American Society of Hematology

Figure 4.

$Hematoxylin (HEM)-induced apoptosis and disrupted CALR-MPL interaction in CALR-mutated cells. (A) Dose-response test of HEM in Ba/F3-MPL cell lines transduced with pMSCVpuro retroviral constructs carrying CALR WT, CALR del52, CALR del52 K111G, and CALR del52 D317G (n = 3). (B) Annexin V/PI staining of Ba/F3-MPL cells carrying GBD-mutated CALR del52 after HEM treatment for 24 hours. Annexin V+ cells were quantified as the apoptotic cells. Data are shown as mean ± standard deviation (SD; n = 3). The statistical test was conducted by 1-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison tests (n = 3). (C) Dose-response test of HEM in Ba/F3-MPL cell lines transduced with retroviral constructs carrying JAK2 WT and JAK2V617F (n = 4). (D) Dose-response test of HEM in Ba/F3 cell lines that were cotransduced with retroviral constructs carrying erythropoietin receptor (EPOR) and CALR WT or CALR del52 (n = 3). (E) The interaction of mutant CALR and MPL was detected by the NanoBRET assay. EBNA cells were transfected with CALR del52-HaloTag and NanoLuciferase-MPL/EPOR constructs. Cells were treated with HEM for 24 hours at respective concentrations. The BRET ratio was calculated by dividing the donor bioluminescent signal (460 nm) by the acceptor bioluminescent signal (618 nm). EPOR-CALRdel52 was used as negative control for background signal. (F) Quantification of the NanoBRET assay result. Data are shown as mean ± SD. The statistical test was calculated by 1-way ANOVA followed by Tukey’s multiple comparisons test. (G) HEM binding with mutant and WT CALR tested by the cellular thermal shift assay. HEK293T cells were transfected with pcDNA3.1 CALR ins5/CALR WT/MPL constructs. HEM or DMSO was added to cell lysates before heating at respective temperatures for 6 minutes. Western blot was used to detect the soluble fraction of the protein lysates. The lysates were treated with DMSO/1 mM of HEM and incubated with a gradient of temperatures (left). The lysates were treated with multiple concentrations of HEM and incubated with a gradient of temperatures (bottom). *P < .05, **P < .01, ****P < .0001.$

Hematoxylin (HEM)-induced apoptosis and disrupted CALR-MPL interaction in CALR-mutated cells. (A) Dose-response test of HEM in Ba/F3-MPL cell lines transduced with pMSCVpuro retroviral constructs carrying CALR WT, CALR del52, CALR del52 K111G, and CALR del52 D317G (n = 3). (B) Annexin V/PI staining of Ba/F3-MPL cells carrying GBD-mutated CALR del52 after HEM treatment for 24 hours. Annexin V⁺ cells were quantified as the apoptotic cells. Data are shown as mean ± standard deviation (SD; n = 3). The statistical test was conducted by 1-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison tests (n = 3). (C) Dose-response test of HEM in Ba/F3-MPL cell lines transduced with retroviral constructs carrying JAK2 WT and JAK2V617F (n = 4). (D) Dose-response test of HEM in Ba/F3 cell lines that were cotransduced with retroviral constructs carrying erythropoietin receptor (EPOR) and CALR WT or CALR del52 (n = 3). (E) The interaction of mutant CALR and MPL was detected by the NanoBRET assay. EBNA cells were transfected with CALR del52-HaloTag and NanoLuciferase-MPL/EPOR constructs. Cells were treated with HEM for 24 hours at respective concentrations. The BRET ratio was calculated by dividing the donor bioluminescent signal (460 nm) by the acceptor bioluminescent signal (618 nm). EPOR-CALRdel52 was used as negative control for background signal. (F) Quantification of the NanoBRET assay result. Data are shown as mean ± SD. The statistical test was calculated by 1-way ANOVA followed by Tukey’s multiple comparisons test. (G) HEM binding with mutant and WT CALR tested by the cellular thermal shift assay. HEK293T cells were transfected with pcDNA3.1 CALR ins5/CALR WT/MPL constructs. HEM or DMSO was added to cell lysates before heating at respective temperatures for 6 minutes. Western blot was used to detect the soluble fraction of the protein lysates. The lysates were treated with DMSO/1 mM of HEM and incubated with a gradient of temperatures (left). The lysates were treated with multiple concentrations of HEM and incubated with a gradient of temperatures (bottom). *P < .05, **P < .01, ****P < .0001.

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