Figure 7: ECM-mediated mechanosignaling controls OxSR via HSF1 and YME1L1.

A. Confocal microscopy of indicators of apoptosis with cleaved caspase 3 staining (red) and nuclear condensation (dapi) of MECs cultured on 400 or 60k Pa ECM for 24 h with subsequent 24 h +/− paraquat treatment [10 mM]. 100k cells/well of 24 well plate. (n=4 replicates, repeated 3 separate times)

B. Quantitation of cells from 16 field views depicted in G for condensed nuclei and cleaved caspase-3 positive cells (1653–575 cells counted per condition, repeated 3 times).

C. Western blot of YME1L1 and β-actin from 5 μg of protein derived from cells cultured on soft-to-stiff ECM for 24 h. (2 biological replicates shown, repeated 3 times)

D. Western blot of YME1L1 and β-actin from 5 μg of protein derived from cells cultured on 60k Pa ECM for 24 h +/− KRIBB11 [2 μM] (3 biological replicates shown, repeated 2 times)

E. Graphical representation of the conceptual paradigm pertaining to this figure.

F. Quantitation of MECs with YME1L1 knockdown via CRISPR-I compared to CRISPR-I and empty guide vector expressing cells on 400 Pa (dashed lines) or 60k Pa ECM, 11 field views quantified for condensed nuclei and cleaved caspase-3 positive cells (923–2880 cells counted per condition, repeated 3 times).

G. C. elegans survival in 50 mM paraquat, with C. elegans overexpressing hsf-1 (sur-5p::hsf-1) compared or control line (N2) grown on either empty vector or ymel-1 RNAi from hatch (n=80 animals per condition, repeated 3 times).