Characteristics of CRHR1-KO COLO320 cells. (A) Target sequences of sgRNAs for CRISPR/Cas9 gene KO in human CRHR1 gene exon 2. (B) Nucleotide sequences of part of CRHR1 exon 2 in WT COLO320 cells and CRHR1-KO COLO320 cells (left). Schematic membrane topology of CRHR1 protein in WT COLO320 cells and three CRHR1-KO COLO320 clones (#1, #2, #3) obtained with TMHMM Server version 2.0 (right). (C) Quantitative RT-PCR analysis of POMC gene expression after 48-h treatment in normal pH 7.4 (N) and acidic pH 6.8 (A) medium in WT COLO320 cells and three CRHR1-KO COLO320 clones (#1, #2, #3). (D) Representative Western blots for POMC, CRHR1, p-CaMKII, CaMKII, and β-actin in normal pH 7.4 (N) and acidic pH 6.8 (A) medium in WT COLO320 cells and three CRHR1-KO COLO320 clones (#1, #2, #3). Results are mean ± SD. *P < 0.05 vs pH 7.4; **P < 0.01 vs pH 7.4.