Fig 2: The miR-145-5p inhibits HepG2 cell growth by activating TP53A. Growth kinetics of HepG2 cells transiently transfected with either miR-145-5p precursor or scramble sequence (NC2) or vehicle of transfection (Lipofectamine). B. Cell morphology of HepG2 cells at 72 hours after transfection with either miR-145-5p or NC2. C. TP53 dependent transcriptional activity measured by the TP53 responsive luciferase reporter vector, pP53-TA-luc, in HepG2 transfected with either miR-145-5p or NC2 or an expression vector carrying the human wild type TP53 cDNA (P53). Firefly luciferase activity was normalized on Renilla luciferase acitivity generated by the co-transfected vector pRL-TK. D. CDKN1A and BBC3 expression by RT-qPCR and E. Luminescent cell viability assay of HepG2 cells treated (48 hours) with miR-145-5p alone or in combination with siRNA against TP53 (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001).

Fig 3: ABT-263 fails to disrupt the interaction between BCL-XL and BAK in the absence of activator BH3s.(a) H2171 and SW1271 cells were treated with vehicle, 1 µM ABT-263 or 1 µM ABT-199 for 48 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). (b) H2171 cells, transfected with scrambled siRNA (siSCR) or siRNA against BIM, were untreated or treated with ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). The expression of BIM was assessed by an anti-BIM immunoblot. (c) SV40-transformed wild-type or Bid-/-Bim-/-Puma-/-Noxa-/- QKO MEFs, infected with retrovirus expressing shRNA against luciferase or Mcl-1, were untreated or treated with ABT-263 or ABT-199 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). (d) SV40-transformed wild-type or Bid-/-Bim-/-Puma-/-Noxa-/- QKO MEFs stably expressing GFP, HA-BCL-XL or HA-BCL-2, untreated or treated with ABT-263, were subjected to anti-HA immunoprecipitation. The input (5%) and immunoprecipitates were assessed by immunoblot analysis. (e) H2171 and SW1271 cells, untreated or treated with ABT-263, were subjected to anti-HA immunoprecipitation. The input (5%) and immunoprecipitates were assessed by immunoblot analysis. Arrow indicates a cross-reactive band. (f) H2171 cells stably expressing GFP, HA-BCL-2, HA-BCL-XL, or HA-BCL-XL BH1 mutant were untreated or treated with 1 µM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). EC50 of ABT-263 in H2171 cells was assessed by CellTiter-Glo assays at 48 h. The expression of HA-tagged BCL-2 and BCL-XL protein was assessed by immunoblot analysis. (g) DMS53 cells stably expressing GFP, HA-BCL-2, HA-BCL-XL or HA-BCL-XL BH1 mutant were untreated or treated with 1 µM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n=3). EC50 of ABT-263 in H2171 cells was assessed by CellTiter-Glo assays at 48 h. The expression of HA-tagged BCL-2 and BCL-XL protein was assessed by immunoblot analysis. ***P<0.001 (Student’s t-test). Unprocessed original scans of blots are shown in Supplementary Fig. 11.

Fig 4: FBXL20 regulates PUMA and BAX at the proteasomal level through SCF complex.A and B, quantitative real-time RT-PCR monitoring the mRNA levels of PUMA and BAX expression in MCF7 cells expressing either vector or FBXL20. GAPDH was used as a normalizing control. Experiment was repeated three times. C, immunoblot monitoring the levels of PUMA and BAX in MCF7 cells transfected either vector or DDK-FBXL20. After, 42 h of transfection, cells were treated 5 µM MG132 for additional 6 h before harvesting. D, cycloheximide chase/immunoblot assay monitoring PUMA and BAX turnover in MCF7 cells expressing either NS or FBXL20 shRNA treated with cycloheximide (100 µg/ml) for the indicated time. The experiment was repeated three times. E and F, quantitative analysis of PUMA and BAX (panel D) using Image J. The graphs show the ratio of the relative levels of PUMA and tubulin (loading control) or BAX and Tubulin (loading control) at each time; The expression level was set to 100% at time “0”. Data are presented as the mean ± SD from three independent experiments. G, immunoblot analysis monitoring PUMA and BAX levels in MCF7 cells stably expressing NS or p53 shRNA following ectopic expression of either vector or DDK-FBXL20. The experiment was repeated two times. H, immunoblot monitoring PUMA and BAX in MCF7 cells expressing either vector or DDK-FBXL20 or F-box deleted FBXL20 (DDK-?F-FBXL20). I, densitometric analysis of (H) using Image J quantifying relative proteins levels of PUMA and BAX. Data are presented as the mean ± SD from three independent experiments. For all immunoblot analysis tubulin was monitored as a loading control. **p < 0.01, *p < 0.05.

Fig 5: A subset of HNSCC cell lines and side population (SP) cells exhibit increased expression of CSC-related markers. A. Basal mRNA levels for Nanog, Oct4, and Sox2 were quantified by qRT-PCR in HEKA and nine UM-SCC cell lines, including 3 lines with deficient expression of wt TP 53 (UM-SCC-1, -6, -9), 2 lines lacking mtTP53 (UM-SCC-11A, -11B) and 4 lines overexpressing TAp73 with mt TP53 (UM-SCC-22A, -22B, -38 and -46)(16). Higher relative expression of Sox2 in UM-SCC was due to low signal in control HEKA, verified in an independent experiment and consistent with low Sox2 protein in control in panel B by Western blot. B. Basal expression of Nanog, Oct4, and Sox2 proteins was analyzed by Western blot in whole cell lysates of control HOK and 9 UM-SCC cell lines, with ß-actin used as control for loading. C. UM-SCC-46 cell lines were labeled with Hoechst 33342 dye and analyzed by flow cytometry without and with treatment with verapamil. HoechstLow SP cells highlighted within the window indicated are quantified. D. The mRNA expression of CSC related gene markers Nanog, Oct4, Sox2, BMI1, ABCG2, and pro-apoptotic gene PUMA were assessed in non-SP and SP sub-populations sorted by FACS, using QRT-PCR. The expression level in non-SP cells is normalized to 1.0. Columns, mean between triplicate samples; bars, SD. *, Student t test, P < .05.