Fig 2: CDC34 is required for proliferation of NSCLC cells in vitro and in vivo. (a) A549 and H1975 cells were transfected with siCDC34 (1# to 4#) and lyzed 72 h later for the detection of CDC34 expression by Western blot assays (left). Cell viability was assessed by the CellTiter-Glo Reagent (right). Numbers under the Western blot bands are the relative expression values to Actin determined by densitometry analysis. Error bars, sd. (b) A549, H1975, and H460 cells were transfected with siCDC34 (1# and 4#), and cell proliferation was assessed by trypan blue exclusion analysis. The relative expression of CDC34 was detected by Western blot, and the numbers under the Western blot bands are the relative expression values to Actin determined by densitometry analysis. Error bars, sd; P values, Student's t-test. (c) Foci formation and soft-agar assays of A549, H460, and H1975 cells transfected with siCDC34. Error bars, sd; P values, Student's t-test. (d–f) The H460 (d, f) or A549 (e) cells were transfected with Flag or HA-tagged CDC34 and cell proliferation was assessed by trypan blue exclusion analysis (d, e) or colony formation assays (f). The etopic expression of exogenous proteins was shown by Western blot bands. Inat: inactive; NC, negative control. Error bars, sd; P values, Student's t-test. *P< 0.05, **P< 0.01.

Fig 3: CDC34 regulates Cyclin D1 and promotes lung cancer via EGFR oncoprotein. (a) The cells were transfected with siCDC34 and cell cycle distribution was determined by propidium iodide staining for DNA content followed by flow cytometric analysis. Error bars, sd; P values, Student's t-test. (b) The cells were transfected with siCDC34, lyzed 48 h later, and the expression of CDC34, EGFR, and Cyclin D1 were tested by Western blot. (c, d) H1975 cells were transfected with siCDC34, lysed 48 h later, and the cytoplasm and nucleus proteins were harvested for immunoblotting using indicated antibodies (c). The expression of CDC34 and CCND1 at mRNA level was tested by qRT-PCR (d). Error bars, sd; P values, Student's t-test. (e–i) H460 cells were transfected with siCDC34-1#, siEGFR, and Flag-EGFR, lyzed, and the lysates were subjected to immunoblot using indicated antibodies (e). The cell cycle distribution (f), cell viability (g), and colony forming activity (h, i) were tested. Error bars, sd; P values, Student's t-test.

Fig 4: CDC34 positively regulates EGFR. (a) A scatter diagram of reverse phase protein array (RPPA) data showing a negative correlation between the mRNA level of CDC34 and protein level of p27, and a positive correlation between the mRNA level of CDC34 and protein level of EGFR. The data are from TCGA datasets. P values, Student's t-test. (b) IHC analysis of CDC34 and EGFR in NSCLCs of our setting. (c) The expression of CDC34 in EGFR WT and mutant NSCLCs in datasets and our setting. P values, Student's t-test. (d) Western blot analysis of EGFR/pEGFR in cells transfected with siCDC34 in H460, H226, and H1975 cells with or without EGF co-incubation. Numbers under the Western blot bands are the relative expression values to Actin determined by densitometry analysis. (e) H460 cells transfected with siCDC34 were lysed, the lysates were immunoprecipitated with an anti-EGFR antibody and assayed for tyrosine kinase activity. Error bars, sd. P values, Student's t-test. (f) Western blot analysis of EGFR/pEGFR and downstream molecules in EGF-stimulated, siCDC34-transfected H460 cells. (g) Immunoblotting of lysates of Flag-CDC34-expressing A549 (upper) or HA-CDC34-transfected H226 (lower) cells co-incubated with or without EGF. Numbers under the Western blot bands are the relative expression values to Actin determined by densitometry analysis. (h) H460 cells were transfected with Flag-CDC34, co-incubated with or without EGF, lysed, and the cytoplasm and nucleus proteins were harvested for immunoblotting using indicated antibodies. (i) A549 cells were transfected with Flag-CDC34, lysed 24 h later, and the lysates were immunoprecipitated with an anti-EGFR antibody and assayed for tyrosine kinase activity. Error bars,sd. P values, Student's t-test. (j – l) H460 cells were transfected with siCDC34 and/or CDC34res, which is mutated to resist siCDC34 targeting. Immunoblot assays were conducted using indicated antibodies (j), cell viability was analyzed by MTT assay (k), and colony forming activity was evaluated by foci formation and soft-agar assays (l). Error bars, sd; P values, Student's t-test. P values for indicated comparison: Group 1 vs group 2: < 0.05; group 1 vs group 3: < 0.01; group 2 vs group 4: < 0.05; group 3 vs group 5: < 0.05.

Fig 5: CDC34 inhibits the ubiquitination of EGFR and protects it from proteolytic degradation. (a) qRT-PCR analysis of CDC34 and EGFR in A549 and H1975 cells 72 h after siCDC34 transfection. Error bars, sd; P values, Student's t-test. (b) Cycloheximide (CHX) chase assay to measure EGFR half-life in H460 (in the absence and presence of EGF) and H1975 cells transfected with siCDC34-1# (lower panel). The relative expression values of EGFR were the results determined by densitometry analysis normalized to Actin. Error bars, sd. (c) HCC827 cells were transfected with 50 nM siCDC34-1# for 24 h, treated with erlotinib for 24 h, and lyszed for Western blot assays using indicated antibodies. (d) Western blot analysis of the EGFR expression in H460 cells transfected with siCDC34-1# in the presence or absence of MG132. (e) A549 and H1975 cells were transfected with siCDC34 and HA-Ub, lysed, and the lysates were subjected to immunoprecipitation (IP) and immunoblot using indicated antibodies. (f) A549 cells were transfected with Flag-CDC34 and HA-Ub, and lysed for IP and immunoblot assays. (g) H460 cells were transfected with indicated constructs, treated with MG132, and lysed for IP and immunoblot assays. (h) The cells were transfected with CDC34 and HA-Ub, and lysed for IP and immunoblot assays using indicated antibodies. (i) Western blot analysis of EGFR in #1 siCDC34-transfected H460 cells in the presence or absence of chloroquine (CQ). (j) HCC827 cells were transfected with siCDC34 and HA-Ub for 24 h, treated with or without chloropuine for additional 24 h, lyzed, and subjected to IP and immunoblot using indicated antibodies. (k) H460, A549, and H1975 cells transfected with siCDC34 were lysed and subjected to Co-IP and immunoblotting using indicated antibodies. (l) H460 cells were transfected with CDC34 (left) or CBL (right) in the absence or presence of EGF for 48 h, and lysed for Co-IP and immunoblot using indicated antibodies. (m) In vitro Co-IP experiments using Flag-EGFR purified from 293T cells, His-CDC34 (from E. coli), and increasing amount of purified His-c-Cbl (from E. coli) proteins and indicated antibodies. (n) In vitro Co-IP experiments using Flag-EGFR purified from 293T cells, His-c-Cbl (from E. coli), and increasing amount of purified His-CDC34 (from E. coli) proteins and indicated antibodies. (o) 293T cells were transfected with the wild type or mutant (Y1045F) Flag-EGFRICD for 48 h, lyzed, and subjected to IP and immunobloting using indicated antibodies. (p) siCDC34-mediated loss of EGFR is rescued by knockdown of CBL. A549 cells were transfected with siCDC34 and/or siCBL for 72 h. Before lysis for immunoblot assays, the cells were serum-starved for 3 h and then stimulated by 50 ng/mL EGF for 15 min.