Figure 3.

Lipin1-deficient myoblasts display alterations in Rab7 positioning and activation and impaired lysosomal function

(A) The distribution of endogenous Rab7 in 30 individual patient and control myoblasts was determined by calculating the intensity of Rab7 fluorescence along a longitudinal axis traversing the nucleus.

(B) The interaction between Rab7 and FYCO1 determined by fluorescence resonance energy transfer (FRET) after staining with Rab7 (green) only or for Rab7 (donor) and FYCO1 (acceptor) in red was depicted by line graphs (ns, nanoseconds).

(C) Myoblasts transfected with RFP-RILP were immunostained for Rab7. Box and whisker plots (30 images/condition) show the percentage of proximity of Rab7 with RFP-RILP (unpaired t test).

(D and E) Myoblasts transfected with a construct encoding RFP-Armus were stained for Rab7 or PI3P. Box and whisker plots (50 images/condition) show the percentage of proximity of Rab7 (D) or PI3P (E) with RFP-Armus (Mann-Whitney U test).

(F) Dot plot (mean of 2 technical replicates/dot) with lines depicts the relative level of residual EGFR as compared to β-actin upon incubation with EGF for indicated times.

(G) Myoblasts were loaded with DQ-OVA, and fluorescence resulting from the cleavage of DQ-OVA substrate was analyzed by flow cytometry. Dot plots (mean of 2 technical replicates/dot) show the means ± SDs of the value of the mean fluorescence intensity (MFI) of DQ-OVA 60 and 120 min after uptake (mean effect of interaction F(1,6) = 14.43, p = 0.009, of time F(1,6) = 53.46, p = 0.0003, of subjects F(1,6) = 11.23, p = 0.0154).

(H) Levels of immature and mature cathepsin D relative to β-actin expression in primary myoblasts were determined by immunoblot.

(I) Myoblasts cultured in GM or EBSS were loaded with the Lysotracker Red dye, then stained for endogenous Rab7, LAMP1, and LC3. Box and whisker plots (30 images/condition) show the percentage of proximity of Rab7 (mean effect of interaction F(1,116) = 22.01, p < 0,0001, of stimulus F(1,116) = 16.55, p < 0.0001, of subjects F(1,116) = 47.86, p < 0.0001), LAMP1 (mean effect of interaction F(1,116) = 0.2088, p = 0.6486, of stimulus F(1,116) = 4.252, p = 0.0414, of subjects F(1,116) = 0.7115, p = 0.4007), or LC3 (mean effect of interaction F(1,116) = 1.786, p = 0.1840, of stimulus F(1,116) = 7.002, p = 0.0093, of subjects F(1,116) = 11.76, p = 0.0008) with Lysotracker Red.

Scale bars, 10 μm (A–E and I). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001: adjusted p values as determined by within-subjects (G) or between-subjects (I) 2-way ANOVA and post hoc Sidak’s correction for multiple comparisons. Images and graphs show typical staining in myoblasts from 1 of 2 (B), 3 (C–F, H, and I), or 4 (A and G) patients and controls. Results are from 1 of at least 3 independent experiments.

See also Figure S2.