Journal: bioRxiv

Article Title: Identification of MYC synthetic lethal genes and networks

doi: 10.1101/2024.04.25.590465

Figure Lengend Snippet: A heatmap showing some of the top scoring MYC SL genes from the CRISPR screen. Gene names in red are validated in this figure or accompanying figures. B. Diagram of the multicolor competition assay (MCA) to validate MYC SL genes. Non-fluorescent TRE- MYC cells were mixed with GFP expressing parental cells at a 20%:80% (TRE- MYC:parental) ratio. Cell mixtures were then infected with Cas9/MYC SL gene sgRNA vectors, selected with puromycin, treated with 100 ng/mL doxycycline, and allowed to proliferate for 1 week. After 1 week, cell mixtures were examined by flow cytometry to determine the ratio of TRE-MYC:parental cells. C. MCL1 is a MYC SL gene. Representative flow cytometry and MCA data from 3 MCL1 sgRNAs and an AAVS1 targeting control. Disruption of MCL1 reduces the growth of MYC overexpressing non-fluorescent cells resulting in an increase in GFP+ WT MYC contribution to the overall cell population. D. Small molecule inhibition of MCL1 is more toxic to MYC overexpressing cells. Cells were mixed as in panel B and treated with the MCL1 inhibitor S63845 for 4 days. Cell mixtures were examined by flow cytometry after 4 days. E. The transcription factor HSF1 is a MYC SL gene. Cells were mixed as in panel B and infected with HSF1 or an AAVS1 control sgRNA. Following 1 week, cell mixtures were examined by flow cytometry. F. Choline kinase (CHKA) targeting using CRISPR. TRE-MYC cells were infected with CHKA targeting or AAVS1 control sgRNAs. Cells were treated with vehicle or 100 ng/mL dox for 7 days and cell lysates were western blotted with the indicated antibodies to determine CHKA protein disruption. G. Choline kinase (CHKA) depletion is toxic to MYC High cells. Cells were mixed as in panel B and infected with CHKA or an AAVS1 control sgRNA. Following 1 week, cell mixtures were examined by flow cytometry. Numbers above bar graphs indicate the p- value from an unpaired Student’s t-test to compare sgRNA or treatment groups to sgControl or Vehicle, respectively.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% milk in TBST (Santa Cruz, cat. # sc-362311) and incubated with the following antibodies at the indicated dilutions in 5% BSA (VWR, cat. # VWRV0332) in TBST either overnight at 4C or 1 h at room temperature: Vinculin (1:10,000, Sigma cat. # V9131), MYC (1:1,000, Cell Signaling, cat. # 5605), CHKA (1:1,000, Cell Signaling, cat. # 13422), CS (1:1,000, ProteinTech, cat. # 16131-1-AP), SLC25A1 (1:1,000, ProteinTech, cat. # 5235-1-AP), PGD (1:1,000, ProteinTech, cat. # 14718-1-AP), ATF4 (1:1,000, Cell Signaling, cat. # 11815), CHOP (1:1,000, Cell Signaling, cat. # 2895), PARP (1:1,000, Cell Signaling, cat. # 9542), Caspase 3 (1:1,000, Cell Signaling, cat. # 9662), HSF1 (1:1,000, Cell Signaling, cat. # 4356), β-actin(1:10,000, Cell Signaling, cat. # 3700), and GAPDH (1:10,000, Santa Cruz, cat. # sc-365062).

Techniques: CRISPR, Competitive Binding Assay, Expressing, Infection, Flow Cytometry, Control, Disruption, Inhibition, Western Blot

Journal: bioRxiv

Article Title: Identification of MYC synthetic lethal genes and networks

doi: 10.1101/2024.04.25.590465

Figure Lengend Snippet: Further validation of a selection of MYC SL genes. A. MYC overexpression increases sensitivity to the MCL1 inhibitor S63845. HCEC cells with TRE-MYC were treated vehicle or 100 ng/mL doxycycline and with the indicated doses of S63845. After 72 h, cell viability was measured by CellTiter Blue and the GR50 was determined using the online GR calculator ( http://www.grcalculator.org/grcalculator/ ) which can correct for the proliferation rate difference in cells with low and high MYC expression. B. Treatment of MYC overexpressing cells with S63845 induces visual apoptosis. Cells were treated with 1 μM of S63845 for 72 h and images were taken. Cells treated with dox to induce high levels of MYC expression were noticeably more apoptotic (i.e. round cells that detached from the plate) than those not treated with dox. C. Treatment of MYC overexpressing cells with S63845 induces apoptosis as determined by western blot. HCEC cells with TRE-MYC were treated vehicle or 100 ng/mL doxycycline and either DMSO, 100 nM, or 1 μM S63845 for 24 h. Cell lysates were analyzed for cleaved caspase 3 and cleaved PARP to monitor apoptosis induction. D. CRISPR disruption of HSF1 expression. HSF1 targeting sgRNAs used in the MCA in were expressed in HCEC TRE-MYC cells. Following 1 week of sgRNA expression, HSF1 protein levels were determined by western blot. E. Inhibition of CHKA via MN58b is more toxic to MYC overexpressing cells. Non- fluorescent HCEC TRE-MYC cells were mixed with GFP+ HCECs at an 80:20 ratio. Cell mixtures were treated with the indicated concentrations of the CHKA inhibitor MN58b or DMSO vehicle control. Changes in cell populations were determined after 48 h of treatment by flow cytometry. F. CRISPR disruption of PPAT is toxic to MYC overexpressing cells. Non-fluorescent HCEC TRE-MYC cells were mixed with GFP+ HCECs at an 80:20 ratio. Cell mixtures were treated with the indicated sgRNAs. After 1 week in 100 ng/mL dox, cell populations were examined by flow cytometry. Numbers above bar graphs indicate the p-value from an unpaired Student’s t-test to compare sgRNA or treatment groups to sgControl or Vehicle, respectively.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% milk in TBST (Santa Cruz, cat. # sc-362311) and incubated with the following antibodies at the indicated dilutions in 5% BSA (VWR, cat. # VWRV0332) in TBST either overnight at 4C or 1 h at room temperature: Vinculin (1:10,000, Sigma cat. # V9131), MYC (1:1,000, Cell Signaling, cat. # 5605), CHKA (1:1,000, Cell Signaling, cat. # 13422), CS (1:1,000, ProteinTech, cat. # 16131-1-AP), SLC25A1 (1:1,000, ProteinTech, cat. # 5235-1-AP), PGD (1:1,000, ProteinTech, cat. # 14718-1-AP), ATF4 (1:1,000, Cell Signaling, cat. # 11815), CHOP (1:1,000, Cell Signaling, cat. # 2895), PARP (1:1,000, Cell Signaling, cat. # 9542), Caspase 3 (1:1,000, Cell Signaling, cat. # 9662), HSF1 (1:1,000, Cell Signaling, cat. # 4356), β-actin(1:10,000, Cell Signaling, cat. # 3700), and GAPDH (1:10,000, Santa Cruz, cat. # sc-365062).

Techniques: Biomarker Discovery, Selection, Over Expression, Expressing, Western Blot, CRISPR, Disruption, Inhibition, Control, Flow Cytometry

Journal: Journal of Virology

Article Title: Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation

doi: 10.1128/JVI.00960-16

Figure Lengend Snippet: Assessment of the interaction of hCKα with NS5A. (A) Huh7 cells coexpressing T7 RNA polymerase and NS3-NS5B were lysed and subjected to co-IP with an anti-NS5 MAb or an isotype-matched control mouse IgG. The precipitates were analyzed by SDS-PAGE, followed by immunoblotting analysis to detect each of the indicated proteins. A portion of aliquots containing 5% of the total proteins in the lysates used for precipitation was loaded as the input control. (B) 293T cells were cotransfected with plasmids encoding each of HCV genotype 1b Con1 strain NS proteins, as indicated, with a pCMV6 plasmid encoding hCKα-Flag. Cell lysates were pulled down with rabbit anti-HA, and the precipitated proteins were subjected to immunoblot analysis with the indicated MAb antibodies. (C) Lysates from Huh7 cells expressing the indicated proteins were precipitated with rabbit anti-Flag, and the coprecipitated proteins were subjected to immunoblot detection with the indicated MAbs. (D) Lysates from Huh7 cells coexpressing GFP-tagged hCKα and different Myc-tagged NS5A domains were pulled down with rabbit anti-GFP, followed by immunoblot analysis using the indicated MAbs.

Article Snippet: Plasmids pCMVΔR8.91 and pMD.G and the pLKO-based short hairpin RNA (shRNA) constructs TRC0005 and TRCN0000284352 were obtained from the National RNAi Core Facility at Academia Sinica. pCMV6-hCKα (RC207209), which encodes hCKα tagged with Myc and DDDK (or Flag) tags at the C terminus, was purchased from Origene, while pEGFP-C1-hCKα encodes hCKα fused with EGFP at the N terminus.

Techniques: Co-Immunoprecipitation Assay, SDS Page, Western Blot, Plasmid Preparation, Expressing

Journal: Oncology Letters

Article Title: MicroRNA-367-3p induces apoptosis and suppresses migration of MCF-7 cells by downregulating the expression of human choline kinase α

doi: 10.3892/ol.2021.12444

Figure Lengend Snippet: Features of miR-367-3p and chka mRNA 3′-UTR pairing. The shaded sequence represents Watson-Crick base pairing at the seed region of miRNA. miR-367-3p binds the chka mRNA transcript (NM_001277.2) at nucleotides 1804–1825. miR, microRNA; chka , choline kinase α; UTR, untranslated region.

Article Snippet: The firefly luciferase reporter construct containing the 3′-UTR of the chka gene (pMirTarget- chka −3′-UTR) was purchased from OriGene Technologies, Inc. (cat. no. SC212759).

Techniques: Sequencing

Journal: Oncology Letters

Article Title: MicroRNA-367-3p induces apoptosis and suppresses migration of MCF-7 cells by downregulating the expression of human choline kinase α

doi: 10.3892/ol.2021.12444

Figure Lengend Snippet: Target validation of miR-367-3p. MCF-7 cells transfected with the pMirTarget- chka −3′-UTR were treated individually or in combination with the specified miRNAs and miRNA inhibitors. Error bars indicate standard error of the mean from triplicate experiments. *P<0.05, compared with cells treated with the negative control miRNA. miR, microRNA; chka , choline kinase α; UTR, untranslated region.

Article Snippet: The firefly luciferase reporter construct containing the 3′-UTR of the chka gene (pMirTarget- chka −3′-UTR) was purchased from OriGene Technologies, Inc. (cat. no. SC212759).

Techniques: Transfection, Negative Control