Fig 2: KLF16 regulates MAGT1 through transcriptional activation. (A) The JASPAR database (https://jaspar.genereg.net/) predicted that there were two potential KLF16 responsive elements (E1 and E2) binding to MAGT1 promoter. MCF-7 cells were first transfected with sh-KLF16-1/2 and sh-NC, respectively, and the expression level of KLF16 was then detected using (B) RT-qPCR and (C) western blot analysis. (D) MCF-7 cells were transfected with sh-KLF16 and sh-NC, and the expression level of MAGT1 was detected using RT-qPCR. (E) MAGT1 promoter transcriptional activity upon KLF16 silencing was evaluated by luciferase reporter assay. ***P<0.001 vs. sh-NC. (F) The relative luciferase activity of MAGT1 promoter deletion mutants (E1 Del and E2 Del) upon KLF16 overexpression was detected using luciferase reporter assay. All experiments were performed in triplicate. ***P<0.001 vs. Oe-NC. (G) Immunoprecipitated chromatin fragments were analyzed using RT-qPCR. ***P<0.001 vs. IgG. KLF16, Kruppel-like factor 16; MAGT1, magnesium transporter protein 1; RT-qPCR, reverse transcription-quantitative PCR.

Fig 3: The inhibitory effect of KLF16 on tumor growth is diminished by MAGT1 overexpression. (A) Male BALB/c nude mice were subcutaneously injected with MCF-7 cells transfected with sh-KLF16 or co-transfected with sh-KLF16 and pcDNA-MAGT1. After sacrifice, the mice with tumors were imaged. (B) The tumors were removed and imaged. (C) The tumor weight was recorded. Before sacrifice, the (D) body weight and (E) tumor volume was recorded every 3 days. (F) The protein expression of Ki67, PCNA, MMP2 and MMP9 in tumor tissues of mice was examined using western blot analysis. All experiments were performed in triplicate. **P<0.01 and ***P<0.001 vs. shRNA-NC; #P<0.05 and ###P<0.001 vs. sh-KLF16 + pcDNA-NC. Kruppel-like factor 16; MAGT1, magnesium transporter protein 1; PCNA, proliferation cell nuclear antigen.

Fig 4: The inhibitory effects of KLF16 on the cell proliferation, migration and invasion are diminished by MAGT1 overexpression. (A) The expression level of KLF16 in multiple breast cancer cell lines (MCF-7, MDA-MB-231, SK-BR-3, and SUM190PT cells) and MCF-10A cells was determined using RT-qPCR. ***P<0.001 vs. MCF-10A cells. MCF-7 cells were transfected with pcDNA-NC or pcDNA-MAGT1, and the expression level of MAGT1 was measured using (B) RT-qPCR and (C) western blot analysis. MCF-7 cells were transfected with shRNA-NC or shRNA-KLF16 alone, or co-transfected with shRNA-KLF16 and pcDNA-NC/pcDNA-MAGT1. The expression level of MAGT1 was measured using (D) RT-qPCR and (E) western blot analysis. (F) Cell viability was then evaluated using MTT assay. (G) Cell colony formation assay was conducted. (H) Protein expression of Ki67 and PCNA was detected using western blot analysis. (I) Wound healing and Transwell assays were performed to examine cell migration and invasion, respectively. (J) Quantification of cell migration rate. (K) Quantification of cell invasion rate. (L) Protein expression of MMP2 and MMP9 was determined using western blot analysis. All experiments were performed in triplicate. **P<0.01 and ***P<0.001 vs. shRNA-NC; #P<0.05, ##P<0.01 and ###P<0.001 vs. sh-KLF16 + pcDNA-NC. Kruppel-like factor 16; MAGT1, magnesium transporter protein 1; RT-qPCR, reverse transcription-quantitative PCR; PCNA, proliferation cell nuclear antigen.