Fig. 4: BRCA1/2 transcriptionally regulate MGAT5 expression. | Nature Communications

Fig. 4: BRCA1/2 transcriptionally regulate MGAT5 expression.

From: Targeting branched N-glycans and fucosylation sensitizes ovarian tumors to immune checkpoint blockade

Fig. 4

a BRCA1 ChIP-seq tracks in the MGAT5 gene locus in the indicated HepG2 and Hela-S3 cells in the ENCODE database. b The association of BRCA1 (left) and BRCA2 (right) with the MGAT5 gene promoter in the indicated cells was examined by ChIP–qPCR analysis. An isotype-matched IgG was used as a negative control. P-values were calculated using two-tailed t-test. n = 3 biologically independent samples. Error bars represent mean with SD. c HR-proficient cell lines KPCA and ID8 expressing shBRCA1, shBRCA2 or shControl were validated for BRCA1/2 knockdown and examined for MGAT5 expression by qRT-PCR. P-values were calculated using two-tailed t-test. n = 3 biologically independent samples. Error bars represent mean with SD. d HR-proficient cell line OVCAR3 expressing shBRCA1, shBRCA2 or shControl were validated for BRCA1/2 knockdown and examined for MGAT5 expression by immunoblot. The experiment was repeated independently for 3 times with similar results. e Levels of PHA-L binding to OVCAR3 expressing shBRCA1, shBRCA2 or shControl. Two-tailed P-values (q-values) were calculated using ANOVA tests, corrected using the Benjamini and Hochberg method. n = 3 biologically independent samples. Error bars represent mean with SD. f Validation endogenous BRCA1 knockdown by RT-qPCR using primers targeting the 3’ UTR region of the BRCA1 gene as well as ectopic expression of the indicated full length or truncated BRCA1 by immunoblot using an antibody against the N-terminus of BRCA1. P-values were calculated using two-tailed t-test. n = 3 biologically independent samples. Error bars represent mean with SD. g MGAT5 gene promoter luciferase activity in control and BRCA1 knockdown OVCAR3 cells with full length BRCA1 and truncated BRCA1 re-expression. P-values were calculated using two-tailed t-test. n = 3 biologically independent samples. Error bars represent mean with SD. h HR-proficient cell line OVCAR3 treated with 0.5 μM and 1 μM cisplatin (Selleckchem, Cat#: S1166) for 48 h were examined for BRCA1 foci by immunofluorescence. The experiment was repeated independently for 3 times with similar results. i HR-proficient cell line OVCAR3 treated with 0.5 μM and 1 μM cisplatin (Selleckchem, Cat#: S1166) for 48 h were examined for MGAT5 expression by immunoblot. The experiment was repeated independently for 3 times with similar results. Source data are provided as a Source Data file.

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