Figure 1

Expression of CMTM3 was upregulated in B-ALL patients and cell lines A, analysis of ImmuSort database (http://immusort.bjmu.edu.cn/Account/Immusort.html); B, CMTM3 mRNA level of the bone marrow cells from healthy donors (n=2) and primary B-ALL patients (n=5) was analyzed by semi-quantitative PCR; C, real-time RT-PCR; D, CMTM3 mRNA level of SUP-B15 and BV173 cells were analyzed by semi-quantitative PCR; E, CMTM3 protein levels were analyzed by western blot. B-ALL, B-cell acute lymphoblastic leukemia; CMTM3, CKLF-like MARVEL transmembrane domain-containing member 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Data (B to E) were representative of three independent experiments. One-way ANOVA and two-way ANOVA with Dunnett's multiple comparisons test and presented as the $\bar x \pm s$ (# P < 0.01)."

Figure 2

Knockdown of CMTM3 in B-ALL cell lines inhibited cell proliferation A, SUP-B15 and BV173 cells were infected with CMTM3 shRNA (sh391, sh393) or the matching control non-targeting shRNA (shN), and CMTM3 mRNA level of SUP-B15 and BV173 cells were analyzed by semi-quantitative PCR; B, real-time RT-PCR; C, CMTM3 protein levels were analyzed by Western blot; D, analysis of cell proliferation by CCK-8; E, analysis of cell proliferation by cell counting. CMTM3, CKLF-like MARVEL transmembrane domain-containing member 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Data were representative of three independent experiments. One-way ANOVA and two-way ANOVA with Dunnett's multiple comparisons test and presented as the $\bar x \pm s$ (*P < 0.05, #P < 0.01, ★P < 0.001)."

Figure 3

Knockdown of CMTM3 in B-ALL cell lines increased cell apoptosis, but not affected cell cycle A, SUP-B15 and BV173 cells were infected with CMTM3 shRNA (sh391, sh393) or the matching control non-targeting shRNA (shN), and SUP-B15 cell cycle was analyzed by flow cytometry; B, BV173 cell cycle was analyzed by flow cytometry; C, annexin V/PI-staining of SUP-B15 cells was analyzed by flow cytometry; D, annexin V/PI-staining of BV173 cells was analyzed by flow cytometry. 7-AAD, 7-aminoactinomycin D. Data were representative of three independent experiments. One-way ANOVA and two-way ANOVA with Dunnett's multiple comparisons test and presented as the $\bar x \pm s$ (*P < 0.05)."

Figure 4

Knockdown of CMTM3 increased the sensitivity to imatinib A, SUP-B15 and BV173 cells were infected with CMTM3 shRNA (sh391, sh393) or the matching control non-targeting shRNA (shN), and cell counting assays of SUP-B15 cells treated with imatinib for 0, 24, 48 and 72 h; B, cell counting assays of BV173 cells treated with imatinib for 0, 24, 48 and 72 h; C, analysis of cell death of SUP-B15 cells after 2.5 μmol/L imatinib for 24, 48 and 72 h; D, analysis of cell death of BV173 cells after 2.5 μmol/L imatinib for 24, 48 and 72 h. 7-AAD, 7-aminoactinomycin D. Data were representative of three independent experiments. One-way ANOVA and two-way ANOVA with Dunnett's multiple comparisons test and presented as the $\bar x \pm s$ (*P < 0.05, # P < 0.01, ★P < 0.001)."

Figure 5

Knockdown of CMTM3 inhibited Wnt signaling pathway. A, SUP-B15 and BV173 cells were infected with CMTM3 shRNA (sh391, sh393) or the matching control non-targeting shRNA (shN), and Western blot analysis of Ph+B-ALL cell lines to detect the expression of BCR-ABL; B, Erk and Akt levels treated with imatinib for 24 h were analyzed by Western blot; C, go enrichment analysis (shN/sh391) (1, L-alpha-amino acid transmembrane transport; 2, regulation of non-canonical Wnt signaling pathway; 3, regulation of double-strand break repair via nonhomologous end joining; 4, amino acid transmembrane transport; 5, tau-protein kinase activity; 6, cellular response to oxygen-containing compound; 7, cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript; 8, positive regulation of double-strand break repair via nonhomologous end joining; 9, positive regulation of non-canonical Wnt signaling pathway; 10, negative regulation of collateral sprouting); D, knockdown of CMTM3 regulates some molecules by mass spectrometry analysis. Erk, extracellular signal-regulated kinase; Akt, protein kinase B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RNF213, E3 ubiquitin ligase ring finger protein 213; FERMT1, fermitin family member 1; AES, amino-terminal enhancer of split; ZEB2, zinc finger E-box binding homeobox 2; RhoGTP4, Rho guanosine triphosphate 4; CK1δ, casein kinase 1δ; FAM53B, family with sequence similarity 53, member B. Data (A to B) were representative of three independent experiments. One-way ANOVA and two-way ANOVA with Dunnett's multiple comparisons test and presented as the $\bar x \pm s$ (*P < 0.05, # P < 0.01, ★P < 0.001)."