Fig 2: Cholesterol stimulates Shh release in human PDAC cells. (A) Table highlights the primary driver mutations in each PDAC cell line along with tumor differentiation status where PDEC and Capan-2 are the most differentiated, BxPC-3 is moderately differentiated, and MIA PaCa-2 are poorly differentiated. (B) RT-qPCR data measuring relative gene expression of Shh in PDAC cells normalized to PDEC control cells. (C) ELISA results measuring fold-change differences in the amount of Shh released in PDAC cells stimulated with HBSS + cholesterol relative to HBSS alone. Statistical significance denoted by asterisks were *p < 0.05, ***p < 0.001, and ****p < 0.0001. Lack of asterisks indicates data were not significant. ELISA; HBSS, Hank's buffer saline solution; PDAC, pancreatic ductal adenocarcinoma; PDEC, pancreatic ductal epithelial cells; RT-qPCR, quantitative reverse transcriptase polymerase chain reaction; Shh, Sonic Hedgehog; WT, wild-type.

Fig 3: OLFM4 protein directly interacts with SHH protein and reduces SHH protein level in the culture media of PC-3 cells.(a) Immunofluorescent staining of PC-3 cells transfected with OLFM4-V5 tag (PC-3OLFM4 clones) or vector control (PC-3V), using anti-V5 (green) anti-SHH (red) antibodies. Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. (b) Coimmunoprecipitation analysis of OLFM4 and SHH. Cell lysates of PC-3 vector control-transfected cell clones (PC-3V), PC-3 cell clones stably expressing OLFM4-V5 tag (PC-3W), or PC-3 cell clones expressing OLFM4-N (a truncated deletion of OLFM4)-Flag tag (PC-3N) were immunoprecipitated with anti-V5 or anti-Flag (or normal IgG) antibody. Immunoprecipitates were subjected to Western-blot analysis with anti-SHH (upper panel) or anti-OLFM4 (middle panel) antibody. Total lysate subjected to Western-blot analysis with anti-SHH antibody was used as a loading control (lower panel). IgG indicates a normal IgG used as a negative control in the immunoprecipitation assays presented. (c) Time course of SHH protein secretion into the culture media of vector control-transfected PC-3 (PC-3V) and OLFM4-transfected PC-3 (PC-3O) cell clones. The cell-culture media (RPMI 1640 containing 0.5% FBS) was harvested from 3 individual wells of 12-well plates after culturing for 6, 18, 24, 30, 42, or 54 h. SHH secretion was determined by ELISA. Data represent the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. The significance of differences between experimental groups was determined by ANOVA.

Fig 4: Interfering CHOP of AECII abrogates HH activation of fibroblast. MRC5 was incubated with AECII CM for 72 h. A Expression of GLI1, GLI2, and PTCH1 was examined by Western blotting. Shh (100 ng/mL) was used as the positive control. ß-actin was used as the loading control. Each group has 3 replicates. B Quantification of protein level relative to control. C Expression of nuclear GLI1 and GLI2 by Western blotting. Lamin B was used as the loading control. D Quantification of protein level of nuclear GLI1 and GLI2 relative to control. E mRNA levels of GLI1, GLI2, and PTCH1 in MRC5 relative to control

Fig 5: Schematic model of the CHOP induced Shh secretion by AECII and activation of fibroblast during IPF. Multiple factors contribute to ER stress of AECII during IPF. The UPR induced by ER stress upregulates CHOP. Then, CHOP regulates the expression and secretion in an undefined mechanism. Secreted Shh activates HH signaling of fibroblast and promotes ECM synthesis, thus accelerates the development of pulmonary fibrosis