American Society of Hematology

Figure 2.

$Calreticulin surface exposure is AMPK dependent in AML cells. (A) Western blot showing knockdown efficiency of AMPKα in MOLM14 and OCI-AML2 cells transduced with a nontargeting control sgRNA guide (NTG) or PRKAA1 sgRNA. (B) Representative dot plots of MOLM14 and OCI-AML2 surface CALR. Percentages represent the fraction of CALR+ among 4′,6-diamidino-2-phenylindole (DAPI)-negative cells. (C) Control (NTG) or AMPK KO MOLM14 and OCI-AML2 cells were treated with GSK621 (30 μM) or vehicle for 24 hours, and surface exposure of CALR was measured by flow cytometry. Results are MFI of CALR on live (DAPI negative) cells. Baseline fluorescence intensity of the isotype control stained or unstained DMSO treated cells are shown on the same graphs. Ordinary one-way ANOVA with Šidák multiple comparisons test. (D) MOLM14 AMPK KO or control cells (MOLM14 NTG) were treated for 24 hours with DMSO, GSK621 (30 μM), idarubicin (IDA; 25 nM), venetoclax (VEN; 500 nM), or AraC (2 μM), and surface CALR was measured by flow cytometry. The MFI of 3 independent replicates is shown. Ordinary two-way ANOVA with Dunnett multiple comparisons test for comparison to DMSO within each cell line group (NTG and AMPK KO), and ordinary two-way ANOVA with Tukey multiple comparisons test to compare GSK621 treatment in the NTG vs AMPK KO cell line. (E) Western blot showing the expression of a V5-tagged truncated form of AMPKα1 lacking its regulatory region (AMPK 1-312; selected in blasticidin; predicted size: 37.2 kDa). (F) MFI of surface CALR on AMPK KO MOLM14 cells with or without AMPK (1-312) rescue after 24 hours treatment with DMSO, GSK621, VEN, or AraC. Ordinary two-way ANOVA with Šidák multiple comparisons test. (G) MOLM14 cells lacking endogenous PERK expression were generated by CRISPR/Cas9 editing of EIF2AK3 at an intron-exon junction, compared with NTG cells. Cells were rescued with doxycycline-inducible expression of wild-type (WT) PERK bearing a Myc 9E10 tag. Western blot showing the indicated proteins in PERK knockdown and rescue cells, with and without treatment with GSK621 (30 μM). (H) Surface exposure of CALR was measured by flow cytometry before and after induction of PERK rescue, with or without GSK621 treatment. Ordinary two-way ANOVA with Šidák multiple comparisons test.$

Calreticulin surface exposure is AMPK dependent in AML cells. (A) Western blot showing knockdown efficiency of AMPKα in MOLM14 and OCI-AML2 cells transduced with a nontargeting control sgRNA guide (NTG) or PRKAA1 sgRNA. (B) Representative dot plots of MOLM14 and OCI-AML2 surface CALR. Percentages represent the fraction of CALR⁺ among 4′,6-diamidino-2-phenylindole (DAPI)-negative cells. (C) Control (NTG) or AMPK KO MOLM14 and OCI-AML2 cells were treated with GSK621 (30 μM) or vehicle for 24 hours, and surface exposure of CALR was measured by flow cytometry. Results are MFI of CALR on live (DAPI negative) cells. Baseline fluorescence intensity of the isotype control stained or unstained DMSO treated cells are shown on the same graphs. Ordinary one-way ANOVA with Šidák multiple comparisons test. (D) MOLM14 AMPK KO or control cells (MOLM14 NTG) were treated for 24 hours with DMSO, GSK621 (30 μM), idarubicin (IDA; 25 nM), venetoclax (VEN; 500 nM), or AraC (2 μM), and surface CALR was measured by flow cytometry. The MFI of 3 independent replicates is shown. Ordinary two-way ANOVA with Dunnett multiple comparisons test for comparison to DMSO within each cell line group (NTG and AMPK KO), and ordinary two-way ANOVA with Tukey multiple comparisons test to compare GSK621 treatment in the NTG vs AMPK KO cell line. (E) Western blot showing the expression of a V5-tagged truncated form of AMPKα1 lacking its regulatory region (AMPK 1-312; selected in blasticidin; predicted size: 37.2 kDa). (F) MFI of surface CALR on AMPK KO MOLM14 cells with or without AMPK (1-312) rescue after 24 hours treatment with DMSO, GSK621, VEN, or AraC. Ordinary two-way ANOVA with Šidák multiple comparisons test. (G) MOLM14 cells lacking endogenous PERK expression were generated by CRISPR/Cas9 editing of EIF2AK3 at an intron-exon junction, compared with NTG cells. Cells were rescued with doxycycline-inducible expression of wild-type (WT) PERK bearing a Myc 9E10 tag. Western blot showing the indicated proteins in PERK knockdown and rescue cells, with and without treatment with GSK621 (30 μM). (H) Surface exposure of CALR was measured by flow cytometry before and after induction of PERK rescue, with or without GSK621 treatment. Ordinary two-way ANOVA with Šidák multiple comparisons test.

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