Fig 2: Dual effects of heterozygous GBA mutation on mitophagy. (A) GBA protein levels of GBA knockout (KO), MYC-WT GBA (WT) and MYC-L444P (LP) mutant GBA-overexpressing SH-SY5Y cell lines (left panel). The overexpression of MYC-WT and MYC-GBAL444P, and the deletion of endogenous GBA were validated by western blotting using anti-MYC and anti-GBA antibodies (middle panel), and by measuring GBA enzyme activity from lysosomal-enriched fractions (right panel). Compared to Mock controls (C), **, p < 0.01, One-way ANOVA, Bonferroni post hoc test; (B) MitoTracker Red, MitoTracker Green, and MitoSOX Red labeling in control, KO, WT and L444P SH-SY5Y cells. All 4 cell lines were loaded with MitoTracker Red (MitoRed, 200 µM, 20 min) and MitoTracker Green (MitoGreen, 200 µM, 20 min), or MitoSOX Red (3 µM, 10 min) prior to live cell imaging. Scale bar: 10 µm; (C-D) Quantification of MitoTracker fluorescence intensity in KO- (C) and L444P- (D) SH-SY5Y cells. MitoGreen and MitoRed fluorescence estimates the amount of total mitochondria and functional mitochondria, respectively. The ratio of MitoRed to MitoGreen fluorescence intensity was calculated to estimate the levels of functional mitochondria (n = 60–80 cells per condition from 3 independent experiments). (E) Quantification of MitoSOX Red fluorescence intensity in control, KO, WT and L444P SH-SY5Y cells (n = 60–80 cells per condition from 3 independent experiments). Both L444P and KO cells exhibited an increase in MitoSOX Red intensity, indicative of mitochondrial oxidative stress. (F) Western blot analysis of mitochondrial fraction for autophagy receptors in control, KO, WT and L444P cells. Compared to controls, L444P cells showed lower mitochondrial levels of SQSTM1, NBR1, and BNIP3L. (G) Impaired autophagy in KO and L444P cells. Control, KO, WT and L444P cells were treated with (PI+) and without (PI-) lysosome inhibitors leupeptin and pepstatin A (10 µM each). Flux of autophagy including lysosomal degradation was denoted as ‘PI+-PI-’. (H) Western blot analysis of autophagy-activating proteins AMBRA1 and BECN1 in all 4 cell lines. Data represent mean % of mock controls from 3 independent experiments. Compared to controls, L444P cells showed a reduction in protein levels of BECN1 and AMBRA1. *, p < 0.05; **, p < 0.01, One-way ANOVA, Bonferroni post hoc test.

Fig 3: Animal models of RA-ILD were constructed, and the expression of autophagy-related proteins was changed. (A) CFA and chicken collagen type II were subcutaneously injected into the tail root of C57BL/6 mice on day 0 and 21 and then treated with bleomycin on day 25. On day 35, the mice were sacrificed. (B) The lung weight was measured. (C,D) Representative H&E and Masson’s staining images of lungs from the three groups. Scale bars = 50 µm, shown at 100× (top panels) and 200× (bottom panels). (E) The levels of hydroxyproline in the lung were measured. (F) The mRNA levels of IL-1ß, Col1A1, Col3A1, FN-1, and TGF-ß were quantified. (G) The levels of HIF-1a, BECN1, BNIP3L, BNIP3, LC3 I, and LC3 II in the lungs were detected using immunoblotting. The ratios of LC3 II/I were quantified as described above. Values represent the mean ± SEM (n = 3–5). Compared with the Ctrl group, * p < 0.05, ** p < 0.01, and *** p < 0.001; compared with the model group (BLM), # p < 0.05 and ## p < 0.01.