Journal: Science Advances

Article Title: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

doi: 10.1126/sciadv.aea5932

Figure Lengend Snippet: ( A ) Quantification of S9.6 staining in control, C/X3/X2-depleted U2OS cells with or without HU (1 mM, 4 hours) treatment. Data are represented as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥242 cells were analyzed per condition). ( B ) Quantification of S9.6 staining in control, C/X3/X2-depleted U2OS cells with or without Aph (0.4 μM, 16 hours) treatment. Data are represented as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥211 cells were analyzed per condition). ( C ) Quantification of γH2AX intensity in the presence or absence of HU (1 mM, 4 hours) treatment in control, C/X3-depleted cells with or without overexpression of RNH1-Flag. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test, ≥241 cells were analyzed per condition). ( D ) Effect of RAD51C or XRCC3 depletion on fork progression/fork symmetry following 50 nM CPT treatment as indicated in the schematic. Top: Experimental schematic; middle: representative images; and bottom: quantification of sister fork ratio. Sister fork ratios were measured by dividing the longer sister fork by the shorter fork (IdU tracts). Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥40 sister fork pairs were analyzed per condition). P values are indicated.

Article Snippet: Human cell lines U2OS (American Type Culture Collection; HTB-96; RRID: CVCL_0042), HeLa Kyoto (Sachin Kotak laboratory; RRID: CVCL_1922), and hTERT RPE-1 (Sachin Kotak laboratory) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% Glutamax (Gibco) under standard conditions (5% CO 2 , humidified atmosphere) at 37°C.

Techniques: Staining, Control, Two Tailed Test, Over Expression

Journal: Science Advances

Article Title: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

doi: 10.1126/sciadv.aea5932

Figure Lengend Snippet: ( A ) Representative immunoblot indicating levels of WT and R258H RAD51C-Flag after depletion of endogenous RAD51C in U2OS cells. MCM3 levels are shown as loading control. ( B ) Quantification of S9.6 staining after transfection of cells with the indicated DNA constructs, as shown in (A). Relative nuclear S9.6 intensity data are plotted as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥252 cells were analyzed per condition). ( C ) Quantification of RNAPIIS2P + PCNA PLA foci after transfection of cells with the indicated DNA constructs. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥303 cells were analyzed per condition). ( D ) Representative immunofluorescence images (top) and quantification (bottom) of S9.6 + Flag PLA foci in WT and R258H Flag-RAD51C–transfected cells. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥731 cells were analyzed per condition). Scale bar, 5 μm. ( E ) Co-IP of exogenously expressed Flag-tagged WT and R258H RAD51C, followed by immunoblotting with indicated antibodies. Endogenous protein levels were shown in the input. ( F and G ) Representative immunofluorescence images (F) and quantification (G) of S9.6 + FANCM PLA foci in cells transfected with the indicated DNA constructs. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥251 cells were analyzed per condition). Scale bar, 5 μm. P values are indicated. res, shRNA-resistant.

Article Snippet: Human cell lines U2OS (American Type Culture Collection; HTB-96; RRID: CVCL_0042), HeLa Kyoto (Sachin Kotak laboratory; RRID: CVCL_1922), and hTERT RPE-1 (Sachin Kotak laboratory) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% Glutamax (Gibco) under standard conditions (5% CO 2 , humidified atmosphere) at 37°C.

Techniques: Western Blot, Control, Staining, Transfection, Construct, Two Tailed Test, Immunofluorescence, Co-Immunoprecipitation Assay, shRNA

Journal: Science Advances

Article Title: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

doi: 10.1126/sciadv.aea5932

Figure Lengend Snippet: ( A ) Experimental schematic and representative images, and ( B ) quantification of IdU/CldU ratio for fork protection assay in cells expressing the indicated mutants of RAD51C. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, Mann-Whitney test, ≥203 fibers were analyzed per condition). ( C ) Effect of expressing WT, K131A, and K131R RAD51C mutants on replication restart following the treatment of U2OS cells with 2 mM HU for the indicated time. Top: Experimental schematic; and bottom: quantification of stalled forks. Data show percentage of stalled forks as means ± SD ( n = 3, two-tailed unpaired t test). ( D and E ) Representative immunofluorescence images (D) and quantification (E) of S9.6 staining after transfection of cells with indicated shRNA and RAD51C constructs. Relative nuclear S9.6 intensity data are plotted as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥278 cells were analyzed per condition). Scale bar, 5 μm. P values are indicated. EV, empty vector. h, hours.

Article Snippet: Human cell lines U2OS (American Type Culture Collection; HTB-96; RRID: CVCL_0042), HeLa Kyoto (Sachin Kotak laboratory; RRID: CVCL_1922), and hTERT RPE-1 (Sachin Kotak laboratory) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% Glutamax (Gibco) under standard conditions (5% CO 2 , humidified atmosphere) at 37°C.

Techniques: Expressing, MANN-WHITNEY, Two Tailed Test, Immunofluorescence, Staining, Transfection, shRNA, Construct, Plasmid Preparation

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A) Schematic representation of the time-of-drug-addition assay. U2OS cells were treated with (B) 2 µM JG98 or (C) 1 µM JG345 at the indicated time points (conditions 1-8) or with the DMSO control. Virus inoculum (CHIKV-LR at MOI 1) was present for 1.5 h, after which the inoculum was removed, and fresh medium was added with or without the Hsp70 inhibitors or DMSO control. At 9 hpi, the infectious virus particle production was assessed using the plaque assay. Data are presented as mean±SEM from at least three independent experiments. One-way ANOVA was used to evaluate statistical differences from the non-treated control and was presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Control, Virus, Plaque Assay

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A) U2OS cells were treated with increasing concentrations of the Hsp70 inhibitors or the equivalent volume of the DMSO control. The metabolic activity of the cells was assessed after 16 hours (h) of treatment using the MTS assay and was normalized to the DMSO control. A decrease of 15% in metabolically active cells was considered non-toxic (dotted line represents 85%). (B-F) U2OS cells were infected with CHIKV-LR OPY1 at multiplicity of infection (MOI) 1 in the presence of increasing concentrations of the (B) DMSO control (0.002% matches 0.1 µM JG-compound, 0.01% DMSO matches 0.5 µM JG-compound, etc.) or the Hsp70 inhibitors concentrations of (C) JG18, (D) JG40, (E) JG98, (F) JG345. Supernatants were collected at 9 hpi, and the number of infectious CHIKV particles was quantified using plaque assay on Vero-WHO cells. (B-F) Percentage inhibition was determined relative to the DMSO control, and IC50 (which corresponds to a 50% reduction in viral titer) per Hsp70 inhibitor was determined via non-linear regression. Data are presented as mean±SEM from three independent experiments. (B) Statistical differences were determined using One-way ANOVA and were presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Control, Activity Assay, MTS Assay, Metabolic Labelling, Infection, Plaque Assay, Inhibition

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: U2OS cells were infected with the (A) African S27 CHIKV strain or the (B) Caribbean 99659 strain at MOI 1 and simultaneously treated with 10 µM, 10 µM, 2 µM, or 1 µM of JG18, JG40, JG98, or JG345, respectively, or the DMSO control. Production of infectious virus particles was assessed at 9 hpi. (C) U2OS cells were infected with DENV A2 (16681) at MOI 0.5 and treated with 10 µM JG18 or JG40, or the vehicle control. 24 hpi, supernatants were collected and DENV progeny production was analyzed via plaque assay on BHK-21 cells. Data are presented as mean±SEM from three independent experiments, and statistical differences were determined via one-way ANOVA and presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Virus, Plaque Assay

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: U2OS cells were treated with 2 µM JG98, 1 µM JG345, or the DMSO control during CHIKV infection at MOI 10. Virus production was measured at 9 hpi using a plaque assay. Data are presented as mean±SEM from at least three independent experiments. One-way ANOVA was used to evaluate statistical differences from the DMSO control and was presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Control, Infection, Virus, Plaque Assay

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A) The total intracellular vRNA and (B) genomic vRNA copy number were assessed at 4, 6, and 8 hpi in U2OS cells infected with CHIKV at MOI 10 and treated with vehicle control DMSO or 2μM JG98. Intracellular vRNA copies were quantified by RT-qPCR using specific primers against (A) E1 and (B) nsP1. (C) The number of subgenomic RNA copies was determined by subtracting the genomic vRNA copies from the total vRNA copies. Data is presented as mean ± SEM from at least three independent experiments. Student T-test was used to evaluate statistical differences from the DMSO control per timepoint and were presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: Gating strategy to determine the percentage of cells positive for CHIKV E2 and capsid expression in U2OS cells treated with JG98, JG345, or the DMSO control. (A) Gating for cells and exclusion of doublets. (B and C) Gating to determine the percentage of cells positive for (B) capsid and (C) E2 based on mock-infected cells.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Expressing, Control, Infection

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A-D) U2OS cells were infected with CHIKV at MOI 10 and treated with JG98 (2μM), JG345 (1μM), or the DMSO control. At 9hpi, E2 and capsid expression were assessed using flow cytometry to determine the (A and C) percentage of positive cells and (B and D) mean fluorescent intensity (MFI; geometric mean). (E and F) Representative western blot of capsid, E2, or vinculin expression from protein lysates of U2OS cells infected with CHIKV at MOI 10 and treated for 9 h with Hsp70 inhibitor JG-98 (2μM), JG-345 (1μM), or the DMSO control. (G and H) Quantification of Western blots from three independent experiments. Protein levels are normalized to vinculin and are expressed as relative protein level to vehicle control DMSO. Data are presented as mean±SEM from at least three independent experiments. One-way ANOVA was used to evaluate statistical differences from the DMSO control and was presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Expressing, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: U2OS cells were infected with the (A-C, G-I) CHIKV-LR or the (D-F) CHIKV S27 strain for 9 h in the presence of (A-F) 2 µM JG98, 1 µM JG345, (G-I) 20 µM VER-155008, or the DMSO control. Supernatants were collected and the number of (A, D, and G) infectious particles and (B, E, and H) secreted genome equivalent copies (GECs) were measured using plaque assay and RT-qPCR, respectively. (C, F, and I) Specific infectivity is depicted as the ratio between produced infectious particles and GECs. Data are presented as mean± SEM from three independent experiments. One-way ANOVA was used to evaluate statistical differences from the DMSO control and was given when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Plaque Assay, Quantitative RT-PCR, Produced

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: Metabolic activity of the U2OS cells was assessed after 16 hours of treatment with increasing concentrations of the VER155008 or the equivalent volume of the DMSO control. The percentage of metabolically active cells relative to the DMSO control. A decrease of 15% in metabolically active cells was considered non-toxic (dotted line represents 85%). Data are presented as mean±SEM from three independent experiments.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Activity Assay, Control, Metabolic Labelling

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: Metabolic activity of (A) U2OS and (B) HFF-1 cells was assessed after 16 h of treatment with increasing concentrations of JG231 or the equivalent volume of the DMSO control. The percentage of metabolically active cells is presented relative to the DMSO control. A decrease of 15% in metabolically active cells was considered non-toxic (dotted line represents 85%). (C) U2OS cells and (D) HFF-1 cells were infected with CHIKV-LR for 9 h in the presence of 2 µM JG231 or the DMSO control. Supernatants were collected, and the number of infectious particles in the supernatant was determined using the plaque assay. (E and F) Mouse skin explants were infected with 10 6 PFU/mL of the CHIKV 899 strain during treatment with 2 µM JG231 or an equal volume of DMSO. (E) Infectious virus production (tissue culture infectious dose 50% (TCID 50 )) and (F) total virus production (GEC) at 2 dpi is displayed per mg tissue. The dotted line indicates the limit of quantification (LOQ). To evaluate statistical differences from the DMSO control, we used a Student T-test, and differences are presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Activity Assay, Control, Metabolic Labelling, Infection, Plaque Assay, Virus