Fig 2: The expression of C6R mHTT promotes autophagy. a Primary MEF cultures from YAC128, C6R or wt littermate embryos were treated with bafilomycin or DMSO as a control. Levels of p62 and LC3-II were analyzed by Western blot. p62: 2way-ANOVA genotype p < 0.0001, bafilomycin p < 0.0001, LC3-II: 2way-ANOVA genotype p = 0.0239, bafilomycin p < 0.0001. b Primary MEF cultures from YAC128, C6R or wt littermate embryos were seeded onto coverslips and treated with bafilomycin. Cells were fixed and stained for p62 and LC3, Hoechst dye was used for nuclear counterstaining. Samples were imaged on a confocal microscope and the density of punctae, staining intensity as well as the co-localization of LC3 and p62 staining were analyzed. p62 density: 1way-ANOVA p = 0.0167, LC3 density: 1way-ANOVA p = 0.0116, LC3 staining intensity: 1way-ANOVA p = 0.0003, colocalization: 1way-ANOVA p = 0.0051. c Primary MEF cultures from YAC128, C6R or wt littermate embryos were seeded onto coverslips and treated with MG132 or DMSO as a control. Cells were fixed and stained for p62, Hoechst dye was used for nuclear counterstaining. Samples were imaged on a confocal microscope and the density of punctae was analyzed. 1way-ANOVA p < 0.0001. d Primary MEF cultures from YAC128, C6R or wt littermate embryos were seeded onto coverslips and treated with MG132 or DMSO as a control. RNA levels of p62 were determined by quantitative RT-PCR (qPCR), normalized to the expression level of Rpl13a. 2way-ANOVA: genotype p = 0.0461, treatment p < 0.0001. Representative blots/images and pooled quantification data with S.E.M. are shown, 3-5 independent cultures were analyzed. Number of replicates is shown as insets for Western blot and qPCR experiments, for imaging experiments 24-30 cells per condition were analyzed. Statistical significance was determined 2way-ANOVA with post-hoc Bonferroni correction. *: p < 0.05, **: p < 0.01, ***: p < 0.001

Fig 3: Lipid droplet accumulation and autophagy analysis of siTMEM199 and siCCDC15 HepG2 cells. HepG2 cells were treated with siRNAs for non-targeted (control), TMEM199, and CCDC115. (A) After 24 hours of siRNA treatment cells were incubated with 200 µmol BSA conjugated OA in full medium for 24 hours. After incubation with OA cellular neutral lipids and lysosomes were labeled with LipidTox and Lysotracker red DND-99, respectively. (B) Quantification of lipid droplet size, number of lipids per nucleus, and Pearson’s correlation coefficient for LipidTox Green and Lysotracker Red DND-99. (C) Oroboros respiratory capacity in siTMEM199 HepG2 cells compared with controls. (D) HepG2 cells were treated 48 hours with siRNA; Bafilomycin A1 is positive control for V-ATPase mediated induced autophagy inhibition. Panels represent whole cell lysate protein levels of autophagy markers (P62; LC3B) and GAPDH as loading control. (E) Mouse liver lysates analyzed for autophagy marker (p62/LC3). (F) siRNA treated HepG2 cells were analyzed for lysosomal acidification by LysoSensor DND-160; shown are the pH measurements of the acidification capacity of treated cells and pH calibration curve. Bafilomycin A1 and chloroquine were used as positive de-acidification agents. All panels show representative Western blots from 3 experiments with triplicate measurements per experiment.

Fig 4: CSE modulates mitochondrial homeostasis by mitophagy and lysosome activity. (A) Representative Western blots and image analysis of p62, beclin-1, and LC3A/B protein of control and COPD ASMCs; GAPDH served as reference protein; p < 0.05 was considered significant. Statistics were calculated by Student’s t-test. (B) Representative confocal microscopy of co-localization mitochondria-LC3 (60×, green: cytochrome C, red: LC3A/B, blue: DAPI, white arrow shows co-localization). (C) Representative Western blots and image analysis of EEA1 and LAMP-1 protein of control and COPD ASMCs; GAPDH was used as reference protein, p < 0.05 was considered significant. Statistics were calculated by Student’s t-test. (D), Representative confocal microscopy of mitochondria–lysosome co-localization (60×, red: TOMM20, green: LAMP1, blue: DAPI, white arrow shows co-localization.

Fig 5: C6R mHTT preferentially colocalizes with p62 and is degraded faster than cleavable mHTT. a COS-7 cells were seeded onto coverslips, cotransfected with cleavable mHTT1-1212, C6R mHTT1-1212 or mHTT1-586 and p62 as indicated and treated with bafilomycin to block autophagic flux. Cells were fixed and stained for p62 and HTT, Hoechst dye was used for nuclear counterstaining. Samples were imaged on a confocal microscope and the colocalization of HTT and p62 signals was analyzed. 1way-ANOVA p < 0.0001. b COS-7 cells were cotransfected with cleavable mHTT1-1212, C6R mHTT1-1212 and p62 as indicated and treated with MG132 to enforce autophagic degradation. Cycloheximide was added for the indicated periods of time and samples were analyzed by Western blot. Data are graphed in three different versions to better visualize differences between groups: (i) all four conditions, (ii) cleavable mHTT vs. C6R mHTT, 2way-ANOVA mHTT construct p = 0.0146, time p < 0.0001, (iii) cleavable mHTT without/with p62, 2way-ANOVA p62 p = 0.0436, time p < 0.0001. Representative blots/images and pooled quantification data with S.E.M. are shown, for imaging experiments 14-21 cells per condition were analyzed, for Western blotting 3 independent experiments were performed with 3 technical replicates each. Statistical significance was determined by 1way-ANOVA with Tukey’s post-hoc correction for A and 2way-ANOVA with Bonferroni’s post-hoc correction for B. *: p < 0.05, **: p < 0.01, ***: p < 0.001