Fig 1: Knockdown of CCL28 in endometrial stromal cells significantly suppresses cell proliferation and invasion. (A) Following CCL28 knockdown in endometrial stromal cells, cell proliferation was detected using a Cell Counting Kit-8 assay at 0, 12, 24 and 48 h. (B) Cell invasion at 48 h was detected using a Transwell invasion assay (×200, 50 µm). (C) CCL28 levels in endometrial stromal cell supernatants were detected using an ELISA. (D) mRNA expression levels of CCL28, CCR10, MMP2, MMP9 and ITGB1 were examined using reverse transcription-quantitative PCR. (E) Activities of MMP2 and MMP9 were detected using gelatinase zymography. (F) Protein expression levels of CCL28, CCR10, MMP2, MMP9 and ITGB1 were detected via western blotting. (G) Protein expression levels of p-ERK/ERK ratio were detected via western blotting. **P<0.01 and ***P<0.001 vs. shNC. shNC, cells infected with negative control lentivirus; shCCL28-1 and -2, cells transduced with shCCL28 lentivirus-1 and -2; CCL28, C-C motif chemokine ligand 28; CCR10, CC chemokine receptor 10; ITGB1, integrin ß1; sh, short hairpin RNA; p, phosphorylated; OD, optical density.
Fig 2: CCL28 downregulation inhibits tumor infiltration of activated PSCs and angiogenesis. Representative images of immunofluorescence (IF) and quantification for CCL28 (A), a-SMA (B), and CD31 (C) were shown (n = 8). Scale bar = 100 µm; * P < .05, ** P < .01.
Fig 3: CCL28 knockdown impairs pancreatic cancer cell proliferation and migration in vitro. (A) Cell surface expression of CCR3 and CCR10 was detected by flow cytometry in PAN02 cells; (B) immunoblotting image and quantification of CCL28 in PAN02 cell transduced with lentiviral shRNA (n = 3); (C) cell proliferation was measured in shCcl28 (Ccl28KD) cells with or without exogenous mouse recombinant CCL28 and shScr control cells using the CCK-8 assay (n = 4); (D and E) EdU-based cell proliferation assay (D) and cell apoptosis assay (E) were performed in shCcl28 (Ccl28KD) and shScr control cells using flow cytometry (n = 3); (F) immunoblotting images and quantification of Bcl-2 and ß-catenin in shCcl28 (Ccl28KD) and shScr control cells (n = 3); (G) cell migration was evaluated by transwell assay (n = 12); (H) cell invasion ability of shCcl28 cells and shScr control cells was determined (n = 8). Scale bar = 100µm; * P < .05, ** P < .01, *** P < .001, **** P < .0001, n.s. = not significant.
Fig 4: CCL28 may contribute to endometriosis progression by regulating MMP2, MMP9 and ITGB1 expression via activating the ERK signaling pathway. Healthy endometrial stromal cells were pre-treated with 10 µmol/l PD98059 (ERK inhibitor) for 30 min, and then treated with 20 ng/ml CCL28 recombinant protein for 48 h. (A) Cell proliferation was detected using the Cell Counting Kit-8 assay at 0, 12, 24 and 48 h. (B) Cell invasion was detected using the Transwell invasion assay at 48 h (×200, 50 µm). (C) MMP2 and MMP9 activity was detected via gelatinase zymography. Relative protein expression levels of (D) MMP2, MMP9 and ITGB1 and (E) the p-ERK/ERK ratio were analyzed via western blotting. **P<0.01, ***P<0.001 vs. vehicle + DMSO; and ###P<0.001 vs. CCL28 + vehicle. Vehicle, solvent of CCL28 recombinant proteins; DMSO, solvent of PD98059; CCL28, C-C motif chemokine ligand 28; ITGB1, integrin ß1; p, phosphorylated; OD, optical density.
Fig 5: Knockdown of CCL28 expression in endometrial stromal cells by lentiviral transduction. (A) endometrial stromal cells were identified via immunocytochemistry, which was used to analyze CK19 and vimentin expression (×200, 50 µm). (B) CD10 and CD90 were detected using flow cytometry to identify the percentage of EM stromal cells. shCCL28-1, -2 and -3 were constructed to transduce endometrial stromal cells, CCL28 (C) mRNA and (D) protein expression levels were detected to determine the knockdown efficiency of the constructs. **P<0.01 and ***P<0.001 vs. shNC. Control, cells cultured with medium; shNC, cells infected with negative control lentivirus; shCCL28-1, -2 and -3, cells transduced with shCCL28 lentivirus-1, -2 and -3; CCL28, C-C motif chemokine ligand 28; sh, short hairpin RNA; CK19, cytokeratin-19.
Supplier Page from Abcam for Anti-CCL28/MEC antibody