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Supplementary Materials: Validation of CRISPR targeting for proliferation and cytarabine resistance control genes in the AML cell line MOLM-13

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Supplementary figure 1. Control genes’ mRNA expression relative to HPRT in MOLM-13 cells. GAPDH was used as reference gene for data normalization. Data represents mean ± SEM (n = 3).

Supplementary figure 2. p53 depletion induces MOLM-13 cells’ proliferation. (A) Density plot of EdU incorporation analysis of p53 (p53-1, -2 and -3) depleted MOLM-13 cells. LentiCRISPRv2-GFP (EP) and three independent NS sgRNA lentiCRISPRv2-GFP (NS1-3) constructs transduced cells served as controls. EdU untreated MOLM-13 cell sample was used as control to gate EdU positive cells. (B) Bar graph shows relative quantitation of EdU incorporation. Data represents mean ± SEM (n = 6) and are from two independent triplicate experiments. (C) Colorimetric proliferation analysis of p53 depleted cells compared to control cells. EP and three independent NS sgRNA constructs transduced cells served as controls. Data represents mean ± SEM (n = 6) and are from two independent triplicate experiments. (D) Flow cytometric cell count analysis of control versus p53 depleted MOLM-13 cells. EP and NS sgRNA constructs transduced cells served as controls. Data represents mean ± SEM (n = 6) and are from two independent triplicate experiments. Significance testing was performed using student's t-test **p<0.01, ***p<0.001. EP = empty plasmid; NS= non-specific; EP = empty plasmid.

Supplementary figure 3. DCK depletion induces MOLM-13 cells’ resistance to Ara-C. (A) Ara-C kill curve of MOLM-13 cells. Dashed lines’ intersection indicates half maximal inhibitory concentration (IC50) of Ara-C for MOLM-13 cells. (B) Colorimetric survival analysis of DCK depleted MOLM-13 cells treated with Ara-C. lentiCRISPRv2-GFP (EP) and NS sgRNA lentiCRISPRv2-GFP constructs transduced cells served as controls. Data represents mean ± SEM (n = 6) and are from two independent triplicate experiments. (C) Flow cytometric cell count analysis of control (NS) versus DCK depleted MOLM-13 cells subjected to Ara-C treatment. EP and NS sgRNA constructs transduced cells served as controls. Data represents mean ± SEM (n = 6) and are from two independent triplicate experiments. ***p<0.001 Ara-C treated DCK group was compared with Ara-C treated control group. EP = empty plasmid; NS= non-specific sgRNAs.

Supplementary figure 4. MS2 expression in lentiMPHv2 stably transduced MOLM-13 cells. Shown is relative mRNA transcript level of MS2 relative to HPRT in a representative MOLM-13 sample transduced with lentiMPHv2. GAPDH was used as the reference gene for data normalization. Data represents mean ± SEM (n = 3).

Supplementary figure 5. CDA upregulation induces MOLM-13 cells’ resistance to Ara-C. (A) CDA mRNA expression (q-PCR) in activation constructs transduced lentiMPHv2 MOLM-13 cells. LentiSAMv2 (EP) and three independent NSa sgRNA LentiSAMv2 constructs transduced cells served as controls. Data represents mean ± SEM (n = 6) and are from two independent triplicate experiments. (B) Colorimetric survival analysis of activation constructs transduced lentiMPHv2 MOLM-13 cells treated with Ara-C. EP and three NSa sgRNA constructs transduced cells served as controls. Data represents mean ± SEM (n = 6) and are from two independent triplicate experiments. (C) Flow cytometric cell count analysis of control versus CDA upregulated MOLM-13 cells subjected to Ara-C treatment. EP and NSa sgRNA constructs transduced cells served as controls. Data represents mean ± SEM (n = 6) and are from two independent triplicate experiments. ***p<0.0001 Ara-C treated CDA group was compared with Ara-C treated control group. Comparison testing was performed using student's t-test. EP = empty plasmid; NSa = non-specific sgRNAs.

Supplementary Table 1. Supplementary Table 1. Results from experimental comparison testing. Statistics of experimental results and results from paired T-tests performed. Shown are experimental and control group means and standard deviation as well as results from comparative testing performed (t statistic and p-value from paired t-tests).

Supplementary Table 2. Reagents, supplies and equipment table. Information on reagents, supplies and equipment used in described protocol with annotation to respective protocol step.

Protocol for Validation of CRISPR targeting for proliferation and cytarabine resistance control genes in the AML cell line MOLM-13.

Funding

This study was supported by a University of Virginia Cancer Center Trainee Grant to SCP, 5T32CA009109-43 trainee grant and a UVA Double Hoo award to ND and funding from the University of Virginia, UVA Cancer Center through the NCI Cancer Center Support Grant P30 CA44579 and an American Cancer Society Institutional Research Grant to FEG-B.

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