Fig 1: CCNYc could promote cell motility and invasiveness in H1299 cells. (A) Wound healing assay performed with H1299-CCNY KD and control cells over 24 h. The migration activity is shown as mean ± SEM. Bars represent 200 µm. **p < 0.01 vs. NC cells (t-test, N = 3). (B,C) Transwell assay performed with H1299-CCNY KD and the control cells. Cells migrated or invaded through the membrane were calculated and standardized. Bars represent 200 µm. **p < 0.01 vs. NC cells (t-test, N = 3). (D) The upper panel, levels of ß-catenin, vimentin, ZEB1 and snail were measured by WB; the lower panel, the level of proteins was quantified by gray analysis. *p < 0.05 vs. NC cells, **p < 0.01 vs. NC cells (t-test). (E) Expression levels of GFP and CCNY were examined by immunoblotting. The levels of GFP and CCNY were quantified by gray analysis in the lower panel. **p < 0.01 vs. H1299- pEGFP-N1 cells (t-test). (F) Expression of ZEB1, vimentin, ß-catenin and snail were examined by WB. The quantification of the proteins was listed in the lower panel. *p < 0.05 vs. H1299-vector cells, **p < 0.01 vs. H1299-pEGFP-N1 cells (t-test). (G) Wound healing assay performed with CCNYm, CCNYc, and CCNYm G2A upregulation cells. The migration activity is expressed as mean ± SEM. **p < 0.01 vs. H1299 cells transfected with pEGFP-N1 vector (t-test, N = 3). (H,I) Transwell migration assay and invasion assay were used. Bars represent 200 µm. The data are shown as mean ± SEM. **p < 0.01 vs. H1299 cells transfected with pEGFP-N1 vector (t-test, N = 3).
Fig 2: PFTK1 was involved in CCNYc signal pathway. (A) Co-IP assay was done using H1299 CCNYc and H1299 CCNYm cells. Co-IP demonstrated the binding ability of CCNY and PFTK1. (B) Co-localization of PFTK1 and CCNY in human lung cancer cells. PFTK1 and CCNY were marked by immunofluorescence staining (IF). Bars represent 10 μm. (C) The level of PFTK1 in CCNY up-regulated H1299 cells was examined by WB (left panel) and quantified by gray analysis (right panel). (D) H1299 CCNYc cells were infected by recombinant lentivirus carrying shRNA targeting to PFTK1 mRNA. PFTK1 mRNA expression was inhibited by PFTK1-shRNA1-3. The level of PFTK1 was detected by WB (upper panel) and high-content cell analysis (lower panel). *p < 0.05 vs. shControl H1299-CCNYc cells, **p < 0.01 vs. shControl H1299-CCNYc cells (t-test). (E) Wound healing assay performed with PFTK1 downregulation cells. The migration activity is expressed as mean ± SEM. **p < 0.01 vs. H1299-CCNYc cells expressing control shRNA (t-test, N = 3). (F,G) Transwell migration assay and invasion assay of H1299. The data are shown as mean ± SEM. **p < 0.01 vs. H1299-CCNYc cells expressing control shRNA (t-test, N = 3). Bars represent 200 μm. (H) Expression of ZEB1, vimentin, β-catenin and snail and was examined by WB (right panel). The left panel, the level of proteins was quantified by gray analysis. *p < 0.05 vs. H1299-CCNYc cells expressing control shRNA, **p < 0.01 vs H1299-CCNYc cells expressing control shRNA (t-test).
Fig 3: CCNY were highly expressed in NSCLC tissues. Immunohistochemical staining of CCNY. (A) IHC assay detecting the level of CCNY in lung cancer tissue and the paired non-tumoral lung from the TMA. CCNY was mainly localized in the cytoplasm, whereas the non-tumoral lung tissues were negative. Case 1, adenocarcinoma; case 2, squamous cell carcinoma (SCC); case 3, adenocarcinoma-SCC; case 4, large-cell carcinoma; case 5, bronchioloalveolar carcinoma (BAC). The magnification of images in circles is ×40; the magnification of images in rectangles is ×100. (B) Immunohistochemical examination of CCNY protein in primary NSCLC tissue and the matched non-tumoral lung tissues. Upper panel with an original magnification of ×100; lower panel with an original magnification of ×400. (C) IHC examination of CCNY protein in primary NSCLC tissue. CCNY was mainly distributed in the cell membrane. Original magnification, right panel, ×400; left panel, ×100.
Fig 4: CCNYm and CCNYc were highly expressed in lung cancer cells. (A) A simple view of the amino acid sequence for CCNY isoforms. The letter G with red color in the CCNY isoform one sequence is the myristoylation signal motif. (B) The levels of CCNY and loading control GDPAH in lung cancer cells and MRC cells were determined by WB. (C) Western blot was done using cell cytoplasmic extracts, membrane extracts and nuclear extracts, respectively. The CCNY level in cell membrane, cytoplasm and cell nucleus was shown in the figure. (D) Intracellular localization of CCNY in lung cancer. Bars represent 20 µm. The red fluorescence represents CCNY protein, and the blue fluorescence is the nuclear DNA staining with Hoechst 33342.
Fig 5: CCNYc/PFTK1 complex promoted the assembly of F-actin via TPM4. (A–C) F-actin was marked by IF staining. Bars represent 20 μm. (D) Expression level of TPM4 was shown in the figure (upper panel). The quantification of TPM4 level was done by gray analysis (lower panel). **p < 0.01. (E) TPM4 level in PFTK1 down-regulated H1299 CCNYc cells. Upper panel, the picture of WB, lower panel, the quantification of the TPM4 level. **p < 0.01 vs. H1299-CCNYc cells expressing control shRNA. (F) TPM4 level was detected by WB. The quantification of TPM4 was shown in the right panel. **p < 0.01. (G) F-actin was marked by phalloidin conjugating to TRITC. Bars represent 20 μm. (H) RhoA activity was regulated by CCNY. GTPase activity assay was used to determine the activated level of (GTP-) Rac1, RhoA, and Cdc42. The phosphorylated FAK on Y397, as well as the FAK level, were examined by immunoblotting. The protein levels were quantified by gray analysis and showed in the right panel. **p < 0.01 vs. H1299 cells transfected with pEGFP-N1 vector.
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