Fig 2: Myh10 and Myh14 are the NM2 isoforms expressed in the proximal tubular segment. Confocal fluorescence microscopy of the adult mouse kidney sections stained for megalin (proximal tubule marker) and one of the NM2 isoforms revealed that Myh10 and Myh14 are the prominent isoforms of NM2 in the proximal tubules. Myh9 (red) is not expressed in the megalin (green)-positive proximal tubules (arrowheads, A–F); however, Myh9 expression is present in the adjacent tubules and the glomerulus (arrows, D–F). Myh10 localized to apical (arrowhead) and basolateral (arrow) membranes in the megalin-positive proximal tubular segments (G–I). Myh14 was expressed in the megalin-positive tubules on the apical membrane (arrowhead); punctate structures were also observed on the basolateral membrane and within the epithelial cells (J–L). The majority of Myh10 and Myh14 does not colocalize with megalin on the apical membrane, and appear to be above the megalin staining on the membrane. However, there are a few regions (microdomains) on the apical membrane (yellow arrowhead, I&L) where we observe partial colocalization. Sections were mounted with vectashield containing DAPI to stain the nuclei (blue; C, F, I & L). Scale bar = 10 µm.

Fig 3: ß Cardiac Myosin II (ßCMII) Filament Assembly in hiCMs.(A) Endogenous localization of NMIIB (left) and ßCMII in the same hiCM and imaged with SIM. (B) Averaged line-scans of NMIIB (Figure 7B) and ßCMII localization in hiCMs spread for 24 hr. Note the peak fluorescence of ßCMII is more towards the cell body than peak fluorescence of NMIIB. 23 cells from four experiments were used for ßCMII localization. (C) Schematic (top) of NMIIB-ßCMII co-filaments. High-mag views of ßCMII-NMIIB co-filaments (bottom). Endogenous staining of hiCM (bottom, left) for ßCMII (N-terminal motors) and NMIIB (rod domain) and imaged with SIM. Mouse and human tissue (bottom middle, and bottom right, respectively) stained for ßCMII (motors) and NMIIB (rod domain) and imaged with SIM and Zeiss 880 with Airyscan, respectively. (D) Histograms displaying width of NMII filaments (top), ßCMII filaments (middle), and NMIIB-ßCMII co-filaments in hiCMs. Measurements made from motor-domain to motor domain as in Figure 7D and E. (E) Histograms displaying distribution of ßCMII filaments widths with respect to their location in hiCMs. Note ßCMII filaments tend to grow larger as they move towards the center of the cell. Measurements were not taken from ‘mature’ sarcomere structures in highly organized A-bands. (F) Actin and ßCMII of scramble control hiCM (top) and NMIIB KD (siMYH10) hiCM (bottom) spread for 24 hr. Note loss of organized A-bands but presence of ßCMII filaments in NMIIB KD hiCM. (G) Fourier transforms of ßCMII signal from white boxes in Figure 11F from scramble control and NMIIB KD hiCMs (above and below respectively). Yellow arrows indicate sarcomeric periodicity in scramble control hiCMs, which is lacking in NMIIB KD cells. (H) Actin and a-actinin 2 localized in a hiCM after NMIIB KD. (I) High mag views of actin and ßCMII in NMIIB KD (siRNA MYH10) hiCM imaged with SIM. ßCMII filaments localize to residual actin filaments. (J) ßCMII in hiCM spread for total of 24 hr, with the final 6 hr in 5 µM Latrunculin B and imaged with SIM. Notice lack of ßCMII A-bands in the periphery of hiCM, and large ßCMII filament aggregates (yellow box). (K) Percentage of scramble control, NMIIB KD (siRNA MYH10), and 5 µM Latrunculin B hiCMs with ßCMII A-bands. Control: 26 cells, three experiments; NMIIB KD: 26 cells, two experiments; Latrunculin B: 11 cells, three experiments. Cell bodies were not analyzed in the latrunculin experiment due to the density of ßCMII localization. Scale Bars; (A) 10 µm, (C) 200 nm, (F), (G) 5 µm, (H) 1 µm. (I) 10 µm low mag, 5 µm high mag insets. (J) 10 µm low mag, 5 µm high mag inset. P-values denoted in graphs.

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 induces changes in actomyosin structure organization in a DRP-1-dependent manner.A Mitochondrial fission was detected by TOM20 staining in CAFs treated or not with S63845 500 nM or A1331852 100 nM for 18 h in the presence or not of Mdivi-1 50 µM. Scale bar = 50 µm. B Quantification of mitochondrial network length was performed by measuring the size of TOM-20 labeled structures on deconvolved images in 10 cells/condition in 3 independent experiments. Objects smaller than 0.5 µm were not considered. C Representative co-immunoprecipitation of MCL-1 and DRP-1 and western blotting for MCL-1, DRP1, or NOXA in CAFs treated or not with S63845 in presence or not of Mdivi-1 (n = 3). FT: Flow Through. D qRT-PCR of MYH10, MYH2, ACTA2, and ITGA11 mRNA in CAFs treated or not by S63845 500 nM for 18 h in the presence or not of Mdivi-1. Mean and SEM of three independent experiments are represented as fold-change of mRNA level related to untreated control. Two-way ANOVA, *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant. E Quantification (percentage of cells positive for nuclear YAP or nuclear and cytosolic YAP) (Bottom) and confocal image (Top) of YAP staining (red) and FITC-phalloidin (green) in CAFs treated or not with S63845. Randomly generated images set names were randomized for analysis. Around sixty to one-hundred cells were analyzed per condition. Counting events were done manually through NIS-Elements software (Nikon software). Fluorescence intensity and positivity staining were determined by positive and negative control comparison, dependent on the experiment. The nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. Two-way ANOVA, *P < 0.05. F Quantification (Bottom) and confocal image (Top) of Myosin IIB staining (green) and immunofluorescence of TOM-20 (red) in CAFs treated or not with different BH3 mimetics (as indicated) in the presence or not of Mdivi-1. Randomly generated images set names were randomized for analysis. Around sixty to one-hundred cells were analyzed per condition. Counting events were done manually through NIS-Elements software (Nikon software). Fluorescence intensity and positivity staining were determined by positive and negative control comparison, dependent on the experiment. The nuclei were counterstained with DAPI (blue). Scale bar = 50 µm, Two-way ANOVA, ###P < 0.001, ##P < 0.01, #P < 0.05 for myosin IIB-, **P < 0.01; *P < 0.05 for fragmented mitochondria.