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. 2022 Apr 8;3(4):298–315. doi: 10.1158/2643-3230.BCD-21-0144

Figure 4.

Figure 4. Type I mutant CALRdel52-expressing cells are dependent on depleted ER Ca2+ to activate IRE1α/XBP1, which promotes cell survival via upregulation of BCL-2. A, Total viable cell number at 48 hours post IL3 withdrawal in Ba/F3-MPL cells expressing calreticulin (CALR) variants and either empty vector or P+C rescue construct. Ba/F3-MPL-CALRwt cells grown in the presence of IL3 was included as a control. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05). B, Top, Western blot analysis for IRE1α and XBP1s in Ba/F3-MPL cells expressing CALR variants and either a scrambled shRNA or shRNA against IRE1α. Bottom, Western blot analysis for XBP1 in Ba/F3-MPL cells expressing CALR variants and either a scrambled shRNA or shRNA against XBP1. C, Total viable cell number at 48 hours post IL3 withdrawal in Ba/F3-MPL cells expressing CALR variants and either a scrambled shRNA or shRNA against IRE1α (left) or XBP1 (right). Ba/F3-MPL-CALRwt cells grown in the presence of IL3 were included as a control. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01; ***, P < 0.001). D, Left, GSEA plots for BH domain binding in Ba/F3-MPL-CALRdel52 cells versus Ba/F3-MPL-CALRwt cells. Right, Heat map displaying relative expression levels of BCL-2 family genes BCL-2, BCL-xL, and MCL-1 in Ba/F3-MPL cells expressing CALR variants. E, qPCR for BCL-2 expression in Ba/F3-MPL cells (left) and UT-7-MPL cells (middle) expressing CALRwt, CALRdel52, and CALRins5, and in peripheral blood mononuclear cells (PBMC) from a healthy donor (HD) or patients with myeloproliferative neoplasms (MPN; patient number depicted below each bar) with CALRdel52 or CALRins5 mutations (right). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05; ***, P < 0.001). F, Western blot analysis for BCL-2 in UT-7-MPL cells expressing CALR variants. β-Actin was used as a loading control. G, qPCR for BCL-2 expression in Ba/F3-MPL cells expressing CALR variants and either empty vector or P+C rescue construct. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01). H, Left, qPCR for BCL-2 expression in Ba/F3-MPL cells expressing CALR variants and a scramble shRNA or shRNA against XBP1. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (***, P < 0.001). Right, Western blot analysis for BCL-2 in Ba/F3-MPL cells expressing CALR variants and a scramble shRNA (−) or shRNA against XBP1 (+). I, Quantification of flow cytometric analysis for Annexin V/PI double positivity in Ba/F3-MPL cells expressing CALR variants and treated with or without venetoclax (1 μmol/L for 24 hours). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (***, P < 0.001).

Type I mutant CALRdel52-expressing cells are dependent on depleted ER Ca2+ to activate IRE1α/XBP1, which promotes cell survival via upregulation of BCL-2. A, Total viable cell number at 48 hours post IL3 withdrawal in Ba/F3-MPL cells expressing calreticulin (CALR) variants and either empty vector or P+C rescue construct. Ba/F3-MPL-CALRwt cells grown in the presence of IL3 was included as a control. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05). B, Top, Western blot analysis for IRE1α and XBP1s in Ba/F3-MPL cells expressing CALR variants and either a scrambled shRNA or shRNA against IRE1α. Bottom, Western blot analysis for XBP1 in Ba/F3-MPL cells expressing CALR variants and either a scrambled shRNA or shRNA against XBP1. C, Total viable cell number at 48 hours post IL3 withdrawal in Ba/F3-MPL cells expressing CALR variants and either a scrambled shRNA or shRNA against IRE1α (left) or XBP1 (right). Ba/F3-MPL-CALRwt cells grown in the presence of IL3 were included as a control. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01; ***, P < 0.001). D, Left, GSEA plots for BH domain binding in Ba/F3-MPL-CALRdel52 cells versus Ba/F3-MPL-CALRwt cells. Right, Heat map displaying relative expression levels of BCL-2 family genes BCL-2, BCL-xL, and MCL-1 in Ba/F3-MPL cells expressing CALR variants. E, qPCR for BCL-2 expression in Ba/F3-MPL cells (left) and UT-7-MPL cells (middle) expressing CALRwt, CALRdel52, and CALRins5, and in peripheral blood mononuclear cells (PBMC) from a healthy donor (HD) or patients with myeloproliferative neoplasms (MPN; patient number depicted below each bar) with CALRdel52 or CALRins5 mutations (right). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05; ***, P < 0.001). F, Western blot analysis for BCL-2 in UT-7-MPL cells expressing CALR variants. β-Actin was used as a loading control. G, qPCR for BCL-2 expression in Ba/F3-MPL cells expressing CALR variants and either empty vector or P+C rescue construct. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01). H, Left, qPCR for BCL-2 expression in Ba/F3-MPL cells expressing CALR variants and a scramble shRNA or shRNA against XBP1. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (***, P < 0.001). Right, Western blot analysis for BCL-2 in Ba/F3-MPL cells expressing CALR variants and a scramble shRNA (−) or shRNA against XBP1 (+). I, Quantification of flow cytometric analysis for Annexin V/PI double positivity in Ba/F3-MPL cells expressing CALR variants and treated with or without venetoclax (1 μmol/L for 24 hours). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (***, P < 0.001).