Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A-B) Generation of HLA-A2 + Luciferase + human oligodendrocytic cell line MO3.13-A2-Luc. (A-B) FACS analysis of the (A) HLA-A2 expression on wildtype (WT) MO3.13 cells (left), MO3.13-A2 cells 2 weeks after transfection with HLA-A2 expression vector (middle), and MO3.13-A2 cells after the FACS sorting for HLA-A2 (right). (B) GFP expression on MO3.13-A2-Luc cells after lentiviral transduction (left), GFP expression on FACS sorted MO3.13-A2-Luc cells (middle) and HLA-A2 expression on MO3.13-A2-Luc cells (right). Grey histograms represent the isotype control and untransduced cells for the analysis of HLA-A2 and GFP expression respectively. (C-E) Optimization of siRNA reverse transfection protocol for MO3.13-A2-Luc cells. (C) Real-time live cell imaging of MO3.13-A2-Luc cells transfected either with a non-targeting siRNA control (Scr) or with a siRNA cocktail targeting genes essential for cell survival (cell death siRNA, siCD). Transfected cells were imaged via the IncuCyte SX5 system for 72 h and real-time % cell confluency was quantified. (D) RT-qPCR analysis of PDL1 mRNA expression in MO3.13-A2-Luc cells transfected either with Scr siRNA or pool of 4 non-overlapping siRNAs targeting PDL1 (siCD274). Results are presented as fold change compared to the Scr after β-actin mRNA normalization. (E) FACS analysis of PD-L1 surface expression on MO3.13-A2-Luc cells transfected either with Scr or PD-L1 specific siRNA pool. Left: representative histograms indicating % PD-L1 expression, right: mean fluorescent intensity (MFI) of PD-L1 from 2 independent FACS data. (F) Phenotypic characterization of FluTC. The expression of CD4 and CD8 was determined on CD3 + FluTC, whereas the expression of effector and memory markers CD45RO and CD62L and co-inhibitory immune checkpoint molecules PD-1, LAG-3 and TIM-3 was determined on CD3 + CD8 + FluTC. (Tn: naïve T cells, Tcm: central memory T cells, Teff: terminal effector T cells, Tem: effector memory T cells). Grey histograms represent the isotype control. (G) Luciferase-based cytotoxicity assay to optimize the co-culture conditions for MO3.13-A2-Luc and FluTC. MO3.13-A2-Luc were pulsed with serial dilutions of the flu-peptide and co-cultured with FluTC. After 20 h of co-culture, remaining relative luciferase units (RLU) were measured. RLU measured in pulsed samples were normalized to RLU of unpulsed (no peptide) control. Statistical significance was calculated compared with no peptide control. (H) Selection of cytotoxicity and viability controls for the HTP screen. MO3.13-A2-Luc cells were transfected with the indicated siRNAs. After 72 h of transfection cells were pulsed with 0,01 µg/ml flu-peptide and co-cultured either with FluTC or treated with control T cell medium (CM). 20 h following co-culture, remaining RLU was measured. The RLU of each sample was normalized to the Scr1 control in the cytotoxicity (FluTC) or viability (CM) setting to determine the impact of gene knockdown. (A-G) Representative data of at least 2 independent experiments. (H) Cumulative data of three independent experiments each performed with four replicates per sample. (D, G-H) Graphs show mean +/- SD. p-values were calculated using two-tailed student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001.

Article Snippet: MO3.13-A2-Luc cells were generated after transduction of MO3.13-A2 cells with lentiviral particles encoding firefly luciferase and GFP (GenTarget Inc – Amsbio, #LVP020 (CMV-Luciferase (firefly)-2A-GFP (Puro)) and FACS sorting.

Techniques: Luciferase, Expressing, Transfection, Plasmid Preparation, Transduction, Control, Live Cell Imaging, Quantitative RT-PCR, Cytotoxicity Assay, Co-Culture Assay, Cell Culture, Selection, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: ATGL-catalyzed lipid catabolism promotes DNA repair

doi: 10.64898/2026.04.03.716381

Figure Lengend Snippet: A) AML12 cells transduced with ATGL or GFP control adenoviruses were treated with 30 μM etoposide for the indicated times and assayed for γH2Ax. B) Quantification of A. *p<0.05, **p<0.01. C) AML12 cells transduced with ATGL or GFP control adenoviruses were irradiated (10 Gy) and harvested at the indicated times post-IR. D) Quantification of C. **p<0.01. E) Primary MEFs were isolated from WT or ATGL knock-in (AKI) mice, followed by etoposide incubation for 6 hours. Immunofluorescence was performed for γH2Ax, and cells were imaged using the Agilent Gen5 Cytation. Representative γH2Ax images and foci distribution are shown. F) Cells were treated with DGAT inhibitors for 24 hours, followed by ATGL adenoviral transduction, followed by 24 hours of etoposide treatment. Western blotting for γH2Ax, β-actin, and ATGL. G) AML12 cells were treated as in F. The Comet assay was performed on cells after 24 hours of etoposide. Comets were quantified with OpenComet software. A minimum of 180 comets were quantified per condition. Statistics: one-way ANOVA with Tukey’s post hoc test. ****p<0.0001. H) γH2Ax results and quantification from tissues harvested from E2A Cre-ATGL (AKI) mice irradiated with 7.5 Gy + 4-hour recovery. Mice were of mixed sexes, n=4 mice. P-values from one-way ANOVA are displayed

Article Snippet: For adenoviral transductions, ATGL (Lot: 20160819, mouse ATGL) was obtained from VectorBiolabs, TrackGFP (adGFP) was obtained from UNC Gene Therapy Center, and Null adenovirus was obtained from VectorBiolabs (#1300).

Techniques: Transduction, Control, Irradiation, Isolation, Knock-In, Incubation, Immunofluorescence, Western Blot, Single Cell Gel Electrophoresis, Software