Fig 1: Mutated MCT4 without lactate transportation activity does not promote migration or invasion of L929 cells. A panel of three clones with high expression of MCT4-R278Q was obtained, as evidenced by (A) flow cytometry and a (B) western blotting. (C) Co-expression of MCT4-R278Q and CD147 was observed with clone 9G2. Scale bar, 30 µm. (D) Normalized concentration of lactate in the culture medium of AZD3965-treated cells compared with that of the corresponding cells that are treated with only the solvent. MCT4-R278Q-L929 cells lost the ability to compensate for the inhibition mediated by AZD3965 in L929 cells compared with MCT4-L929 cells. (E) Wound healing assay. MCT4-R278Q-L929 (9G2) cells showed a similar migration rate as EGFP-L929 (1H9) cells, which was much slower compared with that of wild-type MCT4-L929 (3E10) cells. Scale bar, 300 µm. The average percentage of wound closure at 48 h is shown to the right. (F) Representative images of EGFP-L929 (1H9), MCT4-L929 (3E10) and MCT4-R278Q-L929 (9G2) cells that crossed through migration filters 36 h post seeding are shown. The average number of cells that adhered to the lower chamber is shown to the right. (G) Representative images of EGFP-L929 (1H9), MCT4-L929 (3E10) and MCT4-R278Q-L929 (9G2) cells that invaded through the Matrigel-coated filters 36 h after seeding are shown. The average number of cells that adhered to the lower chamber is presented to the right. The average is from three independent experiments that use three different clones, and each error bar represents one standard deviation. P-values shown in (D), (E), (F), and (G) were calculated using one-way ANOVA with a Tukey's post hoc test. *P<0.05, **P<0.01 vs. EGFP. EGFP, enhanced green fluorescent protein; MCT, monocarboxylate transporter.
Fig 2: Overexpression of EGFP, MCT1 and MCT4 in L929 cells. (A) Overexpression of EGFP, MCT1 and MCT4 was confirmed using flow cytometry for EGFP-L929, MCT1-L929 and MCT4-L929 clones. (B) Overexpression of EGFP, MCT1, and MCT4, as well as expression of endogenous CD147 were confirmed using western blotting. (C) Immunofluorescence was performed to investigate the expression of transfected MCT1 or MCT4. Co-expression of MCT and CD147 was observed in clone 5B8 of MCT1-L929 and clone 3E10 of MCT4-L929 cells. Scale bar, 30 µm. (D) Lactate concentration in the culture medium of MCT1-, MCT4- and EGFP-transfected cells. No significant differences were observed among different panel of clones. Data were analyzed using a two-tailed Welch's t-test. (E) Normalized lactate concentration in the culture medium of AZD3965-treated cells to respective control cells that received only AZD3965 solvent (DMSO). The MCT1/2 inhibitor, AZD3965, reduced lactate secretion of EGFP-L929 cells in a dose-dependent manner, but MCT1-L929 and MCT4-L929 cells were partially or completely resistant to AZD3965. The results in (D) and (E) were the average of three independent experiments using three different clones, and each error bar indicates one standard deviation. P-values shown in (E) were calculated using one-way ANOVA with a Tukey's post hoc test. *P<0.05, **P<0.01 vs. EGFP. EGFP, enhanced green fluorescent protein; MCT, monocarboxylate transporter.
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