Journal: bioRxiv

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1101/2025.08.05.668752

Figure Lengend Snippet: a , Schematic of sensitized UBAL degradation CRISPR-Cas9 screen. b , Gene effects and gene scores calculated for individual genes analyzed in the targeted degradation screen. c , STRING network map of the identified genes with a 10% FDR cutoff, where each bubble color represents an enriched GO term functional cluster. d , Fluorescence histograms of FSP1 KO cells expressing FSP1-GFP in the indicated cell lines. e , Quantification of median fluorescence intensity (MFI) change in GFP from ( d ). f , Immunoblot of lysates from ( d ). g-h , Kinetics of GFP fluorescence decay of FSP1 KO cells expressing FSP1-GFP in control or RFK KO cells with or without the loss of RNF8 ( g ) or RFK/RNF8 DKO (double knock-out) cells expressing the indicated RNF8 variants ( h ) following doxycycline washout. i , FSP1-GFP or RNF8 S-tag immunoprecipitation from FSP1 KO cells expressing FSP1-GFP in the indicated cell lines and blotting for RNF8 (top) or FSP1 (middle). j , Model illustrating the mechanism by which vitamin B2 metabolism and FAD synthesis impacts FSP1 activity and stability to prevent ferroptosis.

Article Snippet: For immunoprecipitation of FSP1 and RNF8 interactions, 1 mg of pre-cleared lysates were incubated with 10 μL of ChromoTek GFP-Trap Magnetic Agarose or 10 μL of S-protein Agarose (Millipore Sigma, no. 69704) for 1 h at 4°C with end-over-end rotation.

Techniques: CRISPR, Functional Assay, Fluorescence, Expressing, Western Blot, Control, Knock-Out, Immunoprecipitation, Activity Assay

Journal: Nucleic Acids Research

Article Title: PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling

doi: 10.1093/nar/gkae918

Figure Lengend Snippet: RNF168 is recruited to DNA replication and DSB via separable mechanisms. ( A ) U2OS cells were transiently co-transfected with expression constructs encoding HA-PCNA and FLAG-RNF168 WT or FLAG-RNF168 ΔMIU2. Forty-eight hours post-transfection, cells were treated conditionally with 2 mM HU or with 10 Gy IR. Cells were fixed 2 h following HU or 30 min following IR treatments, then stained with anti-HA and anti-FLAG antibodies prior to analysis by immunofluorescence confocal microscopy. Panel ( A ) shows images of representative HA-PCNA and FLAG-RNF168 co-expressing cells from all experimental conditions. ( B ) Bar chart showing enumeration of cells containing HA-PCNA-co-localizing FLAG-RNF168 foci. Each data point represents the mean of results from three separate experiments and the error-bars represent the standard deviation. ( C ) U2OS cells were transiently co-transfected with constructs encoding FLAG-RNF168 WT or FLAG-RNF8 WT. Forty-eight hours post-transfection, cells were treated conditionally with 2 mM HU. Cells were fixed 2 h following HU or treatments, then stained with anti-HA and anti-PCNA antibodies prior to analysis by immunofluorescence confocal microscopy. The individual images are of representative cells. Cells transfected with FLAG-RNF168 plasmids co-expressed both PCNA and RNF168 proteins. Unexpectedly, however, in cultures co-transfected with FLAG-RNF8 plasmid, immunoreactivity with anti-PCNA and anti-FLAG antibodies was mutually exclusive (suggesting that FLAG-RNF8 is not expressed at detectable levels in PCNA-positive S-phase cells). ( D ) U2OS cells were transiently transfected with constructs encoding FLAG-RNF168 WT or FLAG-RNF8 WT. Twenty-four hours post-transfection, cells were treated conditionally with 300 ng/ml NCS. Cells were fixed 1 h following NCS or treatments, then stained with anti-FLAG and anti-γH2AX antibodies prior to analysis by immunofluorescence confocal microscopy. The individual images are of representative cells showing that both RNF8 and RNF168 co-localize with γH2AX. ( E ) Parental RNF8 +/+ U2OS cells, RNF8 −/− U2OS cells or RNF8 −/− cells transiently transfected with an RNF8 expression plasmid (for 24 h) were treated with 300 ng/ml NCS. One hour after NCS treatment, cells were fixed and stained with 53BP1 antibodies prior to analysis by immunofluorescence confocal microscopy. The individual images are of representative cells showing RNF8-dependency of 53BP1 focus formation. ( F ) RNF8 +/+ cells or RNF8 −/− derivatives were transiently transfected with an RNF168 expression plasmid. Twenty-four hours post-transfection, cells were pulse-labeled with 10 mM EdU for 1 h to label sites of ongoing DNA synthesis. Cells were fixed and stained with antibodies against EdU and FLAG prior to analysis by immunofluorescence confocal microscopy. The individual images are of representative cells showing RNF8-independent co-localization of RNF168 and EdU. ( G ) Bar chart showing enumeration of cells containing EdU-co-localizing FLAG-RNF168 foci in RNF8 +/+ and RNF8 −/− cells. Each data point represents the mean of results from three separate experiments and the error-bars represent the standard deviation. Unpaired Student’s t -test demonstrated no statistically significant co-localization between RNF8 and EdU incorporation ( P = 0.5867).

Article Snippet: For immunoblotting, cell extracts or immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated overnight with the following primary antibodies: PCNA (sc-56), Chk1 (sc-7898), β-actin (sc-130656), GAPDH (sc-32233), RNF8 (SC-134492), pRPA32 S4/8 (A300-245A) and RAD18 (A301-340A) from Bethyl Laboratories (Montgomery, TX); p-Chk1 S317 (2344) and H2A (2578) antibodies were purchased from Cell Signaling Technology; γH2AX (05–636) and H2A K15ub (MABE11119) were from Millipore; H2B (Ab1790) antibody was from Abcam.

Techniques: Transfection, Expressing, Construct, Staining, Immunofluorescence, Confocal Microscopy, Standard Deviation, Plasmid Preparation, Labeling, DNA Synthesis

Journal: Nucleic Acids Research

Article Title: PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling

doi: 10.1093/nar/gkae918

Figure Lengend Snippet: RNF168 contains a DPIP domain. ( A ) Domain organization of RNF168 indicating key functional domains and a degenerate PIP-like sequence residing in a disordered C-terminal region. The protein disorder profile was generated using the Protein Disorder Prediction (PrDOS) tool at http://prdos.hgc.jp/cgi-bin/top.cgi . ( B ) AlphaFold model of the PCNA–RNF168 DPIP complex reveals a similar binding model to that of the PCNA-Pol Kappa PIP complex. Left panel shows the top predicted model of the trimeric PCNA–RNF168 DPIP complex. Right panel shows the overlay of the AlphaFold model of the trimeric PCNA–RNF168 DPIP complex with the crystal structure of the trimeric PCNA-Polκ PIP (PDB: 2zvl), with the PCNA and Polk PIP in the crystal structure colored in cyan and yellow, respectively. ( C ) H1299 cells were transiently co-transfected with expression constructs encoding HA-PCNA and FLAG-RNF168 WT or FLAG-RNF168 ΔDPIP. Forty-eight hours post-transfection cells were fixed, then stained with anti-HA and anti-FLAG antibodies prior to analysis by immunofluorescence confocal microscopy. The photographs are of representative cells co-expressing HA-PCNA and WT or ΔDPIP forms of FLAG-RNF168. The bar chart shows enumeration of cells containing HA-PCNA-co-localizing FLAG-RNF168 foci. Each data point represents the mean of results from three separate experiments and the error-bars represent the standard deviation. ( D ) RNF168 −/− U2OS cells were infected with adenoviral vectors encoding FLAG-RNF168 WT or FLAG-RNF168 ΔDPIP. Twenty-four hours post-infection cells were fixed and subject to PLA to compare proximities of WT and ΔDPIP RNF168 with endogenous PCNA. The photographs are of representative DAPI-stained nuclei from each experimental condition. The immunoblots show levels of ectopically expressed WT and ΔPIP RNF168 in this experiment. The bar chart showing enumeration of cells containing eight or more PCNA / FLAG-RNF168 PLA foci. Each data point represents the mean of results from three separate experiments and the error-bars represent the standard deviation. Ordinary one-way analysis of variance (ANOVA) demonstrated statistically significant difference between RNF168 WT and PIP ( P = 0.0049). ( E ) Replicate plates of H1299 cells were co-infected with adenovirus vectors encoding HA-PCNA and RNF168 WT or RNF168 ΔDPIP. Twenty-four hours post infection, some cultures were treated with HU (2 mM, 2 h), camptothecin (CPT, 100 nM, 2 h), ATM inhibitor KU55933 100 nM, 2 h) or the WEE1 inhibitor MK1775 (10 μM, 2 h). Chromatin extracts from the treated and untreated (control) cells were normalized for protein content and immunoprecipitated with anti-HA antibodies. Anti-HA immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies. ( F ) Replicate plates of RNF8 −/− U2OS cells were transiently infected with adenovirus vectors encoding FLAG-RNF168 WT, FLAG-RNF168 ΔDPIP or FLAG-RNF168 ΔMIU2, or with a control ‘empty’ adenoviral vector. Forty-eight hours post-infection chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-FLAG antibodies. Anti-FLAG immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies.

Article Snippet: For immunoblotting, cell extracts or immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated overnight with the following primary antibodies: PCNA (sc-56), Chk1 (sc-7898), β-actin (sc-130656), GAPDH (sc-32233), RNF8 (SC-134492), pRPA32 S4/8 (A300-245A) and RAD18 (A301-340A) from Bethyl Laboratories (Montgomery, TX); p-Chk1 S317 (2344) and H2A (2578) antibodies were purchased from Cell Signaling Technology; γH2AX (05–636) and H2A K15ub (MABE11119) were from Millipore; H2B (Ab1790) antibody was from Abcam.

Techniques: Functional Assay, Sequencing, Generated, Binding Assay, Transfection, Expressing, Construct, Staining, Immunofluorescence, Confocal Microscopy, Standard Deviation, Infection, Western Blot, Control, Immunoprecipitation, SDS Page, Plasmid Preparation

Journal: bioRxiv

Article Title: Break-induced replication drives large-scale genomic amplifications in cancer cells

doi: 10.1101/2024.08.27.609980

Figure Lengend Snippet: a , Histogram ( right ) showing the impact of increasing IR doses on the induction of genomic amplifications in control U2OS cells or two independent clones with deletion of DNA-PKcs by CRISPR/Cas9. ( left ) Western blots illustrating the efficiency of deletion of DNA-PKcs in U2OS cells. b , Histogram showing the percentage of U2OS cells with genomic amplifications 72 h following the exposure to 9 Gy of control, DMSO-treated U2OS cells or U2OS cells treated with the DNA-PKcs inhibitor NU7441 for 24 h prior to IR. c, Immunoblot showing deletion of effectors of the c-NHEJ pathway (LIG4, XRCC4, XLF). d , Histogram showing the impact of deletion of the indicated effectors of c-NHEJ on the induction of genomic amplifications by IR. e , The impact of inhibiting DNA LIG4 on the induction of genomic amplifications by IR in U2OS cells. f , g , Deletion of KTM4B (coding for SUV4-20H1) but not deletion of KTM4C (coding for SUV4-20H2) inhibits the recruitment of 53BP1 to DSBs. f , Representative immunofluorescent images of 53BP1 showing formation of 53BP1 foci following exposure of control (pX330) U2OS cells or U2OS cells deleted of KTM4B or KTM4C to 5 Gy analysed 1 h post-exposure. g , Quantitation of the number of 53BP1 foci per cell in control (pX330) cells or U2OS deleted of KTM4B or KTM4C (two independent clones each). The results represent the average of a minimum of 100 nuclei in each of three independent experiments for each condition ± S.D. ns: non-significant, *** p < 0.001. h , i , Histograms showing the percentage of cells undergoing genomic amplifications following the exposure of control or ATM-deleted U2OS cells (inset: immunoblotting of ATM levels) to 9 Gy ( h ), or following pharmacological inhibition of ATM in parental U2OS cells by KU55933 24 h prior to IR ( i ). Genomic amplifications was monitored 72 h post-IR. j , Histogram showing the percentage of control (pX330) U2OS cells or U2OS cells deleted of RNF8 or RNF168 and exposed to 9 Gy. Genomic amplifications was monitored 72 h post-IR. k , l , Histograms showing the percentage of control (pX330) U2OS cells or U2OS cells deleted of 53BP1 (in two independent clones; immunoblots shown in inset ( k )) or RIF1 (immunoblots shown in inset ( l )) with genomic amplifications following the exposure to 9 Gy. Genomic amplifications was monitored 72 h post-IR. Data in all the histograms represent the average of three independent experiments ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies recognizing the following proteins were used: ATM (1:1000, ab78; Abcam), tubulin (1:1000, sc-53646; Santa Cruz), SET8 (1:1000, C18B7; Cell Signaling), mono-methyl Histone H4 (K20) (1:500, #9724; Cell Signaling), di-methyl Histone H4 (K20) (1:500, #9759; Cell Signaling), Tri-methyl Histone H4 (K20) (1:500, #5737; Cell Signaling), DNA-PKcs (1:1000, ab44815; Abcam), 53BP1 (1:5000, N100-304; Novus Biologicals) POLD3 (1:1000, H00010714-M01; Abnova), POLQ (1:1000, H00010721-M09; Abnova), RIF1 (1:1000, A300-569A-M; Bethyl Laboratories), RNF8 (1:1000, sc-2711462; Santa Cruz), RNF168 (1:1000, ABE367; Millipore), CtIP (1:1000, sc-271339, Santa Cruz), EXO1 (1:1000, ab95012; Abcam), XRCC4 (1:1000, sc-271087; Santa Cruz), XLF (1:1000, A300-730A-M; Bethyl Laboratories), NBS1 (1:1000, ab32074; Abcam), MDC1 (1:25,000, ab11171; Abcam), LIG1 (1:1000, 18051-1-AP; Proteintech), and LIG4 (1:1000, HPA001334; Sigma Aldrich).

Techniques: Control, Clone Assay, CRISPR, Western Blot, Quantitation Assay, Inhibition

Journal: bioRxiv

Article Title: Break-induced replication drives large-scale genomic amplifications in cancer cells

doi: 10.1101/2024.08.27.609980

Figure Lengend Snippet: a , ATM suppresses the induction of genomic amplifications by DSBs. The histogram shows the percentage of A si SI-ER-U2OS cells with genomic amplifications as determined by PI-FACS 72 h following treatment with 4-OHT. Where indicated, the cells were pre-treated with the ATM specific inhibitor KU3355 5 or 24 h prior to treatment with 4-OHT. The results represent the average of three independent experiments ± S.D. *** p < 0.001. b , ATM inhibition does not stimulate rereplication induction by MLN4924. The histogram shows the percentage of control (pX330) U2OS cells or U2OS cells with ATM deletion (1ATM) with rereplication as determined by PI-FACS 48 h following treatment with 0.1 μM MLN4924. The results represent the average of three independent experiments ± S.D. ns: non-significant. c , d , Deletion of RNF8, RNF168, or RIF1 suppresses 53BP1 recruitment to DSBs. c, Representative immunofluorescence images of 53BP1 showing formation of 53BP1 foci following exposure of control (pX330) U2OS cells or U2OS cells deleted of RNF8, RNF168, or RIF1 to 5 Gy and analysed 1 h post-exposure. d , Quantitation of the number of 53BP1 foci per cell in control (pX330) cells or U2OS deleted of RNF8, RNF168, or RIF1. The results represent the average of a minimum of 100 nuclei in each of three independent experiments for each condition ± S.D. ** p < 0.01, *** p < 0.001. g , MDC1 and RNF168 suppress IR-induced genomic amplifications. Histograms showing the percentage of control (pX330) 293T cells or 293T cells deleted of MDC1 or RNF168 with genomic amplifications as determined by PI-FACS 48 h following the exposure to 5 Gy. The results represent the average of three independent experiments ± S.D. ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies recognizing the following proteins were used: ATM (1:1000, ab78; Abcam), tubulin (1:1000, sc-53646; Santa Cruz), SET8 (1:1000, C18B7; Cell Signaling), mono-methyl Histone H4 (K20) (1:500, #9724; Cell Signaling), di-methyl Histone H4 (K20) (1:500, #9759; Cell Signaling), Tri-methyl Histone H4 (K20) (1:500, #5737; Cell Signaling), DNA-PKcs (1:1000, ab44815; Abcam), 53BP1 (1:5000, N100-304; Novus Biologicals) POLD3 (1:1000, H00010714-M01; Abnova), POLQ (1:1000, H00010721-M09; Abnova), RIF1 (1:1000, A300-569A-M; Bethyl Laboratories), RNF8 (1:1000, sc-2711462; Santa Cruz), RNF168 (1:1000, ABE367; Millipore), CtIP (1:1000, sc-271339, Santa Cruz), EXO1 (1:1000, ab95012; Abcam), XRCC4 (1:1000, sc-271087; Santa Cruz), XLF (1:1000, A300-730A-M; Bethyl Laboratories), NBS1 (1:1000, ab32074; Abcam), MDC1 (1:25,000, ab11171; Abcam), LIG1 (1:1000, 18051-1-AP; Proteintech), and LIG4 (1:1000, HPA001334; Sigma Aldrich).

Techniques: Inhibition, Control, Immunofluorescence, Quantitation Assay