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Fig. 7 | Breast Cancer Research

Fig. 7

From: CGRRF1, a growth suppressor, regulates EGFR ubiquitination in breast cancer

Fig. 7

CGRRF1 regulates EGFR stability. a, b CGRRF1 was knocked out by CRISPR in MCF7 cells (a) and in MDA-MB-231 cells (b). The #number in sgVector denotes the ID of individual clones. The first number in sgCGRRF1 #number-number indicates the ID of sgCGRRF1 (e.g., #2-3 and #2-6 were generated from sgCGRRF1 oligos 2R and 2F), and the second number indicates the ID of the individual clones generated from each sgCGRRF1. EGFR expression in the CRISPR control (sgVector) and CGRRF1-knockout clones was examined by western blot analysis. c The expression of CGRRF1 in the CGRRF1-knockout MCF7 (sgCGRRF1#2-4) cell line was rescued by infecting with CGRRF1-expressing lentivirus. Rescue of CGRRF1 in these cells was confirmed by western blot analysis (lower panel). The growth rate of these cell lines was determined by MTT assay. Error bars represent mean ± SD (n = 5). **p < 0.001. d EGFR levels in CGRRF1-overexpressing (upper panel) and CGRRF1-knockdown (lower panel) MCF7 cell lines were examined by western blot analysis. e CGRRF1-knockout MCF7 cell lines were treated with 1 μM MG132 for 24 h. Lysates were harvested for western blot analysis to examine the expression of EGFR. f Correlation between CGRRF1 mRNA levels (RNA-seq) and EGFR protein levels (RPPA) in the TCGA breast cancer cohort. R = −0.32, p < 0.001, n = 747. The data in the TCGA breast cancer (BRCA) study were accessed from g Establishment of EGFR overexpression by pcDNA6-EGFR in pInducer-CGRRF1 (wild-type) MDA-MB-231 cells. h The growth rate of pInducer-CGRRF1 (wild-type) MDA-MB-231 cells that overexpressed with and without pcDNA6-EGFR was determined by MTT assay. Cells were treated with different doses of doxycycline for 5 days. Error bars represent mean ± SD (n = 8). *p < 0.01, **p < 0.001

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