Fig 1: MYO1B promotes the activation of RhoA and the formation of migratory focal adhesions in CRC cells. (A) Representative figures of the co-immunoprecipitation assay on MYO1B to CDC42, RAC1, and RhoA. (B) Representative figures of the co-immunoprecipitation assay on RhoA to MYO1B. (C) Representative figures of the immunofluorescence assay on the co-localization of MYO1B and RhoA in CRC cells. (D) Representative figures of the western blots for RhoA expression in cells transfected with lentivirus-MYO1B or lentivirus-shMYO1B. The values under the membrane represent the expression of genes normalized to the expression of the reference gene GAPDH. (E,F) Representative figures of the co-immunoprecipitation assay for the expression of RhoA and GTP-binding RhoA (RhoAGTP). (G) Representative figures of the western blots for the expression of the FAK/paxillin axis signatures in cells transfected with lentivirus-MYO1B or lentivirus-shMYO1B. The values under the membrane represent the expression of genes normalized to the expression of the reference gene GAPDH. Shown at ×10,000 original magnification. (H) Representative figures of the immunofluorescence assay on the formation of migratory focal adhesions. Shown at ×10,000 original magnification. (F) The fibronectin adhesive assay of the adhesive property of CRC cells. Representative figures are in the left panel, and bars in the right panel are expressed as mean ± SEM. (I) fibronectin adhesive assay showed the correlation between MYO1B expression and adhesive property in SW480 cells and HCT116 cells. Cells were fixed and stained with 1% crystal violet. Scale bar: 100 µm. **, P<0.01 vs. control; ##, P<0.01. CRC, colorectal cancer; Ctrl, control; Veh, vehicle.
Fig 2: miR-142-3p blocks EMT by regulating RAC1/ERK1/2 signaling in colon cancer SW620 cells. (A) Representative results of western blot analysis of EMT markers and signaling proteins after transfection with miR-142-3p mimics and treatment with RAC1 inhibitor. (B) Relative expression of EMT markers and signaling protein after transfection of miR-142-3p mimics and treatment with RAC1 inhibitor. *P<0.05; **P<0.01. miR, microRNA; EMT, epithelial-to-mesenchymal transition; RAC1, RAC family small GTPase 1; KD, miR-142-3p mimic cells; NC, miR-142-3p mimic negative control cells; CON, control cells without any treatment; NSC, RAC1 inhibitor NSC23766; MMP, matrix metalloproteinase; p-, phosphorylated-.
Fig 3: Effect of miR-28-3p overexpression and knockdown on p-Erk1/2 and Rac1. (A) Western blotting was performed to determine the effect of miR-28-3p overexpression and knockdown on p-Erk1/2 and Rac1 expression. Protein expression levels of (B) Rac1, (C) Erk1/2 (molecular weight at 44 and 42 kDa, respectively) and (D) p-Erk1/2 (molecular weight at 44 and 42 kDa, respectively) in DU145 cells following transfection with the miR-28-3p mimic or inhibitor. **P<0.01, ***P<0.001. miR, microRNA; p-, phosphorylated; NC, negative control.
Fig 4: MEG3 was overexpressed and miR-130a-5p was knocked down in CCI rats. (A, B) The expression of MEG3 in the L4-L6 dorsal spinal cord of CCI rats and astrocytes was assayed by RT-qPCR 0, 1, 3, 5, 7, and 14 days after CCI and 6 hours, 12 hours, 1 day, 3 days, 7 days, and 14 days after LPS induction of astrocytes, respectively. (C, D) Expression of miR-130a-5p in CCI rats and astrocytes was measured by RT-qPCR at 0, 1, 3, 5, 7, and 14 days after CCI and 6 hours, 12 hours, 1 day, 3 days, 7 days, and 14 days after LPS induction of astrocytes, respectively. (E, F) Expression of CXCL12/CXCR4 and its downstream Rac1 and NF-κB in the dorsal spinal cord of CCI rats and astrocytes at 0, 1, 3, 5, 7, and 14 days and LPS-induced astrocytes at 6 hours, 12 hours, 1 day, 3 days, 7 days, and 14 days, respectively, was measured using WB. Data were expressed as mean±SD. n=5. NSP>0.05, *P<0.05, **P<0.01, ***P<0.001 (vs. sham group).
Fig 5: Analysis of the influence of CAV1, ARF6, Rac1 or CLTC upon inhibiting pkh67 labeled milk-exosome/cis internalization via knockdown technology. (A) Western Blot analysis of A2780CP cells transfected with siRNA against key endocytic regulators caveolin-1 (CAV1), ARF6 and Rac1 and clathrin heavy chain (CLTC), or negative control siRNA (NC). (B,C) Pkh67 labeled milk-exosome/cis (green spots) uptake by A2780CP cells (blue fluorescence DAPI labeled nuclei) pretreated with indicated siRNAs, as visualized and quantified by confocal microscopy (C). Values represent mean ± S.E.M. ****, P < 0.0001. ns, no significance.
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