Fig 1: Stabilization of PINK1 in severe asthmatic fibroblasts.(A) The fibroblasts were cultured in DMEM complete medium for 4 hours post serum-starvation. Under basal conditions, mRNA expression of mitophagy markers, PINK1 and PRKN, in control and S-As fibroblasts was analysed by qRT-PCR and expressed as fold expression change relative to control fibroblasts post normalization to housekeeping gene 18s rRNA. (B) The fibroblasts were cultured in complete medium for the indicated time points post serum-starvation. Whole cell lysates were subjected to immunoblot analysis of PINK1 protein. β-actin was used as loading control. (C) Representative immunoblots depicting mitophagy related proteins, PINK1, Parkin, BNIP3, BNIP3L, NDP52, and optineurin in control and S-As fibroblasts. β-actin was used as loading control. (D) The fibroblasts were cultured in the presence of lysosomal protease inhibitors, E64d and pepstatin A, for 6 hours post serum-starvation. Cell lysates were subjected to immunoblot analysis of PINK1. β-actin was used as loading control. (E) The fibroblasts were cultured in DMEM complete medium for 48 hours post serum-starvation for immunofluorescence measurements at baseline. Representative images depicting control and S-As fibroblasts transduced with LC3B-GFP (green) and immunostained with PINK1-PE (red). (F) Representative images depicting fluorescent staining of mitochondria using MitoTracker Green (green) and lysosomes using LysoTracker Deep Red (red). Graphical data are represented as mean ± SEM from 3–4 independent experiments with at least 3 unique donors in each group. *p < 0.05, **p < 0.01, determined by unpaired two-tailed Student t-test.
Fig 2: IL-17RB alters mitochondrial turnover and IL-6 secretion. (A) IL-17RB OE PSCs stably express IL-17RB without activation. IL-17RB KD PSCs do not express IL-17RB; (B) IL-17RB OE PSCs increase α-SMA. IL-17RB and α-SMA Western blot of IL-17RB OE and IL-17KD cells with corresponding control of IL-17RB VEC OE and IL-17RB VEC KD cells; (C) IL-17RB overexpression increases PSCs proliferation. MTT assay comparing IL-17RB OE, IL-17RB KD PSCs with respective controls (IL-17RB VEC OE and VEC KD, p < 0.0001, Mann–Whitney U test). Data represent the mean of three independent experiments conducted in sextuplicate; (D) IL-17RB overexpression decreases IL-6 expression. IL-6 Western blot of IL-17RB OE and IL-17KD PSCs with corresponding controls; (E) IL-17RB overexpression decreases IL-6 secretion, while IL-17RB knockdown induces no changes. ELISA of cell culture supernatant (n = 3, OE vs. VEC OE, p = 0.0167, Mann-Whitney U test); (F) Auto-/mitophagy Western blot of IL-17RB OE and IL-17KD PSCs with corresponding controls. p62—Autophagy receptor p62, PRK8—Ubiquitin E3 ligase Parkin; (G) IL-17RB overexpression reduces mitochondrial fission and fragmentation. Western blot of IL-17RB OE and IL-17RB KD PSCs with corresponding controls. pMMF—phospho-mitochondrial fission factor, DRP1—phospho-Dynamin related protein 1; (H) IL-17RB overexpression induces elongated mitochondria with tubular cristae (white arrows). Electron microscope images of IL-17RB OE PSCs compared to IL-17RB VEC OE controls. Scale bar = 250 nm. * p < 0.05, ** p < 0.01.
Fig 3: Increase in basal autophagy in severe asthmatic fibroblasts.The control and severe asthmatic (S-As) fibroblasts were cultured in DMEM complete medium for 4 hours post serum-starvation to measure autophagy at baseline. (A) To measure autophagosomal levels, the fibroblasts were stained with Autophagosome Detection Reagent for 30 minutes at 37°C in the dark and fluorescent readings were taken using a fluorescence microscope and plate reader. Representative images showing fluorescent staining of autophagosomal vacuoles (blue) in control and S-As fibroblasts (left panel). Quantitative representation of autophagosomal levels in relative fluorescence units (RFU) (right panel). (B) Under basal conditions, mRNA expression of autophagy markers, ATG5, LC3B, SQSTM1/p62 and LAMP2, in control and S-As fibroblasts was analysed by qRT-PCR and expressed as fold expression change relative to control fibroblasts post normalization to housekeeping gene 18s rRNA. (C) Representative immunoblots depicting protein levels of LC3B, p62 and LAMP2A in control and S-As fibroblasts. β-actin was used as loading control. (D) Densitometric analysis of LC3B lipidation represented as the ratio of LC3BII to LC3BI, p62 and LAMP2A levels in control and S-As fibroblasts. (E) The fibroblasts were cultured in DMEM complete medium for 48 hours post serum-starvation for immunofluorescence measurements at baseline. Representative images depicting fluorescent staining of autophagosomes using LC3B-GFP (green) and lysosomes using LysoTracker Deep Red (red). (F) The fibroblasts were cultured in the presence of lysosomal protease inhibitors, E64d and pepstatin A, for 6 hours post serum-starvation. Cell lysates were subjected to immunoblot analysis of autophagy proteins LC3B and p62. β-actin was used as loading control (top panel). Densitometric analysis of LC3B lipidation represented as the ratio of LC3BII to LC3BI and p62 levels in control and S-As fibroblasts upon treatment with E64d and pepstatin A (bottom panel). Graphical data are represented as mean ± SEM from 2–4 independent experiments with at least 3 unique donors in each group. *p < 0.05, determined by unpaired two-tailed Student t-test (Control vs Severe Asthma) or unpaired t-test with multiple comparisons using the Holm-Sidak method (Cmplt vs Cmplt+E64d+PepA).
Fig 4: MALAT1 Knockdown Impairs Mitophagy(A) Mitophagy biomarkers were detected by western blot. β-Actin was used as the control. (B) Quantitation of mitophagy biomarker western blot results. ∗∗∗∗p < 0.0001, ∗∗p < 0.01 compared with the shCT control group. (C) The LC3B II/I ratio. Autophagy was assessed by LC3B-II/I ratio. After knockdown of MALAT1, the LC3B-II/I ratio was significantly decreased. ∗∗∗∗p < 0.0001 compared with the shCT control group. (D) Acidic lysosome by Lysotracker staining. The intensity of the fluorescence stained by lysotracker was measured. ∗∗∗∗p < 0.0001 compared with the shCT control group.
Fig 5: High-glucose treatment promotes mitophagy and autophagy flow in primary hepatocytes. A Relative expression of mitophagy-related proteins (LC3II, Pink1 and Parkin) in cells (n = 3). B GFP-LC3 staining was used to assess autophagy. Scale bar, 10 μm. C The cells were stained using Lyso-Tracker Red (50 μmol/L; red) and transfected with GFP-LC3 (green) to detect colocalization of autophagosomes and lysosomes. Scale bar, 10 μm. D Mito-RFP (red) and GFP-LC3 (green) were co-transfected to detect the binding of autophagosomes to mitochondria. Scale bar, 10 μm. E The cells were stained using Lyso-Tracker Red (50 μmol/L; red) and Mito-Tracker Green (50 nmol/L; green) to detect the binding of lysosomes to mitochondria. Scale bar, 10 μm
from Cell Signaling Technology for Mitophagy Antibody Sampler Kit