Fig 1: Frequencies of CXCL14 and CXCL16 positive stained cells in primary colon cancer tissue (CC) compared to normal colon tissue and in H&E(+) and H&E(-) lymph nodes from CC patients as determined by immunomorphometric analysis according to Weibel (1979). (A) CC tumor cells (black bars) compared with normal epithelial cells (open bars). (B) CC tumor stroma (black bars) compared with lamina propria in normal colon (open bars). (C) Metastatic tumors cells (black bars) and other cells of H&E(+) lymph nodes (grey bars) compared to cells of H&E(-) lymph nodes (open bars). Bars represent mean +1 SEM. P-values for comparison between tumor and normal tissue by two-sided Mann–Whitney t-test are given. Ten primary CC tumors and 10 normal colon tissue samples were analyzed in A and B. Twelve lymph nodes were analyzed in C.
Fig 2: (A,B) Kaplan–Meier cumulative survival curves for CC patients divided into two groups CXCL16(-) and CXCL16(+) according to the median of mRNA expression in the highest lymph nodes in the CC patients in TNM stages III and IV (7.2 mRNA copies/ 18S rRNA unit). In (B) the Kaplan–Meier cumulative survival curves for CXCL16(-) and CXCL16(+) patients are restricted to the CEA(+) plus CEA(int) subgroup of CC patients. (C,D) Kaplan–Meier cumulative survival curves for CC patients divided into two groups CXCL16(--) and CXCL16(++) according to the median of mRNA expression in lymph nodes from CC patients in the CEA(+) group (11.4 mRNA copies/ 18S rRNA unit). In (D) the Kaplan–Meier cumulative survival curves for CXCL16(--) and CXCL16(++) patients are restricted to the CEA(+) plus CEA(int) subgroup of CC patients. The patients were followed for 12 years. Differences in disease-free survival time after surgery between the two groups are given as a ?-value in months and statistical significance as P-values. n = number of patients in the respective group.
Fig 3: CXCL16 and PDE5 correlation in the stroma of breast cancer patients. (A) Real-time RT-PCR assay for CXCL16 mRNA expression in CAFs treated with vehicle (-) or the PDE5 inhibitors: sildenafil (S, 10 µM), tadalafil, (T, 10 µM), and vardenafil (V, 10 µM) for 24 h. (B) ELISA and (C) immunofluorescent staining for CXCL16 protein levels in CAFs treated as indicated for 24 h. DAPI staining was used for nuclei detection (inset). Scale bar = 5 µm. The values represent the mean ± SEM of three different experiments, each performed in triplicate. * p < 0.05; *** p < 0.0005. (D) Left panel: human CXCL16 immunohistochemical staining of MCF-7/CAF xenograft tumor sections. Scale bar = 25 µm. Right panel: immunohistochemistry scores. Cases were scored according to Allred immunohistochemistry (IHC) score [28] which includes both the proportion and intensity scores (range from 0 to 8). * p < 0.05. (E) Gene expression levels of CXCL16 in normal (N) and breast cancer (T) stroma samples. (F) Kaplan–Meier survival analysis relating stromal CXCL16 levels and overall survival (OS) in breast cancer patients. (G) Kaplan–Meier survival analysis relating high stromal PDE5-CXCL16 levels and OS in breast cancer patients.
Fig 4: Role for C-X-C motif chemokine 16 (CXCL16) in mediating tumor-promoting features of PDE5-overexpressing fibroblasts. (A) Mouse cytokine arrays for the detection of secreted proteins in conditioned medium derived from vector (V) and PDE5-overexpressing (PDE5 2) MEFs. Left panels: membranes, 62 targets detected. Right panels: raw numerical densitometry data were extracted, the background subtracted, and the data normalized to the positive control signals. Results are shown as fold change of PDE5 2 versus V stable clones. (B) Real-time RT-PCR assay for CXCL16 mRNA expression in V, PDE5 1, and PDE5 2 stable clones. (C) ELISA for CXCL16 protein secretion in V, PDE5 1, and PDE5 2 stable clones. (D) Immunofluorescent staining of CXCL16 protein expression in V, PDE5 1, and PDE5 2 stable clones. DAPI staining was used for nuclei detection (inset). Scale bar = 5 µm. (E) Trypan blue cell count assays in V, PDE5 1, and PDE5 2 stable clones treated with vehicle (-) or CXCL16 blocking antibody (Ab CXCL16, 1.5 µg/mL) for 72 h. (F) Boyden chamber transmigration and (G) invasion assays in cells treated with vehicle (-) or Ab CXCL16. (H) Soft agar growth (upper panel) and wound healing (lower panel) assays in MCF-7 breast cancer cells exposed with conditioned medium (CM) derived from V, PDE5 1, and PDE5 2 stable MEFs treated with vehicle (-) or Ab CXCL16. Inset, time 0. Pictures are representative of three independent experiments. The values represent the mean ±SEM of three different experiments, each performed in triplicate. * p < 0.05; ** p < 0.005; *** p < 0.0005.
Fig 5: Knockdown of ERK1/2 expression in podocytes overexpressing CXCL16 could prevent the inhibition of podocyte proliferation, the increase of podocyte apoptosis rate and migration ability. (A) Western blotting detected ERK1/2 expression in CXCL16 overexpressed HPC with ERK1/2 knockdown (referred to as: CXCL16-ERK1/2 shRNA). (B) CCK8 assay was used to detect the growth of CXCL16-ERK1/2 shRNA. (C) Apoptosis was detected by flow cytometry. (D) Cell migration in CXCL16-ERK1/2 shRNA was analyzed using Transwell assay (magnification, ×200). **P<0.01, ***P<0.001. ERK1/2, extracellular signal-regulated kinases 1 and 2; CXCL16, CXC motif chemokine ligand 16; HPC, immortalized human podocytes; sh, short-hairpin RNA.
Supplier Page from Abcam for Anti-CXCL16 antibody