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. 2020 Jan 31;10:1409. doi: 10.3389/fgene.2019.01409

Figure 4.

Figure 4

LUCRC was required for the expression of genes involved in ER stress response, including BIP. (A) HCT116 cells transfected with control siRNA (siCTL) and siRNA specifically targeting LUCRC (siLUCRC) for 3 days were subjected to RNA-seq analysis, and genes positively- and negatively-regulated by LUCRC were shown by pie chart (Fold change (FC) > 1.5). (B, C) Heat map (B) and box plot (C) representation of the expression levels (FPKM, log2) for genes positively- and negatively-regulated by LUCRC in HCT116 cells as shown in (A). Re1: replicate 1; Re2: replicate 2. (D, E) GO (D) and KEGG (E) analysis for genes positively-regulated by LUCRC in HCT116 cells as shown in (A). (F) UCSC genome browser view of RNA-seq as described in (A) for BIP was shown. (G) HCT116 cells were transfected with siCTL or siLUCRC for 3 days followed by RT-qPCR analysis to examine the expression of BIP (± s.e.m., **P < 0.01). (H) HCT116 cells were infected with shCTL or shLUCRC for 3 days followed by RT-qPCR analysis to examine the expression of BIP (± s.e.m., *P < 0.05, ***P < 0.001). (I) HCT116 cells were transfected with siCTL or siLUCRC for 3 days and then treated with or without tunicamycin (TM) (1μg/mL) for 8 h, followed by RNA extraction, reverse transcription and PCR analysis using primers targeting XBP1 or ACTIN. Splicing of XBP1 was presented as PSI (percentage of inclusion: XBP1u/(XBP1u + XBPIs)). XBP1u: unspliced XBP1; XBP1s: spliced XBP1. DNA fragment size was indicated on the left. bp: base pair. (J) HCT116 cells as described in (I) were subjected to immunoblotting analysis by using antibodies as indicated. Molecular weight was indicated on the left. KDa: kilodalton. ATF6-90: full length ATF6; ATF6-55: partial ATF6. (K) Colorectal tumor tissues and the corresponding adjacent normal tissues were collected from a group of colorectal cancer patients (n = 14) and subjected to RNA extraction and RT-qPCR analysis to examine the expression of BIP (± s.e.m., **P < 0.01). (L) The expression of BIP in a cohort of clinical colorectal tumor (n = 647) and normal (n = 51) samples from TCGA (The Cancer Genome Atlas).