Figure 4. KRTAPs Are a Family of Candidate Proliferation Drivers.
(A) Individual validation of selected HMEC ORF GO genes by multi-color competition assay (MCA). Bar plot shows the percent of ORF-expressing cells after 6 PDs of culture from a starting mixture of 50:50 with EV cells. Error bars ± SD. Asterisk represents an ORF that was not in the screening library but was included in the validation experiment.
(B) Distribution of HMEC average Log2FC of KRTAP ORFs (top) and neutral gene family Olfactory Receptor ORFs (bottom).
(C) Distribution of average log2FC of KRTAP ORFs in HPNE (top) and IMR90 (bottom) screens.
(D) HMEC average log2FC values for KRTAP ORFs arranged by sub-family. GO gene sub-families are labeled in red, and selected neutral subfamilies are labeled in gray.
(E) Bar plots of normalized expression of KRTAP (GO or neutral) genes in breast cancer subtypes and normal mammary gland tissue. Normal, normal tissue; ER/PR, ER or PR positive; HER2, HER2-positive; TPBC, triple positive breast cancer (ER/PR positive and HER2 positive); TNBC, triple negative breast cancer. Tumor type and p value (Wilcoxon rank sum test) are indicated above each plot.
(F) Heatmap of top enriched gene sets among most overexpressed and under-expressed genes in each sample, based on GSEA analysis of RNA-seq data. The heatmap depicts GSEA normalized enrichment score (NES) for each pathway in each sample and is annotated to reflect some of the predominant trends in indicated clusters.
(G) E2F1 mRNA levels (from RNA-seq) after overexpression of the indicated ORF (and in TP53KO) in HMECs as compared to empty vector (EV). Error bars ± SD.
(H) Western blot for E2F1 protein levels in cells expressing indicated ORF or empty vector (EV). Vinculin (VINC) was used as a loading control. Normalized expression levels are shown below the blot.
(I) Western blot for E2F1 protein levels in cells utilized for (J). Vinculin (VINC) is used as a loading control.
(J) Bar plot showing proliferation rate (shown as percentage change compared to EV control) of HMECs expressing empty vector (EV), KRTAP10-6 (KAP10-6), or KRTAP4-5 (KAP4-5). Cells were transduced with shRNAs targeting E2F1, or with a control shRNA targeting OR11L1. Cells without any shRNA were also tested. Error bars ± SD.