Skip to main content
Figure 6 | Breast Cancer Research

Figure 6

From: Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer

Figure 6

sXBP1 is involved in PRNP gene expression in MCF-7 and MDA-MB-231 cells. (A) Western blot analyses of PrP with the 3F4 antibody, cleaved ATF6α (ΔATF6α), phosphorylated eIF2α (peIF2α), total eIF2α, and β-actin. Lower panels represent XBP1, spliced XBP1 (sXBP1) and β-actin amplified by RT-PCR. Protein or mRNA extracts were from MCF-7 cells treated with ER stressors for 0 to 6 hrs. The increase of PrP and ΔATF6α levels compared to β-actin levels and the ratios of peIF2α/eIF2α and sXBP1/XBP1 are indicated. (B) MCF-7 cells transfected with siATF6α or siXBP1 and proteins immunoblotted with 3F4 PrP and β-actin antibodies. RT-PCR shows levels of ATF6α and XBP1 mRNAs. (C) Western blot analyses of PrP, HA tag and β-actin in protein extracts from MCF-7 cells transfected for 6 hrs with pCGN-IRES-EGFP (Ctl), pCGN-HA-sXBP1-IRES-EGFP, and pCGN-HA-ATF4-IRES-EGFP constructs. (D) ChIP assays performed on DMSO (Ctl)- or BFA-treated MCF-7 cells with IgG control, XBP1 or ATF6α antibodies. PCR amplification of PRNP and β-actin gene promoters (ACTB) was done on immunoprecipitated and non-immunoprecipitated (input) DNA. (E) Western blot of PrP (top panel) and β-actin (bottom panel) and ethidium stained agarose gel containing ATF6α and XBP1 amplicons from MDA-MB-231 or HS578T cells transfected with siCtl, siATF6α or siXBP1. NT indicates non-transfected, D indicates the Dharmacon siRNAs and SC indicates the Santa Cruz siRNAs. (F) Levels of PRNP mRNA detected by qRT-PCR in siATF6α or siXBP1-transfected cells.

Back to article page