Fig 2: MAGEC2 inhibition suppresses ASC proliferation.(A) Both ADs and ASCs were transfected with siControl or siMAGEC2 at a concentration of 20 ng/mL for 48 hours. The efficiency was evaluated by immunoblot assay. (B) Cells were plated in a 6-well plate at a density of 2 × 105 per well. Cell numbers were counted at 24 and 48 hours post-treatment with siRNAs (20 ng/mL). Cell counting was performed in triplicate. Bars represent the mean ± standard deviation. One-way ANOVA and Student's t-test were performed to assess the statistical significance (p = 0.004 at the time point of 24 hours, while p < 0.001at 48 hours, one-way ANOVA).MAGEC2, melanoma-associated antigen C2; ASC, adapted suspension cell; AD, adherent cell; ANOVA, analysis of variance.*p < 0.05, †p < 0.01, ‡p < 0.001, Student's t-test.

Fig 3: Melanoma antigen family C2 promoted tumor growth, angiogenesis, and metastasis of NSCLC in vivo. A, Photograph of tumors taken from the mice. B, Tumor volume of MAGEC2 cDNA group was significantly bigger while MAGEC2 shRNA group was significantly smaller, compared with NC group. C, Tumor weight of MAGEC2 cDNA group was significantly bigger while MAGEC2 shRNA group was significantly smaller, compared with NC group. D, Enzyme-linked immunosorbent assay: Mice serum VEGF concentration of MAGEC2 cDNA group was significantly higher while MAGEC2 shRNA group was significantly lower, compared with NC group. E, Vascular endothelial growth factor immunohistochemical staining of the tumor tissues (100× magnified): MAGEC2 cDNA group had a higher VEGF positive rate while MAGEC2 shRNA group had a lower positive rate, compared with NC group. F, Hematoxylin and eosin staining of lung tissues (200× magnified) of MAGEC2 cDNA group had more pulmonary nodules while MAGEC2 shRNA group had less, compared with NC group (**P < .01). cDNA indicates complementary DNA; MAGEC2, melanoma antigen family C2; NC, negative control; NSCLC, non-small cell lung carcinoma; NC, negative control; VEGF, vascular endothelial growth factor.

Fig 4: Melanoma antigen family C2 was highly expressed in non-small cell lung carcinoma. A, Volcano plot of differentially expressed messenger RNAs in NSCLC based on the data of The Cancer Genome Atlas (log2(Foldchange) > 2, P <.001). B, Heatmap of top 10 highly expressed mRNAs in NSCLC (P <.001). C, Quantitative reverse transcription-polymerase chain reaction: MAGEC2 mRNA was extremely highly expressed in SK-MES-1, A549, and HCC827 cell lines compared with normal lung epithelial cells BEAS-2B. D, Western blot: MAGEC2 protein was extremely highly expressed in SK-MES-1, A549, and HCC827 cell lines compared with normal lung epithelial cells BEAS-2B (**P <.01, ***P <.001). MAGEC2 indicates melanoma antigen family C2; NSCLC, non-small cell lung carcinoma; mRNAs, messenger RNAs.

Fig 5: MAGEC2 contributes to STAT3 hyperactivation in ASCs.(A) Levels of total STAT3 and pY-STAT3 in the basal ADs and ASCs were evaluated by immunoblot assay. (B) Cells were lysed in the presence or absence of MG132 20 µM for 10 hours and immunoprecipitated with pY-STAT3. Endogenous levels of poly-ubiquitinated pY-STAT3 and pY-STAT3 were measured by immunoblotting. (C) Cells were transfected with siControl or siMAGEC2 for 48 hours and levels of STAT3, pY-STAT3, and pS-STAT3 were evaluated. (D) Protein levels of STAT3 downstream target genes, cyclin D1, MMP9, and survivin, were examined by immunoblot assays in cells treated with siControl and siMAGEC2. (E) Depletion of siMAGEC2 efficiency in ADs was evaluated by over-exposure immunoblotting.MAGEC2, melanoma-associated antigen C2; ASC, adapted suspension cell; AD, adherent cell; STAT3, signal transducer and activator of transcription 3; MMP-9; matrix metallopeptidase 9.