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

Journal: Nucleic acids research

Article Title: A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

doi: 10.1093/nar/gkae563

Figure Lengend Snippet: Figure 1. RAD18 and UBC13 mediate fork recovery in BRCA1- but not BRCA2-deficient cells, and stalled replication forks are targeted for nucleolytic degradation. ( A ) Western blot of whole extract in U2OS cells depleted of BRCA1, RAD18, and / or UBC13 (left), Western blot of nuclear extract in U2OS cells depleted of BRCA1, RAD18 and / or UBC13 (RIGHT). ( B ) Fork recovery fiber assay scheme (TOP). Cells were labeled with 20uM IdU for 15 min, treated with HU for 2 h, and released into CldU for 15 min. Quantification of stalled (red bars) and restarted (blue bars) replication forks upon BRCA1, RAD18 and / or UBC13 knockdown in U2OS cells (bottom). Mean with SEM shown, N = 3, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: unpaired t -test, ** P < 0.01, *** P < 0.001. ( C ) Fork reco v ery fiber assay scheme (top). Quantification of stalled (red bars) and restarted (blue bars) replication forks upon BRCA2 and / or RAD18 knockdown in U2OS cells (bottom). Mean with SEM shown, N = 2, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: unpaired t -test, ns = not significant. ( D ) Fork recovery fiber assay scheme (top). Quantification of stalled (red bars) and restarted (blue bars) replication forks upon BRCA2 and / or UBC13 knockdown in U2OS cells. Mean with SEM shown, N = 2, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: unpaired t -test, ns = not significant. ( E ) Fork degradation fiber assay scheme (top). Cells were labeled with 20 uM IdU for 30 min, followed by incubation with 200 uM CldU for 30 min, and then treated with 4 mM HU for 5 h. IdU tract and CldU tract lengths were measured on contiguous red–green fibers upon BRCA1, SMARCAL1, ZRANB3 and / or HLTF knockdown in U2OS WT (grey) and RAD18 KO (blue) cells (bottom). Each dot represents a CldU / IdU ratio from a single DNA fiber tract, blue line represents median value, N = 3, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: Kruskal–Wallis test f ollo w ed b y Dunn’s multiple comparison test, ** P < 0.0 1, *** P < 0.00 1, **** P < 0.0 0 01. ( F ) Fork degradation fiber assay scheme (top). IdU tract and CldU tract lengths were measured on contiguous red-green fibers upon BRCA1, SMARCAL1, ZRANB3 and / or HLTF knockdown in U2OS WT (gray) or UBC13-depleted (blue) cells (BO TT OM). Each dot represents a CldU / IdU ratio from a single DNA fiber tract, blue line represents median value, N = 3, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: Kruskal–Wallis test followed by Dunn’s multiple comparison test, **** P < 0.0 0 01, ns = not significant.

Article Snippet: Cell culture and cell lines Human bone osteosarcoma U2OS cells (American Type Culture Collection), U2OS cells knocked-out for RAD18 (provided by George-Lucian Moldovan, UPenn Hershey), and MDA-MB-231 breast cancer cells (American Type Culture Collection) were grown in DMEM media supplemented with 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin at 37 ◦C, 5% CO 2 .

Techniques: Western Blot, Labeling, Knockdown, Incubation, Comparison

Journal: Nucleic acids research

Article Title: A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

doi: 10.1093/nar/gkae563

Figure Lengend Snippet: Figure 2. MRE11, but not DNA2, degrades re v ersed replication forks in BRCA1-deficient cancer cells lacking RAD18 or UBC13. ( A ) Fork degradation fiber assay scheme (TOP). Cells were labeled with 20uM IdU for 30 min, followed by incubation with 200 uM CldU for 30 min, and then treated with 4 mM HU for 5 h ± 50 uM Mirin ± 30 uM C5. IdU tract and CldU tract lengths were measured on contiguous red-green fibers upon BRCA1 knockdown in U2OS WT (gray) and RAD18 KO (blue) cells (BO TT OM). Each dot represents a CldU / IdU ratio from a single DNA fiber tract, blue line represents median value, N = 3, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: Kruskal–Wallis test followed by Dunn’s multiple comparison test, ** P = 0.01, **** P < 0.0 0 01, ns = not significant. ( B ) R epresentativ e images of a replication fork (top left) and a reversed replication fork (bottom left) captured with EM. Percentages of re v ersed replication forks were quantified in BRCA1-deficient U2OS WT or RAD18 KO cells upon knockdown of UBC13 under untreated (UT) conditions (gray), with HU alone (red) or with HU + 30 uM Mirin (blue), N = 1. See Supplementary Figure S2 D for the second biological repeat and Supplementary Figure S2 E for quantification.

Article Snippet: Cell culture and cell lines Human bone osteosarcoma U2OS cells (American Type Culture Collection), U2OS cells knocked-out for RAD18 (provided by George-Lucian Moldovan, UPenn Hershey), and MDA-MB-231 breast cancer cells (American Type Culture Collection) were grown in DMEM media supplemented with 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin at 37 ◦C, 5% CO 2 .

Techniques: Labeling, Incubation, Knockdown, Comparison

Journal: Nucleic acids research

Article Title: A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

doi: 10.1093/nar/gkae563

Figure Lengend Snippet: Figure 3. PCNA ubiquitination and PALB2 promote fork recovery in BRCA1-deficient cells. ( A ) Fork recovery fiber assay scheme (top). Quantification of stalled (red bars) and restarted (blue bars) replication forks upon BRCA1 knockdown in 293T WT and K164 mutant cells (bottom). Mean with SEM shown, N = 4, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: unpaired t -test, * P < 0.05, ns = not significant. ( B ) Western blot of chromatin bound RAD18, BRCA1, PCNA and ubiquitinated PCNA upon knockdown of BRCA1 and / or RAD18 under NT conditions or after treatment with 4 mM HU for 2 h. N = 3, Representative blot shown. ( C ) Fork recovery fiber assay scheme (top). Quantification of stalled (red bars) and restarted (blue bars) replication forks upon BRCA1, BRCA2, RAD51 or PALB2 knockdown or treatment with RAD51 inhibitor (RAD51i, B02) at a concentration 27 uM in U2OS WT cells. N = 2, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: unpaired t -test, * P < 0.05, ns = not significant. ( D ) Fork reco v ery fiber assay scheme (top). Quantification of stalled (red bars) and restarted (blue bars) upon BRCA1 and / or RAD52 knockdown. N = 2, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: unpaired t -test, ns = not significant.

Article Snippet: Cell culture and cell lines Human bone osteosarcoma U2OS cells (American Type Culture Collection), U2OS cells knocked-out for RAD18 (provided by George-Lucian Moldovan, UPenn Hershey), and MDA-MB-231 breast cancer cells (American Type Culture Collection) were grown in DMEM media supplemented with 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin at 37 ◦C, 5% CO 2 .

Techniques: Ubiquitin Proteomics, Knockdown, Mutagenesis, Western Blot, Concentration Assay

Journal: Nucleic acids research

Article Title: A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

doi: 10.1093/nar/gkae563

Figure Lengend Snippet: Figure 4. RNF168 promotes fork recovery in BRCA1-deficient cells and mediates RAD18 recruitment to chromatin upon HU treatment. ( A ) Fork reco v ery fiber assay scheme (top). Quantification of stalled (red bars) and restarted (blue bars) replication forks upon BRCA1, RAD18, UBC13, PALB2 or RNF168 knockdown in U2OS WT cells. N = 2, > 150 fiber tracts quantified per sample for each independent experiment, Statistics: unpaired t -test, ** P < 0.01, * P < 0.05, ns = not significant. ( B ) R epresentativ e images of RAD18 foci by immunofluorescence microscopy upon knockdown of BRCA1 and / or RNF168 in U2OS WT cells in non-treated (NT) conditions or treatment with 4 mM HU for 2 h (left). Quantification of RAD18 foci per cell upon knockdown of RAD18, BRCA1, and / or RNF168 in U2OS WT cell under NT conditions (gray bars) or upon HU treatment (red bars). N = 3, > 100 cells quantified per sample for each independent experiment. Statistics: one-way ANO V A, **** P < 0.0 0 01, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant.

Article Snippet: Cell culture and cell lines Human bone osteosarcoma U2OS cells (American Type Culture Collection), U2OS cells knocked-out for RAD18 (provided by George-Lucian Moldovan, UPenn Hershey), and MDA-MB-231 breast cancer cells (American Type Culture Collection) were grown in DMEM media supplemented with 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin at 37 ◦C, 5% CO 2 .

Techniques: Knockdown, Immunofluorescence, Microscopy

Journal: Nucleic acids research

Article Title: A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

doi: 10.1093/nar/gkae563

Figure Lengend Snippet: Figure 5. Loss of RAD18 or mutation of the K164 PCNA residue compromises cell viability in BRCA1-deficient cells. ( A ) Representative images of clonogenic survival assays upon knockdown of BRCA1 and / or UBC13 in U2OS WT or U2OS RAD18 KO cells (left). Quantification of % cell viability by clonogenic survival assay upon knockdown of BRCA1 (gray bars) and / or UBC13 (red bars) in U2OS WT or U2OS RAD18 KO cells (blue bars) (right). Mean with SEM shown, N = 4, Statistics: unpaired t -test, * P < 0.05, ** P < 0.01, ns = not significant. ( B ) Western blot of U2OS WT or U2OS RAD18 KO cells depleted of BRCA1 and / or UBC13. ( C ) Quantification of MTS proliferation assay measuring proliferation relative to Day 0 upon knockdown of BR CA1 (gra y dot ted line) and / or UBC1 3 (red solid and dot ted lines) in U2OS WT (gray solid line) or U2OS RAD18 KO cells (blue solid and dot ted lines). Mean with SEM shown, N = 3, Statistics: tw o-w a y ANO V A f ollo w ed b y B onferroni, **** P < 0.0 0 01, significance sho wn f or comparisons betw een WT versus RAD18 KO siBRCA1; WT versus WT siUBC13; and WT versus WT siBRCA1 / siUBC13.

Article Snippet: Cell culture and cell lines Human bone osteosarcoma U2OS cells (American Type Culture Collection), U2OS cells knocked-out for RAD18 (provided by George-Lucian Moldovan, UPenn Hershey), and MDA-MB-231 breast cancer cells (American Type Culture Collection) were grown in DMEM media supplemented with 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin at 37 ◦C, 5% CO 2 .

Techniques: Mutagenesis, Residue, Knockdown, Clonogenic Cell Survival Assay, Western Blot, Proliferation Assay