Fig. 2: NINJ1 renders the plasma membrane susceptible to rupture under mechanical strain.
From: NINJ1 regulates plasma membrane fragility under mechanical strain
a, LDH release in HEK293T cells transfected with vector or CMV-driven NINJ1-IRES-mCherry was measured 24 h after transfection. n = 3 trials per group. Statistical analysis was performed using two-sided an unpaired Student’s t-test. b, Western blot analysis of NINJ1 in non-transfected, vector-transfected and CMV-NINJ1-transfected HEK293T cells. c, Western blot analysis of NINJ1 in HEK293T cells stably expressing TRE3G-NINJ1-IRES-mCherry induced with 0–300 ng ml−1 doxycycline (Dox) for 24 h; parental cells were used as controls. d, Confocal images of TRE3G-NINJ1-mCherry stable cells with or without 100 ng ml−1 doxycycline induction for 24 h. The arrowheads indicate fluorescent puncta. Scale bar, 10 µm. e,f, LDH release (e; n = 3–5 wells per group) and the DRAQ7+ cell percentage (f; n = 3–4 trials; 300–500 cells per group) 30 min after strain application in parental or NINJ1-inducible HEK293T cells pretreated with doxycycline. The duration of strain application was 5 s. g, Western blot analysis of endogenous NINJ1 in HeLa and ZOS cells. h,i, LDH release (h; n = 8–12 wells per group) and DRAQ7+ percentage (i; n = 3–6 trials; 300–500 cells per group) in HeLa and ZOS cells 30 min after strain. Statistical analysis was performed using two-way ANOVA with Bonferroni correction. j–l, Relative NINJ1 mRNA levels (j; n = 3 per group), LDH release (k; 50% strain; n = 6 trials per group) and the DRAQ7+ cell percentage (l; 50% strain; n = 3 trials; 300–400 cells per group) across human osteosarcoma cell lines. Statistical analysis was performed using one-way ANOVA with Bonferroni correction versus HeLa. m–o, Single-cell clones of 143B cells (n = 18) were analysed for NINJ1 mRNA using qPCR after treatment with 55% strain. m, Schematic of the experiment. LDH release (n) and DRAQ7+ (o) percentages were measured and correlated with NINJ1 expression (300–400 cells per clone). Unless otherwise indicated, data are mean ± s.e.m. The diagram in m was created using BioRender.
