rounds to two rounds 2.5mM 2.5mM thymidine treatment, treatment, then fixed then and fixed stained and stained propidium iodide propidium flow cytometric iodide flow analysis. Compared to untreated asynchronous cells, thymidine treated cells showed cytometric analysis. Compared to untreated asynchronous cells, thymidine treated cells accumulation cells in M1+M2 (<2N DNA content). 24h after release, the cell cycle showed distribution accumulation returns to normal. cells in M1+M2 (<2N DNA content). 24h after release, the cell cycle distribution returns to normal. 2

3 MCF10A-MYCER TAM MCF10A-MYCER TAM -shrna +EtOH (MYC OFF) 50% GFP or RFP +4OHT (MYC ON) FACS analysis FACS analysis FACS analysis Supplementary Figure 3: Schematic fluorescence-based competition assay. Uninfected MCF10A-MYCER cells which express no fluorescent proteins are plated on the same plate MCF10A-MYCER cells infected a single shrna clone choice coexpressing GFP or RFP. At day 1, cells from the plate are subjected to live cell flow cytometry to verify ~50% fluorescence. Subsequently, the cells are split into control or 4OHT treated groups, and passaged every hr. At each passage or desired timepoint, remaining fluorescence on the plate is measured by flow cytometry the duration the experiment. Supplementary Figure 3. Schematic fluorescence-based competition assay. Uninfected MCF10A-MYCER cells which express no fluorescent proteins are plated on the same plate MCF10A-MYCER cells infected a single shrna clone choice co-expressing GFP or RFP. At day 1, cells from the plate are subjected to live cell flow cytometry to verify ~50% fluorescence. Subsequently, the cells are split into control or 4OHT treated groups, and passaged every hr. At each passage or desired timepoint, remaining fluorescence on the plate is measured by flow cytometry the duration the experiment. 3

4 WHOLE CELL EXTRACTS 4OHT MYCER c-jun Notch1 Cyclin E mtor βactin Supplementary Supplementary Figure 4. FBXW7 Figure 4: knockdown FBXW7 knockdown specifically leads specifically to stabilization leads to stabilization MYCER. MCF10A-MYCER MYCER. cells MCF10A-MYCER expressing either cells control expressing or FBXW7 either shrna control were or cultured FBXW7 in the shrna were presence cultured or absence in the 4OHT presence 4 or weeks. absence Whole 4OHT cell lysates 4 weeks. (80µg total Whole protein/lane) cell lysates were (80μg total run on SDS-PAGE protein/lane) and were subjected run on to SDS-PAGE Western blot and analysis subjected to major Western FBXW7 blot targets. analysis major Figure shows FBXW7 a representative targets. Figure image shows replicated a representative experiments. image replicated experiments. 4

5 Supplementary Figure 5. FBXW7 knockdown leads to preferential stabilization MYCER. MCF10A-MYCER cells stably expressing Dox-inducible shrna clones FBXW7 were treated or out 4OHT (MYC OFF or ON) 4 weeks, then cell lysates were analyzed by SDS-PAGE/Western blot. Bands were quantified using ImageQuant v5.2 (Molecular Dynamics) and normalized to loading controls. Figure shows averages from 3 independent biological replicates. Supplementary Figure 5: FBXW7 knockdown leads to preferential stabilization MYCER. MCF10A-MYCER cells stably expressing Dox-inducible shrna clones FBXW7 were treated or out 4OHT (MYC OFF or ON) 4 weeks, then cell lysates were analyzed by SDS-PAGE/Western blot. Bands were quantified using ImageQuant v5.2 (Molecular Dynamics) and normalized to loading controls. Figure shows averages from 3 independent biological replicates. 5

6 A B DNA DAMAGE APOPTOSIS 4OHT P-Chk2-72 4OHT Cleaved Caspase3-28 β actin Cleaved PARP1-95 P-H2AX -28 PUMA -28 HistoneH3 β actin Supplementary Figure 6: Long term FBXW7 knockdown and MYCER activation do not result in significant levels DNA damage or apoptosis. (A) Control or FBXW7 knockdown Supplementary cells Figure were 6. treated Long term FBXW7 or out knockdown 4OHT and MYCER 4 weeks activation and probed do not result the in significant levels DNA damage or apoptosis. (a) Control or FBXW7 knockdown cells presence were treated phosphorylated or out 4OHT Chk2 (in 4 weeks whole and cell probed lysate) or the H2AX presence (in chromatin-bound samples phosphorylated after fractionation) Chk2 (in whole as cell markers lysate) or H2AX checkpoint (in chromatin-bound activation/dna samples damage. after shfbxw7 fractionation) cells as did markers not show checkpoint a significant activation/dna accumulation damage. shfbxw7 either species cells did after not show long term treatment a significant accumulation 4OHT. (B) Similarly either species treated after cells long term were treatment probed 4OHT. the presence (b) Similarly treated cells were probed the presence apoptosis markers in whole cell lysates. apoptosis However, markers we failed in to whole detect any cell significant lysates. However, levels cleaved we failed caspase-3, to detect PARP1, any or significant PUMA in levels the surviving cleaved cells caspase-3, after 4 weeks PARP1, treatment. or PUMA Shown in above the surviving are representative cells after images 4 weeks 3 treatment. independent Shown replicates. above are representative images 3 independent replicates. 6

7 shcontrol shfbxw7-7 MYC OFF MYC ON Supplementary Figure 7: FBXW7 knockdown MYCER activation causes synergistic accumulation Supplementary Figure cells 7. in FBXW7 S and knockdown G2/M phase. MYCER MCF10A-MYCER activation causes cells synergistic stably expressing accumulation cells in S and G2/M phase. MCF10A-MYCER cells stably expressing control or FBXW7 shrna were cultured 4 weeks in the presence or absence control or FBXW7 shrna were cultured 4 weeks in the presence or absence 4OHT 4OHT (MYC OFF (MYC or ON). OFF Cells or ON). were fixed Cells and were stained fixed and propidium stained iodide propidium flow cytometric iodide flow cytometric analysis. Cells analysis. accumulate Cells in accumulate the M3+M4 region in the (<1N M3+M4 DNA content) region only (<1N in the DNA FBXW7 content) only in the knockdown FBXW7 and knockdown MYC ON condition. and MYC Shown ON is condition. a representative Shown image is a representative 3 independent image 3 independent replicates. replicates. 7

8 Supplementary Table 1: List top 78 synthetic lethal targets from MCF10A-MYCER screen. Results were filtered according to fold change (Log2 FC<-1) signal abundance between MYC ON and MYC OFF, and individually associated p-values (p<0.05). Only annotated target genes are shown. Candidates are ranked in order most depleted in the MYC ON population compared to MYC OFF (Log2 FC value). Rank Name p- value Log2 FC Acc. Number shrnai ID 1 ARL5B NM_ v2hs_ C2orf NM_ v2hs_ TTBK AB v2hs_ PSG NM_ v2hs_ NAT NM_ v2hs_ TRPM NM_ v2hs_ MFN NM_ v2hs_ C18orf NM_ v2hs_ DONSON NM_ v2hs_ ZNF NM_ v2hs_ H2AFV NM_ v2hs_ NUPL NM_ v2hs_ ZNF NM_ v2hs_ PSMD NM_ v2hs_ CDC14B NM_ v2hs_ UGT1A NM_ v2hs_ SOX NM_ v2hs_ NUPL NM_ v2hs_ ANKRD NM_ v2hs_ EPHA NM_ v2hs_ ST8SIA NM_ v2hs_ SLC44A NM_ v2hs_ SH3BP NM_ v2hs_ KIAA AB v2hs_ DMKN NM_ v2hs_ STK17A NM_ v2hs_ PHF21A NM_ v2hs_ EYA NM_ v2hs_ C6orf XM_ v2hs_ ZFYVE NM_ v2hs_ LRCH NM_ v2hs_ NR2E NM_ v2hs_ TOP3B NM_ v2hs_ WDSOF NM_ v2hs_ CLDN NM_ v2hs_ CMTM NM_ v2hs_ IRAK NM_ v2hs_

9 38 APOBEC3A NM_ v2hs_ ERGIC NM_ v2hs_ PARP NM_ v2hs_ RAE NM_ v2hs_ LEPREL NM_ v2hs_ ZNF NM_ v2hs_ SLC7A NM_ v2hs_ LAMA NM_ v2hs_ LOC XM_ v2hs_ ATP1B1P NG_ v2hs_ C1orf NM_ v2hs_ KRIT NM_ v2hs_ ASAH NM_ v2hs_ OLFR AJ v2hs_ CCDC NM_ v2hs_ TOP3A NM_ v2hs_ MATK NM_ v2hs_ ST NM_ v2hs_ KIAA AB v2hs_ PERP NM_ v2hs_ FBXW AY v2hs_ APOA NM_ v2hs_ C NM_ v2hs_ SLC36A NM_ v2hs_ PPP3CC NM_ v2hs_ TRIM NM_ v2hs_ ELL NM_ v2hs_ PRMT NM_ v2hs_ PRSS NM_ v2hs_ UBE2I NM_ v2hs_ QRSL XM_ v2hs_ C11orf NM_ v2hs_ EPYC NM_ v2hs_ TNFAIP NM_ v2hs_ PAQR NM_ v2hs_ PREB NM_ v2hs_ CEND NM_ v2hs_ BBOX NM_ v2hs_ CDKL NM_ v2hs_ CMTM NM_ v2hs_ GCN1L XM_ v2hs_

Supplementary Fig. 1. Schematic structure of TRAIP and RAP80. The prey line below TRAIP indicates bait and the two lines above RAP80 highlight the

Supplementary Fig. 1. Schematic structure of TRAIP and RAP80. The prey line below TRAIP indicates bait and the two lines above RAP80 highlight the prey clones identified in the yeast two hybrid screen.