Fig 2: Beclin 1 regulates ALK5 recycling in COS7 cells. Representative images of COS7 cells transiently expressing HA-ALK5 and infected with (a) ctrl or (b) bec shRNA lentivirus. Only infected (GFP+) cells were analyzed. Recycled ALK5 was detected using a mouse anti-HA antibody and a goat-anti-mouse Alexa 647 secondary antibody. Total ALK5 was detected using a rat-anti-ALK5 and goat-anti-rat Alexa 555. c Combined results from three independent experiments. Quantification of recycled/total ALK5 from confocal z-stacks. Data is expressed as the ratio of recycled/total ALK5 summed over the z-stack for each cell where each dot represents one cell (n = 30 fields/group, n > 85 cells/group). ***p = 0.001. Bar graphs are mean ± SEM

Fig 3: Localization of ALK5 with VPS35 is beclin 1-dependent. Representative images of neurons infected with ctrl shRNA (a) or bec shRNA (b) stained with antibodies against ALK5 and VPS35. ALK5 is pseudocolored green and VPS35 is pseudocolored red. Regions in the box are enlarged below. Red arrowheads in (b) indicate ALK5 puncta that don’t colocalize with VPS35. c Quantification of the fraction of ALK5 colocalizing with VPS35 in cells infected with ctrl or bec shRNA, expressed as the colocalization coefficient. Data are combined results of three independent experiments (n = 27 fields/group). Planned comparison of colocalization coefficients in control and beclin 1 shRNA cells were analyzed by unpaired t-test, **p = 0.01. Bar graphs are mean ± SEM.

Fig 4: Localization of ALK5 in Rab11+ recycling endosomes is beclin 1-dependent. Representative images of neurons infected with (a) ctrl shRNA or (b) bec shRNA and stained with antibodies against ALK5 (pseudocolored green) and Rab11 (pseudocolored red). Regions in the box were brightened to ease visualization and enlarged below. Intensity traces (white offset lines) from the original image are plotted at right. Red arrowheads indicate ALK5 puncta that don’t colocalize with Rab11. c Quantification of the fraction of ALK5 colocalizing with Rab11 in cells infected with ctrl or bec shRNA, expressed as the colocalization coefficient. Data are combined results of three independent experiments (n = 27 fields/group). Planned comparison of colocalization coefficients in control and beclin 1 shRNA cells were analyzed by unpaired Student’s t-test, ***p = 0.001. Bar graphs are mean ± SEM.

Fig 5: MHP1-AcN decreased TGF-ß receptor expression via upregulating Bmp7 and Smad7 expression in fibroblasts. (A) qPCR results of Tgfbr1 and Tgfbr2 expression in MRC-5 cells after incubation with MHP1-AcN (10, 30 or 100 µg/mL) for 24 h. **p < 0.01 vs Control. n = 3 in each group. (B) Western blot analysis of TßRI and TßRII levels in MRC-5 cells after incubation with MHP1-AcN (10, 30 or 100 µg/mL) for 24 h. The experiments were repeated at least twice. (C) qPCR results of Bmp7 and Smad7 expression in MRC-5 cells after incubation with MHP1-AcN (10 or 100 µg/mL) for 24 h. *p < 0.05, **p < 0.01 vs Control. n = 3 in each group.