Figure 3: BRCA1 loss impedes replication fork progression by down-regulating RRM2 in GBM cells. | Nature Communications

Figure 3: BRCA1 loss impedes replication fork progression by down-regulating RRM2 in GBM cells.

From: BRCA1-regulated RRM2 expression protects glioblastoma cells from endogenous replication stress and promotes tumorigenicity

Figure 3

(a) DNA fibre assay measuring the replication fork progression speed in GBM cells (GBM01 and GBM02) transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). See also Table 1. (b) Replication fork recovery assay showing the quantification of CldU tract length in GBM01 cells transduced with shCTRL or 2 non-overlapping BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4) and treated or not with 2 mM HU (4 h) prior CldU labelling. See also Table 2; Supplementary Table 1. (c) Immunoblot analysis of RRM2 protein levels in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (d,e) RT-qPCR analysis of BRCA1 and RRM2 mRNA levels in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (f) FACS analysis of RRM2 protein level changes throughout cell cycle in GBM cells (GBM01) transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (g) Partial sequence of the human RRM2 promoter (GenBank accession number AY032750)33, which was used to design Chip primers: P1 primer forward (F)/reverse (R) and P2 primer forward (F)/reverse (R). Positions are numbered from the downstream transcription initiation site (+1). Putative binding sites for transcription factors are color-coded and identified above the sequence. (h) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in GBM01-03 cells using primer set P1 and P2. (i) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in NHA-DRB and BJ cells using primer set P1 and P2. (j) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in PC3, HELA, OVCAR and Cal51 cells using primer set P1 and P2. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test in d–f,h–j or Student’s t test (a,b) and all data are shown as means±s.d. and performed as technical triplicates. (*P<0.05, **P<0.005, ***P<0.005, ****P<0.0001; NS represents non-significance).

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