Fig 2: PACS2–TRPV1 axis is required for ER–mitochondrial tethering during ER stress and lung fibrosis. Cartoon summarizing the results of this study: left side: upon ER stress/CHOP induction, SPC?exon4 overexpression or in IPF AECII where persistent pro-apoptotic ER stress is observed, PACS2 protein is severely decreased and, therefore, a reduction in ER–mitochondrial tethering is observed alongside with severe AECII apoptosis. Upon treatment with CPS, a TRPV1 modulator, PACS2 protein levels are restored, ER–mitochondrial tethering is rescued together with a decrease in AECII apoptosis and COL1A1

Fig 3: Schematic representation of this study. Restoring insufficient BAG3-mediated autophagy in IPF fibroblasts by the indicated drugs. The slope represents the synergistic effect of the indicated drugs on BAG3 inhibition. Small font indicates decreased expression or decreased autophagy flux and vice versa. To the left, highly proliferative IPF fibroblasts (green) are depicted, enwrapped in thick extracellular matrix (brown) with increased BAG3, FLNC and COL1A1 and insufficient BAG3-mediated autophagy. To the right, decreased IPF fibroblast proliferation, decreased FLNC, COL1A1 and BAG3 protein levels and restored autophagy upon treatments with the indicated drugs is shown

Fig 4: Gene-expression profiles of growing and senescent BJ cells after p21 knockdownBJ human fibroblasts (proliferating, G; and DNA damage-induced senescent, DIS) were transduced with either siRNA targeting p21 (sip21) or control siRNA (siCtrl). Cells were harvested and analyzed by Affymetrix PrimeView microarrays (3 replicates). Results are presented as K-means clustering of the microarray data. Probe sets whose abundance was above the mean are shown in red, those below the mean in blue, and those equivalent to the mean in green.Principal component analysis (PCA) scatterplot. Points are colored according to cell type (G, red; DIS, blue). Squares and triangles are drawn for sip21 and siCtrl siRNA groups, respectively.Venn diagram showing the distribution of shared genes among G and DIS cells after p21 knockdown.Enrichment analysis from the WikiPathways database identified pathways affected in 1,545 genes that were uniquely changed in DIS cells after p21 knockdown.K-means clustering of the 1,545 genes that were uniquely changed in DIS BJ cells after p21 knockdown. Probe sets whose abundance was above the mean are shown in red, and those below the mean in blue.mRNA expression levels relative to controls of p21, COL1A1, CDK-1, cyclin A2, Smad-7, andTNF-a genes after transduction with sip21 or control siRNA in G and DIS BJ cells. Data are presented as means ± SEM of three repeats, each performed in triplicate. Data were analyzed using Student's t-test. *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig 5: Therapeutic modulation of BAG3 decreases proliferation and COL1A1 in IPF PCLS. PCLS from explanted IPF patient lungs were treated with drugs, as indicated for 24 h. (A) PCLS lysates were prepared and tested for the indicated proteins via western blotting (top panel). Lower panels represent the quantifications of the indicated proteins after with ß-actin. Values in Veh-treated PCLS were set as one. Treatment and representative analysis and blots from n = 3 IPF patients are shown. (B) Immunofluorescence staining for COL1A1 (red) on IPF PCLS upon Veh or drug treatments for 24 h has indicated. Nuclei were stained with DAPI (blue), scale bar = 200 µm. Lower panel indicates COL1A1 fluorescence intensity after drug treatments. Stainings and analysis were performed from n = 3 IPF PCLS per each group and about three technical replicates were performed. p Value summary: * p = 0.05, ** p = 0.01, *** p = 0.001 show significant difference as compared to Veh-treated PCLS and § p = 0.05, §§ p = 0.01, §§§ p = 0.001 indicate significant difference as compared to Pirf-treated PCLS