Journal: bioRxiv

Article Title: Upregulation of ATP Citrate Lyase Phosphorylation and Neutral Lipid Synthesis through Viral Growth Factor Signaling during Vaccinia Virus Infection

doi: 10.1101/2023.09.21.558916

Figure Lengend Snippet: (A) VACV infection induces lipid droplet formation in HFFs in a VGF-dependent manner. HFFs were infected with the indicated viruses at a multiplicity of infection (MOI) of 5 for 8 h. Lipid droplets were stained with HCS Lipidtox Red, and the nuclei were stained with DAPI and imaged under a confocal microscope. The intensity of the red signal that corresponds to the lipid droplets is quantified in the bar graph. (B) VACV infection increases the lipid droplet-associated protein levels in HFFs in a VGF-dependent manner. HFFs were infected with the indicated viruses at an MOI of 5 for the indicated 8 h. Western blotting analysis was performed to measure the levels of perilipin 2. (C) VACV infection-induced lipid droplet formation is cell-type specific. HEPG2 and A549 cells were infected with VACV at an MOI of 5 for 8 h. Lipid droplets were stained with HCS Lipidtox Red, and the nuclei were stained with DAPI and imaged under a confocal microscope. (D) VACV infection does not increase ACLY phosphorylation and lipid droplet-associated protein levels in HEPG2 and A549 cells. HEPG2 and A549 cells were infected with wild-type VACV at a multiplicity of infection (MOI) of 5 for 8 h. Western blotting analysis was performed to measure the levels of ACLY and perilipin 2. (E) EGFR and Akt pathways are important for the formation of lipid droplets during VACV infection. HFFs were infected with wildtype VACV at a MOI of 5 for 8 h in the presence or the absence of 3 µM afatinib (EGFR inhibitor) or 5µM MK-2206 (Akt inhibitor). Lipid droplets were stained with HCS Lipidtox Red imaged under a confocal microscope. The intensity of the red signal that corresponds to the lipid droplets is quantified in the bar graph.

Article Snippet: Primary HFFs, HeLa cells (ATCC CCL-2), and A549 (ATCC CCL-185) were grown in Dulbecco’s modified Eagle medium (DMEM; Fisher Scientific), supplemented with 10% fetal bovine serum (FBS; Peak Serum), 2 mM glutamine (VWR), 100 U/ml of penicillin, and 100 μg/ml streptomycin (VWR) in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: Infection, Staining, Microscopy, Western Blot

Journal: Cancers

Article Title: Alternative Lengthening of Telomeres in Pediatric High-Grade Glioma and Therapeutic Implications

doi: 10.3390/cancers15123070

Figure Lengend Snippet: TMM in pediatric GBM cell lines and their sensitivity to ATR and CHK1 inhibitor. ( A ) Dot blot of the C-Circle assay to test for ALT activity in different pediatric cell lines. Saos2 cells were used as a positive control and HeLa cells were used as negative control for ALT. ( B ) TRAP assay showing telomerase activity in pediatric GBM cell lines. HeLa cells were used as positive control. ( C ) Evaluation of hTERT expression by qPCR. Error bars represent the standard error of the mean from two different experiments run in triplicates. ( D ) Western blot analysis of CHK1-S345-P (ATR phosphorylation site), CHK1-S296-P (autophosphorylation site), total CHK1, ɣH2AX, and total H3 in pediatric GBM cell lines, HeLa, Saos2, and HFF cells. β-Actin served as a loading control. Irradiated HFF cells with 5 Gy were used as a positive control for CHK1 activation cells. ( E ) Proliferation assay for ATR kinase inhibitor-treated pediatric GBM cells. Cells were treated with the indicated concentrations of ATR inhibitor for 6 days. Proliferation was determined by the WST1 assay. Error bars represent SD; experiment was performed in duplicate. ALT−, Telo+ are ALT negative and telomerase positive cell lines, while ALT+, Telo− are ALT positive and telomerase negative cell lines ( F ) proliferation assay for CHK1 inhibitor-treated pediatric GBM cells. Cells were treated with the indicated concentrations of CHK1 kinase inhibitor for 6 days. Proliferation was determined by the WST1 assay. Error bars represent SD; experiment was performed in duplicate. ALT−, Telo+ are ALT negative and telomerase positive cell lines, while ALT+, Telo− are ALT positive and telomerase negative cell lines. ( G ) Western blot analysis of ATR T1989-P, ATR, CHK1-S345-P (ATR phosphorylation site), CHK1-S296-P (autophosphorylation site), total CHK1 in our pediatric GBM cell lines treated either with ATR or CHK1 kinase inhibitor at predetermined IC 50 for 48 h. β-Actin served as a loading control.

Article Snippet: Primary normal human foreskin fibroblasts (HFF) strains (ATCC CRL-2091), human cervical carcinoma cell lines (HeLa), and the human osteosarcoma cell lines (Saos-2) (ATCC HTB-85) were purchased from ATCC.

Techniques: Dot Blot, Activity Assay, Positive Control, Negative Control, TRAP Assay, Expressing, Western Blot, Irradiation, Activation Assay, Proliferation Assay

Journal: Frontiers in Immunology

Article Title: Suppression of MR1 by human cytomegalovirus inhibits MAIT cell activation

doi: 10.3389/fimmu.2023.1107497

Figure Lengend Snippet: The inhibition of primary MAIT cell activation during HCMV infection is comparable to that achieved by MRI knockout using CRISPR/Cas-9. (A) Parental Cas-9 hTERT HFS and a successful MR1 knockout clone 2 were infected with HCMV strain MER1111 at a MOI of 20. At 48 h.p.i cells were treated with partially fixed E. coli overnight. Cells were washed to remove the E. coli and whole PBMCs were added for 5 h in folate free RPMI medium. In parallel, cells were mock infected and/or not treated with E. coli . Levels of surface CD69 on CD3+CD8+CD161+Vα7.2 MAIT cells were assessed by flow cytometry using MFI (B) and % CD69+ (C) , representative dot plots shown in (D) . n = 4. error bars represent SEM, two-way ANOVA, Sidak's multiple comparisons for MRI KO vs. parental hTERT HFS. Tukey's multiple comparisons for mock vs HCMV vs mock + E. coli vs. HCMV + E. coli ** p<0.01, ***p<0.001.

Article Snippet: Primary human foreskin fibroblasts (HFs) (ATCC), 293-TREX cells (invitrogen), HEK293Ts (ATCC), human telomerase immortalised (hTERT) HFs ( , ) (and CRISPR/Cas-9 derivatives) as well as HF and ARPE-19 cells overexpressing MR1 and MR1-GFP ( ) were grown in DMEM media supplemented with 10% foetal calf serum (FCS) and penicillin streptomycin (100 units/mL).

Techniques: Inhibition, Activation Assay, Infection, Knock-Out, CRISPR, Flow Cytometry