ebss  (Tocris)

Journal: medRxiv

Article Title: Novel variants in ryanodine receptor type 3 predispose to acute rhabdomyolysis due to impaired autophagy

doi: 10.64898/2026.02.27.26345848

Figure Lengend Snippet: RyR3 deficiency induces autophagy defects A. Quantification of control and patients primary myoblasts cell death under basal culture conditions and 4h starvation (EBSS). *p < 0.05, ***p < 0.001, ****p < 0.0001, ns , non-significant from 2-way ANOVA and Sidak’s post hoc test. B-C. Representative images ( B ) and quantification ( C ) of LC3 positive vesicles in control and patients’ primary myoblasts cultured in basal or 2h starvation conditions. ****p < 0.0001, ns , non-significan from two-way ANOVA followed by Sidak’s post hoc test. D-E. Representative western blotting ( D ) and quantification ( E ) of LC3-II in control and RYR3 patients’ primary myoblasts starved (EBSS) or incubated with the autophagosome-lysosome fusion inhibitor bafilomycin A1 (Baf) for indicated time points. Data are represented as mean + SD, *p < 0.05, **p < 0.01, ****p < 0.0001 from two-way ANOVA and Bonferonni post-hoc test. F-G. Representative western blotting ( F ) and quantification ( G ) of LC3-II in control and RYR1 patients primary myoblasts starved (EBSS) or incubated with bafilomycin A1 (Baf) for indicated time points. Data are represented as mean + SD *p < 0.05, **p < 0.01, ****p < 0.0001 from two-way ANOVA and Bonferonni post-hoc test. H-I. Confocal imaging of skeletal muscle ( H ) and autophagy index quantification of the normalized RFP to GFP signal ( I ) of larvae co-injected with the autophagic GFP-LC3-RFP-LC3ΔG probe and control-MO or ryr3-MO. *p < 0.05 from Student’s t-test.

Article Snippet: To mimic environmental stress, the GM was replaced with EBSS medium with or without 100 nM bafilomycin A1 (ref: tlrl-bafA1, InvivoGen) for 1h and 3h.

Techniques: Control, Cell Culture, Western Blot, Incubation, Imaging, Injection

Journal: medRxiv

Article Title: Novel variants in ryanodine receptor type 3 predispose to acute rhabdomyolysis due to impaired autophagy

doi: 10.64898/2026.02.27.26345848

Figure Lengend Snippet: A. Representative immunoblot of LC3 lipidation upon starvation (EBSS: Earle’s Balanced Salt Solution) and Bafilomycin A1 treatment (Baf) in primary myoblasts from RYR3 P1 and RYR3 P2 patients. Due to limited proliferative capacity of primary cells, the experiment could only be repeated twice. GM: Growth Medium. β-actin was used as a loading control. n.r. (non relevant) indicates lanes that were included on the blot but are not relevant for the present study. B. Representative immunoblot of LC3 lipidation upon starvation in siRNA-mediated RYR3 depleted primary myoblasts. β-actin was used as a loading control.

Article Snippet: To mimic environmental stress, the GM was replaced with EBSS medium with or without 100 nM bafilomycin A1 (ref: tlrl-bafA1, InvivoGen) for 1h and 3h.

Techniques: Western Blot, Control