Fig 1: Construction of the PER2 stable knockdown cell line. (A) Western blot analysis of PER2 expression in RKO cells after shRNA transfection; (B) qPCR detection of PER2 mRNA expression in transfected RKO; (C) efficiency of control and shPER-2 lentiviral infection based on GFP fluorescence (approximately >90%) under an optical (left panel) or fluorescence (right panel) microscope, scale bar 50 µm. (D) The numbers of fluorescent cells were counted by ImageJ and are shown in the histogram. PER2, Period 2; RKO, human colorectal adenocarcinoma cells; qPCR: real-time quantitative polymerase chain reaction.
Fig 2: miR-22-3p shuttled by M2-EVs promoted the osteogenic differentiation of AS-BMSCs via the PER2/Wnt/ß-catenin signaling.A Wnt7b mRNA expression in AS-BMSCs transfected with sh-1-Wnt7b or sh-2-Wnt7b determined by RT-qPCR. B miR-22-3p expression in AS-BMSCs treated with M2-EVs (100 µg/mL) and/or sh-Wnt7b measured by RT-qPCR. C The protein expression of PER2, Wnt7b, and Wnt/ß-catenin signaling pathway-related proteins (ß-catenin, C-Myc, and Cyclin D1) in AS-BMSCs treated with M2-EVs (100 µg/mL) and/or sh-Wnt7b measured by Western blot analysis. D The viability of AS-BMSCs treated with M2-EVs (100 µg/mL) and/or sh-Wnt7b detected by CCK-8 assay. E Expression of Runx2 and OCN in AS-BMSCs treated with M2-EVs (100 µg/mL) and/or sh-Wnt7b measured by Western blot analysis. F Mineralization of AS-MSCs treated with M2-EVs (100 µg/mL) and/or sh-Wnt7b detected using Alizarin red staining. G ALP content of AS-BMSCs treated with M2-EVs (100 µg/mL) and/or sh-Wnt7b detected by ALP staining. *p < 0.05, vs. AS-BMSCs treated with sh-NC/NC + sh-NC, #p < 0.05 vs. AS-BMSCs treated with M2-EVs + sh-NC. The cell experiment was repeated three times independently.
Fig 3: Effects of M2 macrophage-derived extracellular vesicles (EVs) on the development of ankylosing spondylitis (AS) through by transferring of microRNA-22-3p.The mechanism diagram illustrating that miR-22-3p shuttled by M2-EVs promoted the osteogenic differentiation of AS-BMSCs and the resultant pathological osteogenesis in AS mice via activation of the Wnt/ß-catenin signaling pathway by targeting PER2.
Fig 4: PER2 silencing inhibited proliferation and ATP production of CCD 841 CoN cells in vitro. (A) qPCR was used to detect the expression of PER2 mRNA in CCD 841 CoN cells after transfection with siRNA (n = 6). (B) Protein expressions of PER2 after transfection with siRNA (n = 6). (C) Immunofluorescence images of Ki67 expression after siRNA transfection with CCD 841 CoN cells. Red fluorescence indicated Ki67, DAPI stained nuclei (n = 6). (D) Quantification of Ki67-positive cells (n = 6). (E) Protein expressions of DRP1 and p-DRP1 after transfection with siRNA (n = 6). (F) The ATP content of CCD 841 CoN cells 72 h after transfection with siRNA (n = 6). (G) Counting of CCD 841 CoN cells 72 h after transfection with siRNA (n = 6). (H) Proliferative activity of CCD 841 CoN cells measured using WST-1 assay 72 h after transfection with siRNA (n = 6). Presented values are mean ± SD. *P < 0.05 vs siCon CCD 841 CoN cells (t-test).
Fig 5: PER2 knockdown promotes RKO cell migration and activates the EMT process. (A) Detect the number of cell migration in the shPER2 and shControl groups by Transwell. (B) The number of migratory cells was counted by ImageJ, scale bar 100 µm (*** P <.001). (C) Determine the cell migration after PER2 knockdown after 48 and 72h by Wound-healing assay. (D) The percentage of wound area closure was analyzed by ImageJ, scale bar 200 µm, shown in the lower panel (**** P <.0001). (E) Mitomycin was added to remove the effect of proliferation on migration. (F) The percentage of wound area closure. (G) The expression of EMT-associated markers was detected by western blotting. EMT, epithelial-mesenchymal transformation.
Supplier Page from Abcam for Anti-PER2 antibody [EPR11381(2)]