Fig 1: PKMYT1 knockdown inhibits the migration, invasion and EMT of SCC-9 cells. (A) Relative cell migration rate was detected using a wound healing assay. (B) Relative cell invasion rate was detected using a Transwell assay. (C) MMP2 and MMP9 expression levels were detected using western blot analysis. (D) The expression of EMT-related proteins was detected using western blot analysis. ***P<0.001. PKMYT1, protein kinase, membrane-associated tyrosine/threonine 1; EMT, epithelial-mesenchymal transition; sh-NC, negative control short hairpin RNA; ZEB1, zinc finger E-box binding homeobox 1.
Fig 2: miR-601 exerts its tumor suppressor function in OS by targeting PKMYT1. (a–d) U2OS/143b cells were transfected with PKMYT1 overexpression plasmid, miR-601 mimic or negative control. (a) The protein level of PKMYT1 were detected by Western blot assay in U2OS and 143b cells. The original blots/gels are presented in Supplementary Dataset File. (b–d) MTT and transwell assays assess the proliferation, invasion and migration of U2OS and 143b cells. (e) IHC assay detected the PKMYT1 protein expression in OS tissues with miR-601-low and miR-601-high. Scale bar: 100 μm.
Fig 3: miR-601 mediates the downregulation of PKMYT1. (a) Venn diagram displayed the targets of miR-601 predicted by the databases of RNA22v2, TargetScam 7.1, miRTarget and miRPathDB. (b) Schematic diagram displayed the putative binding sequence of 3’-UTR-PKMYT1- mRNA and miR-601. The mutated sites of the 3’-UTR-PKMYT1- mRNA was showed. (c, d) The expression of PKMYT1 mRNA in miR-601 upregulated or downregulated U2OS and 143b cells was measured via RT-qPCR experiment. (e, f) The protein of PKMYT1 in miR-601 upregulated or downregulated U2OS and 143b cells was measured via Western blot experiment. The original blots/gels are presented in Supplementary Dataset File. (g) The enrichment of PKMYT1 on biotinylated miR-NC and miR-601 probe was evaluated by biotin-based pulldown assay. The GAPDH mRNA served as control. (h, i) Luciferase activity of U2OS and 143b cells was accessed through luciferase reporter assay.
Fig 4: PKMYT1 knockdown inhibits the proliferation of SCC-9 cells by regulating CCNA2 expression. (A) PKMYT1 was found to be able to bind CCNA2 according to the STRING and GeneMANIA databases. (B and C) Relative mRNA and protein expression levels were detected using (B) RT-qPCR and (C) western blot analysis, respectively. (D) Co-IP assay was used for verification of the binding between PKMYT1 and CCNA2. (E) CCNA2 expression in the sh-PKMYT1 group was detected using western blot analysis. (F) CCNA2 expression in the pcDNA-CCNA2 group was detected using western blot analysis. (G) A Cell Counting Kit-8 assay was used for the determination of cell proliferation. *P<0.05 vs. sh-PKMYT1 + pcDNA. (H) A colony formation assay was used for the detection of cell colony formation. **P<0.01, ***P<0.001. PKMYT1, protein kinase, membrane-associated tyrosine/threonine 1; CCNA2, cyclin A2; RT-qPCR, reverse transcription-quantitative PCR; co-IP, Co-immunoprecipitation; sh, short hairpin; sh-NC, negative control short hairpin RNA; WB, western blotting.
Fig 5: PKMYT1 is upregulated in SCC-9 cells, and its knockdown inhibits cell proliferation. (A) PKMYT1 protein expression was detected using western blot analysis. (B) PKMYT1 mRNA expression was detected using RT-qPCR. (C and D) Relative protein and mRNA expression levels were detected using (C) western blot analysis and (D) RT-qPCR, respectively. Cell proliferation was detected using (E) Cell Counting Kit-8. *P<0.05, **P<0.01, ***P<0.001 vs. sh-NC. (F) Cell colony formation was determined using colony formation assays. **P<0.01, ***P<0.001. PKMYT1, protein kinase, membrane-associated tyrosine/threonine 1; RT-qPCR, reverse transcription-quantitative PCR; sh-NC, negative control short hairpin RNA.
Supplier Page from Abcam for Anti-PKMYT1 antibody