Fig. 5

Depletion of PKMYT1 inhibits tumor growth. a-b Establishment of PKMYT1 knockdown in A549 cells, verified by Real-time RT-PCR (a) and immunoblot (b). c PKMYT1 knockdown dramatically inhibited A549 cell proliferation using growth curve assay. d PKMYT1 knockdown inhibited colony formation ability of A549 and SPC-A1 cells, respectively. Quantification data were also indicated. e-f Effect of PKMYT1 knockdown on the G0/G1 cell cycle transition was examined in A549 cells by PI staining and flow cytometry. f is the quantification data for (e). g PKMYT1 knockdown regulated the expressions of cell cycle transition mediators, including CDK2, CDK6, cyclin D1, p21 and p27. Indicated cell extracts were probed with indicated antibodies. h-i PKMYT1 regulated A549 cell migration examined by wound healing (h) and trans-well (i) assays. Quantification data were also presented, and the OD₅₇₀ values for trans-well assay were indicated below. Scale bar=50 μm. j Indicated cell extracts were probed with indicated antibodies to examine the expression patterns of cell migration regulators, including E-cadherin, N-cadherin, Vimentin and Slug. k Validation of PKMYT1 over-expression by Real-time RT-PCR (top) and immunoblot (bottom) assays. Blue arrow head: exogenous HA-tagged PKMYT1; black arrow head: endogenous PKMYT1. l–r PKMYT1 forced expression overcame PKMYT1AR knockdown effect by cell growth curve (l), wound healing (m-n), trans-well (o, q) and colony formation assays (p, r), (n, q, r) were quantification data for reciprocal assays. Scale bar=50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. MYT1 ove=PKMYT1 over-expression