Fig. 1: High-throughput genetic screen identifies NINJ1 as a regulator of PMR induced by mechanical strain.

a, Schematic of the high-throughput stretch system. A vacuum applied to sealed wells deforms the PDMS membrane, stretching the adherent cells on top. b, The two operating modes of the system: single-well imaging through a microscope and full-plate imaging through a plate reader. c, Images of HeLa-YFP cells immediately before and 120 s after strain application. Scale bar, 150 µm. d, YFP-intensity traces before and after the strain application. Each trace represents data from a single well; 10 wells were analysed and the red trace is the average. e,f, Trypan Blue and DRAQ7/Hoechst staining of the cells after strain application. Scale bars, 100 µm. g, The workflow of the siRNA screen for regulators of strain-induced membrane damage. h, Overview of the primary screen hitpicking, covering 2,726 genes encoding multipass transmembrane proteins. Each dot represents one of 10,843 siRNAs; the red dots indicate primary hits (z score > 1.5). i, Final validation of the top 20 candidate and 4 control genes showed that NINJ1 was the sole hit. j, YFP quenching in HeLa-YFP cells transfected with pooled siRNAs against NINJ1 or scrambled control. n = 4 trials per group. Statistical analysis was performed using a two-sided unpaired Student’s t-test versus the scrambled control. k, Western blot analysis of endogenous NINJ1 in HeLa cells 48 h after siRNA transfection. l, Trypan Blue staining of the control and NINJ1-knockdown cells after mechanical strain. Scale bar, 100 µm. m,n, LDH release (m) and the percentage of DRAQ7⁺ cells (n) in the control and NINJ1-knockdown groups, with or without 50% strain for 5 s. n = 4–8 trials per group. Statistical analysis was performed using using two-way analysis of variance (ANOVA) with Bonferroni correction. Unless otherwise indicated, data are mean ± s.e.m. **P < 0.01 versus scrambled. The diagrams in a, b and g were created using BioRender.

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