Anti bax

Cells were re-suspended in lysis buffer (1% SDS; 10 mM EDTA; 50 mM Tris-Hcl pH 8.1; 1 mM PMSF; 10 μg/ml aprotinin; 10 μg/ml leupeptin; 10 μg/ml pepstatin; 1 mM Na3VO4 and 50 mM NaF). For western blotting, following SDS–PAGE, proteins were transferred to 0.45 µM nitrocellulose membranes using Trans-Blot® Turbo™ Transfer System Cell system (Bio-Rad). The membrane was then blocked in 5% nonfat dry milk TBS 0.05% Tween 20 and incubated with primary antibody overnight at 4 °C. Blots were incubated with the appropriate secondary antibodies for 1 h at room temperature and visualized using the Chemi-Doc XRS + system (Bio-Rad). The used primary antibodies were anti-α-SMA (Invitrogen) (MA5 11547), anti-Myosin-IIB (BioLegend) (909901), anti-MCL-1 (Santa Cruz) (sc-819) anti-Bax (Dako) (A3533), anti-Bak (Cell signaling) (3814), anti-Tom20 (ab186734) (Abcam) anti-β-ACTIN (Millipore) (MAB1501R). Whole uncropped images of the original western blots are presented in Supplementary Fig. 4.

Hydrogels were collected and placed in Tissue-Tek Paraform biopsy cassettes (Sakura Finetek, USA), fixed in 4% formaldehyde and automatically embedded in paraffin (Leica EG1150H; Leica Microsystems; Wetzlas, Germany). Paraffin-embedded samples were cut into 3 μm sections. Hematoxylin-eosin staining (HE) was performed for morphology and MKI studies. MKI was determined by an expert pathologist in accordance with the Shimada classification^{48 (link)}: low MKI (<100 MK cells/5000 cells or <2%); intermediate MKI (100–200 MK cells/5000 cells or 2–4%), or high MKI (>200 MK cells/5000 cells or >4%). Automated IHC and ICC stains (Autostainer Link 48; Dako, Glostrup, Denmark) using anti-Ki67 (prediluted), anti-PTBP1 (dil. 1/400), anti-Bax (dil. 1/50) and anti-Bcl2 (prediluted) antibodies, all from Dako (Agilent Technologies, USA), were quantified by optical microscopy. For IHC and ICC markers, cells stained in blue indicated negative cells while brown staining was considered a positive result. Samples were examined and interpreted using the following criteria: - Negative (<1% positive cells); + Low positive (1–20% positive cells); ++ Intermediate positive (20–50% positive cells); +++ High positive (>50% positive cells).