Figure 4. ERα RNA-binding facilitates XBP1 splicing upon stress.

See also Figure S4.

(A) IGV view of ERα HITS-CLIP showing ERα binding to XBP1 mRNA with indicated cross-link-induced mutation sites (CIMS) representing ERα-bound regions.

(B) Representative RT-PCR analysis of XBP1 splicing in MCF7 cells harboring WT or RBDmut ERα upon DMSO or tunicamycin treatment for 4 h. Upper bands indicate unspliced XBP1 mRNA and the lower indicate spliced XBP1 mRNA. β-actin mRNA (ACTB) is used as an internal control.

(C) Representative western blots for spliced and unspliced XBP1 protein (XBP1s and XBP1u), phosphor-eIF2α (p-eIF2α), eIF2α and GAPDH in MCF7 cells with WT and RBDmut ERα treated with DMSO (−) or tunicamycin (+) for 6 h.

(D) A cartoon demonstrating the processes of XBP1 splicing: cleaved by IRE1 and ligated by RtcB.

(E) Representative western blots for RtcB and ERα immunoprecipitated (IP) by ERα-specific and IgG (negative control) antibodies.

(F) IGV view of ERα (peaks and binding regions in blue) and RtcB [binding regions in red, PAR-CLIP (GSM936508) (Baltz et al., 2012)] association on the XBP1 mRNA.

(G) Left: Representative RT-PCR analysis of XBP1 splicing in MCF7 cells harboring WT and RBDmut ERα, with or without the RtcB silencing (sgRTCB) upon 4 h tunicamycin treatment. Right: Relative ratios of spliced XBP1 (XBP1s) compared to unspliced XBP1 (XBP1u) are quantified.

(H) Representative western blots for ERα hotspot mutants (E380Q, L536H, Y537S and D538G) and WT ERα immunoprecipitated (IP) by an antibody specifically recognizing RtcB.

(I) Relative cell growth of MCF7 cells treated with IRE1 inhibitor (IRE1i, STF-083010) for 72 h compared to DMSO. Cells were cultured in normal cell culture conditions.

(J) Tumor volumes of mice implanted with MCF7 cells in the mammary fat pad of mice dosed with vehicle or 30 mg/kg of IRE1 inhibitor (IRE1i) STF-083010 once per week (N=7 mice per arm).

(K) Representative RT-PCR analysis of XBP1 splicing in tamoxifen-resistant (TamR-1) and its parental tamoxifen-sensitive MCF7 cells without tunicamycin treatment.

(L) Relative cell apoptosis as shown by relative Annexin V+ populations of TamR-1 cells treated with IRE1 inhibitor for 24 h compared to DMSO in stressful cell culture conditions: in ultra-low attachment dishes (Suspension) or serum free media (Serum deprivation).

N=3 biological replicates. Two-sided t-test. All values represent the mean + SD. Otherwise noted, * P < 0.05, ** P < 0.01, *** P < 0.001.