Fig 1: Vimentin expression in HFL-1 cells. HFL-1 cells were transfected with an untagged negative control vector (pCMV3-NC), a plasmid expressing vimentin (VIM-pCMV3), control siRNA (siRNA NC), and vimentin siRNA (VIM-siRNA) as described in the methods section. Representative RT-PCR (A) and immunoblotting (B) demonstrated either the overexpression or suppression of vimentin by VIM-pCMV3 or VIM-siRNA, respectively.
Fig 2: Expression of caspase-3 and Bcl-2 in Jurkat cells lacking vimentin or overexpressing vimentin. Jurkat cells were transfected with control siRNA, vimentin-specific siRNA or a plasmid expressing vimentin as described in the Materials and Methods. Cells were then cultured in serum-free DMEM in the presence or absence of LPS (10 µg/mL) for 24 h. Total cell lysates were analyzed via immunoblotting for caspase-3, Bcl-2 and ß-actin (as a loading control) as described in the Materials and Methods. Panel A: Representative immunoblots. Panel B: Semiquantitative comparison of target proteins with normalization. Vertical axes: protein level expressed as “Ratio versus control”, which was obtained as follows: (1). The densities of target protein versus internal control ß-actin were obtained. (2). The group of cells transfected with Con-siRNA and cultured in SF-DMEM was set as a reference (“Control = 1”), and the ratio of other groups versus this control group were obtained. Horizontal axes: cells transfected with control siRNA (Con-siRNA), vimentin-specific siRNA (Vim-siRNA) or vimentin expressing plasmid (Vim-vector). Open bar: cells were cultured in serum-free DMEM (SF-DMEM); closed bar: cells treated with 10 µg/mL LPS. #p < 0.05; *p < 0.05 compared to cells transfected with Con-siRNA and cultured in SF-DMEM. Data represent an average of 3 separate experiments.
Fig 3: Expression of mitochondrial membrane proteins and apoptosis- or autophagy-associated proteins in the HFL-1 cells lacking vimentin or overexpressing vimentin. The HFL-1 cells were transfected with vimentin-specific siRNA or a plasmid expressing vimentin as described in the methods section. The cells were then cultured in serum-free DMEM in the presence or absence of LPS (1 µg/mL) for 24 h. Total cell lysates were subjected to immunoblotting for COX II, TOM20, TIM 23, caspase-3, Bcl-2, and P62, with GAPDH as the loading control, as described in the methods section. (A) Representative immunoblotting. (B) Semiquantitative comparison of COX II. (C) Semiquantification of TOM20 expression. (D) Semiquantification of TIM23 expression. (E) Semiquantification of Bcl-2 expression. (F) Semiquantification of caspase-3 expression. (G) Semiquantification of P62 expression. (H) Semiquantification of LC3II expression. Vertical axes: grayscale of targeted proteins vs. GAPDH. Horizontal axes: HFL-1 control cells, cells treated with LPS, cells transfected with vimentin-specific siRNA (VIM-siRNA) or vimentin-expressing plasmid (VIM-pCMV3) plus LPS. Data are presented with significance denoted by *P<0.05.
Fig 4: CAF phenotypes following Clec3b/CLEC3B perturbation and effects in trans. A, MEFs were silenced using short hairpin RNA against Clec3b, and qRT-PCR was performed for the transcripts shown. Clec3b knockdown was confirmed as was the downregulation of iCAF.1 marker Ogn. Il6 and Cxcl12 were utilized as iCAF markers, whereas Acta2 and Fn1 were included as myCAF markers. Data are presented as bar plots showing mean ± SD of three replicates. B, Conditioned medium from scrambled control or shClec3b fibroblasts was collected (48 hours) and transferred to PSN1 PDAC cells. Colors correspond to legend in A. FACS for the EMT marker N-cadherin and CXCR4 was performed, and bar plots show mean ± SD. Three biological replicates were included. C, Patient-derived PDAC organoids (P411) from a male donor were cocultured with fibroblasts (1:1 ratio) separated by polycarbonate cell culture inserts (0.4 μm) for 96 hours. Following the coculture, expression of mesenchymal marker N-cadherin was determined in the organoids by flow cytometry. Bars indicate mean ± SD of geometric mean fluorescence intensity of N-cadherin. Colors correspond to legend in A. Parenthesized percentages are fractions of N-cadherin–positive cells. Three biological replicates were included. D, Human fibroblasts (BJ) were silenced using short hairpin RNA against CLEC3B, and qRT-PCR was performed for the transcripts shown. Knockdown was confirmed as was the expression of various CAF markers. Data are presented as bar plots showing the mean ± SD of three replicates. E, Patient-derived PDAC organoids (P067) from a female donor were cocultured with BJ fibroblasts (1:1 ratio) separated by polycarbonate cell culture inserts (0.4 μm) for 96 hours. Following the coculture, expression of EMT and CAF markers was determined in the organoids and fibroblasts, respectively, by flow cytometry. Bars indicate mean ± SD of geometric mean fluorescence intensity (gMFI) of indicated markers. Colors correspond to legend in D. At least three biological replicates were included. E-cad, E-cadherin; N-cad, N-cadherin; Vim, vimentin. F, The expression of CLEC3B was assessed in the indicated primary PDAC sets with their associated subtype labels (GSE36924, ref. 31, and GSE71729, ref. 5). G, As in F, the expression of CLEC3B was assessed in a local metastatic PDAC cohort (CPCT-02) with PurIST subtype labels. H, Kaplan–Meier survival analysis for patients classified to the PurIST (32) and Bailey (31) molecular subtypes and dichotomization for high/low CLEC3B based on median expression (CPCT-02 cohort). I, Association between iCAF proportion and Moffitt subtype gene signatures (z-score) in a human single-cell dataset (PRJA001603; ref. 33). Each dot represents a unique patient. R and P values are indicated on the plots. A, C–E, and G, Statistical significance was assessed by an unpaired t test; B, statistical significance was assessed by one-way ANOVA; F, statistical significance was assessed by comparing expression between all other subtypes and squamous or basal subtypes by an unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Supplier Page from Abcam for FITC Anti-Vimentin antibody [RV202]