Fig 2: Effects of CoPPIX on nuclear translocation and activation of TFEB during LPS treatment in the rat heart.A, Immunohistochemical analysis of TFEB in the left ventricle of the rat heart at 3 h after LPS administration (15 mg/kg). Representative images in each group are shown: control (a), CoPPIX (b), LPS (c), LPS+CoPPIX (d). Bars, 5 µm. B, RT-PCR analysis of the LAMP1 and LAMP2 levels in the heart of LPS-treated and control animals for 1, 3, and 6 h. Amplification of actin cDNA was performed and served as the internal control. Each bar represents the mean ± S.E. of three animals (*p<0.05 vs. none).

Fig 3: Analysis of autophagy process in patients-derived fibroblasts with homocystinuria.(A) Analysis of protein levels by immunoblotting. Equal amounts from control and the five patients were loaded (50 µg of total cell lysates) and subjected to Western Blot with anti-LAMP1 antibody. We used anti-GAPDH antibody to ensure equal amounts of protein loaded in each lane. This result is representative of three independent experiments. (B) Protein quantification was performed by laser densitometry. The ratios between proteins/GAPDH for each cell line were calculated to determine the expression fold-change relative to control. Data represent mean ± standard deviation of three independent experiments. (C) Analysis of autophagosomes formation by fluorescence microscopy. Representative images of the autophagosome monitorization in control and patients-derived fibroblasts. (D) Quantification of autophagosome number per area in patients´cells compared to control. Results were obtained from three independent experiments. Data represent mean ± SEM (standard error of the mean = SD/80) of positive fluorescence points of LC3B in each mm2 of 80 cells analysis. *P<0.05; **P<0.01; ***P<0.001.

Fig 4: Analysis of protein levels involved in ER stress and Ca2+ homeostasis and processing of mRNA XBP1 in control and patients-derived fibroblasts.(A) Equal amounts from controls and patients were loaded (50 µg of total cell lysates) and subjected to Western Blot with anti-Herp, anti-Grp78 and anti-IP3R1 antibodies. We used anti-Hsp60 antibody to ensure equal amounts of protein loaded in each lane. This result is representative of three independent experiments. Protein quantification was performed by laser densitometry. The ratios between proteins/Hsp60 for each cell line were calculated to determine the expression fold-change relative to control. (B) Data represent mean ± standard deviation of three independent experiments. (C) Equal amounts from controls were loaded (50 µg of total cell lysates) and subjected to Western Blot with anti-Herp and anti-Grp78 antibodies. We used anti-GAPDH antibody to ensure equal amounts of protein loaded in each lane. This result is representative of two independent experiments. (D) Equal amounts from control and patients were loaded (50 µg of total cell lysates) and subjected to Western Blot with anti-phospho-PERK antibody. We used anti-GAPDH antibody to ensure equal amounts of protein loaded in each lane. This result is representative of two independent experiments. (E) RT-PCR analysis of the processing of mRNA XBP1 transcription factor. Tm: tunicamycin; u: XBP1 unspliced form; s: XBP1 spliced form.

Fig 5: A schematic representation of relationship between PEDV infection and autophagy flux. PEDV infection upregulated autophagy-related genes such as ATG5 and ATG12, which are important for autophagosome formation. However, it inhibited LAMP1, which mediates autolysosome formation in PEDV life cycles.