Fig 1: COL11A1/Akt signaling regulates the mitochondrial translocation of expressed BCL-2/BAX. (A) The results of immunofluorescence analysis of BAX and BCL-2 expression in BxPC-3 cell following different treatments are shown. Mitochondria were visualized with MitoTracker (red); BAX and BCL-2 were visualized with Alexa Fluor 488 (green), and nuclei were stained with DAPI. Confocal images were taken using a 200× objective. (B) The fluorescence intensity was determined and normalized against the cellular background fluorescence. The levels of BAX and BCL-2 were calculated as a ratio and compared to those in the controls. Data represent the mean ± SD (n=3, *p<0.05).
Fig 2: The COL11A1/a1ß1/DDR2/Akt axis affects apoptosis-related protein function and cancer cell apoptosis. (A) Expression levels of the s-COL11A1, L-COL11A1, BCL-2, BAX, caspase-3/cleaved caspase-3 and caspase-9/cleaved caspase-9 proteins in BxPC-3 cell under serum starvation or treated with GEM (20 µM) and COL11A1 (1 µg/ml) were examined by western blotting. (B) BxPC-3 cell treated with serum starvation or GEM and COL11A1 were stained with PI and FITC Annexin-V and analyzed by flow cytometry. (C) Western blotting was used to determine the protein expression levels of s-COL11A1, L-COL11A1, a1ß1, DDR2, Akt, Erk1/2, Jnk, p-AktSer473, p-Erk1/2 and p-Jnk after stimulation with different treatments. The relative ratios of p-AktSer473/Akt, p-Erk1/2/Erk1/2 and p-Jnk/Jnk expression were shown. The chart shows changes in protein expression. (D) Western blotting analysis of s-COL11A1, L-COL11A1, BCL-2, BAX, caspase-3/cleaved caspase-3 and caspase-9/cleaved caspase-9 protein expression in BxPC-3 cell treated with serum starvation or GEM, COL11A1, siCOL11A1 and/or LY294002. (E) The BCL-2/BAX ratios in BxPC-3 cell after different treatments were determined. (F) Legend for the histograms in (D) and (E) (n=3, *p<0.05).
Fig 3: COL11A1/Akt inhibits the decrease in mitochondrial transmembrane potential. (A) The results of flow cytometry analyses to determine the ??m in BxPC-3 cell after treatment with GEM, COL11A1, siCOL11A1 and/or LY294002 and stained with JC-1 are shown. (B) The relative change in ??m was calculated as a ratio compared to the control. Data represent the mean ± SD. (C) BxPC-3 cell treated with GEM, COL11A1, siCOL11A1 and/or LY294002 and stained with JC-1 (the nuclei were stained with DAPI.) were analyzed by confocal microscopy. Confocal images were taken using a 100× objective. (D) The fluorescence intensity was determined and normalized to the cellular background fluorescence. The proportion of J-monomers was calculated as a ratio compared to the number of J-monomers in the control. Data represent the mean ± SD (n=3, *p<0.05).
Fig 4: COL11A1 promoted the proliferation of pancreatic cancer cells and the resistance to GEM via integrin a1ß1/DDR2 receptors. (A) The mRNA expression of COL11A1, integrin a1ß1 and DDR2 were assessed by RT-qPCR in pancreatic cancer cells (BxPC-3, Capan-1, Mia PaCa-2 and PANC-1). (B) The expression of COL11A1 in cell culture supernatant (s-COL11A1) protein expression levels were assessed by ELISA in BxPC-3, Capan-1, Mia PaCa-2 and PANC-1 cells. (C) s-COL11A1 and L-COL11A1 (the levels of COL11A1 in total cells lysates) of the four pancreatic cancer cell lines (BxPC-3, Capan-1, Mia PaCa-2 and PANC-1) were detected after stimulation with different treatments by western blotting. (D) The quantitative chart of s-COL11A1 and L-COL11A1 protien expression level. (E) BxPC-3, Capan-1, Mia PaCa-2 and PANC-1 cells were treated with COL11A1 (1 µg/ml) or siCOL11A1, and cell proliferation was determined by CCK-8 assay. (F) The IC50 and cytotoxic effects of GEM (20 µM) on BxPC-3, Capan-1, Mia PaCa-2 and PANC-1 cells treated with COL11A1 or siCOL11A1 were evaluated by CCK-8 assay. (G) Cell proliferation, the IC50 and the cytotoxic effects of GEM in BxPC-3 cell following different treatments were determined by CCK-8 assay (n=3, *p<0.05).
Fig 5: COL11A1/Akt modulates molecular signaling in mitochondria-mediated apoptosis. (A) After BxPC-3 cell treated with GEM were stimulated with COL11A1, siCOL11A1 and/or LY294002, the cell lysates were immunoprecipitated with anti-BAX antibody, and immunoblotting were performed with antibodies against BCL-2, BAX, ANT and VDAC. (B) Western blotting analysis of Cyt-C expression in the cytoplasm and mitochondria of BxPC-3 cell treated with COL11A1 or siCOL11A1 and/or LY294002 were carried out. (C) The results of immunofluorescence analysis of Cyt-C expression in BxPC-3 cell following different treatments are shown. Mitochondria were visualized with MitoTracker (red); Cyt-C were visualized with Alexa Fluor 488 (green), and nuclei were stained with DAPI. Confocal images were taken using a 200× objective. The fluorescence intensity was determined and normalized to the cellular background fluorescence. The levels of Cyt-C were calculated as a ratio compared to those in the controls. Data represent the mean ± SD. (D) BxPC-3 cell were treated with COL11A1, siCOL11A1 and LY294002. Cell lysates were immunoprecipitated with anti-Apaf-1 antibody, and immunoblotting was performed with anti-procaspase-9. (E) The cleaved caspase-3/9 protein, L-COL11A1 and s-COL11A1 levels of BxPC-3 cell under different conditions were detected by western blotting. (F) A schematic diagram shows the functions that are altered due to COL11A1.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Human COL11A1 ELISA Kit (Colorimetric)