Fig 2: Knock down of eIF2α prevents the reduction in MCL-1/BCL-XL expression and knock down of Rubicon prevents phosphorylation of YAP. (A) HT1080 and MES cells were transfected with a scrambled control siRNA (siSCR) or with an siRNA to knock down expression of eIF2α. Twenty-four hours after transfection, cells were treated with vehicle control (VEH), [pazopanib (P, 1.0 μM) + entinostat (E, 50 nM)], neratinib (N, 50 nM) or the drugs as indicated in combination for 6 h. Cells were fixed in place and immunofluorescence staining performed to detect the expression and phosphorylation of the indicated proteins. The percentage change in fluorescence intensity compared to vehicle control, defined as 100%, is plotted (n = 3 ± SEM). *p < 0.05 less than vehicle control value; #p < 0.05 greater than vehicle control value; ¶p < 0.05 greater than corresponding value in siSCR cells; £p < 0.05 less than corresponding value in siSCR cells. (B) HT1080 and MES cells were transfected with a scrambled control siRNA (siSCR) or with an siRNA to knock down expression of Rubicon. Twenty-four hour after transfection, cells were treated with vehicle control, [pazopanib (1.0 μM) + entinostat (50 nM)], neratinib (50 nM) or the drugs as indicated in combination for 6 h. Cells were fixed in place and immunofluorescence staining performed to detect the expression and phosphorylation of the indicated proteins. The percentage change in fluorescence intensity compared to vehicle control, defined as 100%, is plotted (n = 3 ± SEM). *p < 0.05 less than vehicle control value; #p < 0.05 greater than vehicle control value; ¶p < 0.05 greater than corresponding value in siSCR cells; £p < 0.05 less than corresponding value in siSCR cells.

Fig 3: MCL1 is a direct target gene of miR-141-3p. (a) Binding region between miR-141-3p and MCL1. (b) MCL1 protein expression and (c) mRNA expression was tested using western blot analysis and RT-qPCR, respectively. ***P < 0.001 vs. CCC-ESF-1. (d) Relative luciferase activities were detected in hHSFs. ***P < 0.001 vs. mimic-NC+MCL1. (e) MCL1 protein level and (f) mRNA level were determined using western blot analysis and RT-qPCR, respectively. ***P < 0.001 vs. shRNA-NC; ##P < 0.01, ###P < 0.001 vs. sh-INHBA-AS1-1+ inhibitor-NC. hHSFs, human hypertrophic scar fibroblasts; sh, short hairpin; NC, negative control; WT, wild-type; MUT, mutant

Fig 4: MCL-1 targeting influences transcriptional programs involved in actomyosin organization/contractility in viable bCAFs.A Three different primary cultures of CAFs were treated with ABT-199 (1 µM), A1331852 (100 nM), ABT-199 (1 µM) +A1331852 (100 nM) or ABT-737 (1 µM) with or without S63845 (500 nM) for 72 h in DMEM containing 1% FBS, apoptosis was measured by Annexin-V flow cytometry. Data are means ± SEM from three independent experiments. Two-way ANOVA, ****P < 0.0001, ns: not significant. B Caspase 3/7 activity was measured by caspase Glo assay and expressed in arbitrary unit. Data are means ± SEM from four independent experiments. Two-way ANOVA. *P < 0.05, ns: not significant C Volcano plot displaying differential expressed genes between S63845 treated and non-treated primary culture of CAFs from luminal B (n = 3) and triple negative (n = 3) breast cancers. The vertical axis (y-axis) corresponds to the mean expression value of log 10 (q-value), and the horizontal axis (x-axis) displays the log2 fold-change value. The red dots represent the upregulated expressed transcripts; the blue dots represent the transcripts whose expression is downregulated. D A hierarchically clustered heatmap showing the expression patterns of the 23 most extreme DE mRNA. Red and blue represent up- and downregulated expression in CAFs treated or not with S63845. Color density indicating levels of fold-change was displayed. E qRT-PCR of MYH10, MYH2, ACTA2 andITGA11 mRNA in CAFs Sg control or sg MCL-1 treated or not by S63845 500 nM for 18 h. Mean and SEM of three independent experiments are represented as fold-change of mRNA level related to untreated Sg control. Two-way ANOVA, *P < 0.1, **P < 0.01, ****P < 0.0001, ns: not significant. F CAFs sg control or silenced for MCL-1 (sg MCL-1) were treated or not with S63845 (500 nM), A1331852 (100 nM) or S63845 (500 nM) + A1331852 (100 nM) for 48 h in DMEM containing 1% FBS. Apoptosis was measured by Annexin-V flow cytometry. Data are means ± SEM from three independent experiments. P-value was determined by two-way ANOVA. ****P < 0.0001, ns: not significant.

Fig 5: MCL-1 targeting in bCAFs does not induce cancer cell migration or proliferation.A qRT-PCR of ESM1, IL33, HAS2 and CCL7 mRNA in CAFs Sg control or sg MCL-1 treated or not by S63845 500 nM for 18 h. Mean and SEM of three to five independent experiments are represented as fold-change of mRNA level related to untreated Sg control. Two-way ANOVA, *P < 0.1, **P < 0.01, ns: not significant. B (Left) Representative images from in vitro scratch wound-healing assays of T47D cancer cells in presence of conditioned media from CAFs Sg control or CAFs Sg MCL-1 at T0 and after 68 h. (Right) Quantification of wound closure expressed in percentage of total wound recovery at indicated time points during the scratch wound assay. Student t-test (ns: not significant). C Proliferation of T47D-eGFP cancer cells in coculture with CAFs pretreated or not with S63845 measured. Confluency was measured using time-lapse video-microscopy (incucyte live cells analysis) during 120 h. Student t-test (****P < 0.0001).