Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: a , Western blots showing ABRAXAS levels in ABRAXAS KO HT-29 cells complemented with WT or S404A/S406A ABRAXAS. GAPDH serves as loading control. b, Representative immunofluorescence images of BRCA1 foci in WT and S404A/S406A ABRAXAS expressing HT-29 cells exposed to CPT (1 µM) alone or in combination with ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. γH2AX marks cells with CPT-induced replication damage. c, Representative immunofluorescence images of HA-ABRAXAS foci in WT and S404A/S406A ABRAXAS expressing HT-29 cells treated with CPT (1 µM) +/- ATMi (250 nM) for 1 h. d, Scatter plot showing quantification of the data as described in (c). Experiments were performed three times with similar results. Data represents mean ± SEM derived from n ≥ 200 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. e, ABRAXAS S404A/S406A mutations restore end resection in ATM-inhibited cells. Representative immunofluorescence images of RPA foci in control (sgROSA), ABRAXAS KO, WT and S404A/S406A ABRAXAS expressing HT-29 cells treated as described in (c). Images were acquired with 100X magnification. Scale bar is shown.

Article Snippet: Images with 100X magnification were acquired using a 100X oil objective lens of Nikon Eclipse Ti2 microscope.

Techniques: Western Blot, Control, Immunofluorescence, Expressing, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells

doi: 10.64898/2026.03.20.713277

Figure Lengend Snippet: (a and b), Representative images of PLA-SIRF experiments showing EXO1 binding to replication forks in control (sgHPRT1) and ABRAXAS KO HT-29 cells treated with CPT (1 µM) +/- ATMi (250 nM) for 40 min. Cells were optionally pretreated with ATMi for 10 min and labeled with EdU during the last 20 min of CPT treatment. ATMi was present throughout the experiment. Images were acquired with 100X magnification. Scale bar is shown ( a ). Scatter plot showing quantification of EXO1-EdU SIRF foci normalized to average EdU-EdU signal in each experimental condition ( b ). Data represent mean ± SEM derived from n ≥ 139 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. (c and d), PLA-SIRF experiments showing increased MRE11 fork binding in ABRAXAS KO cells treated with CPT and ATMi as described in (a). Representative images captured with 100X magnification are shown. Scale bar is shown ( c ). Scatter plot showing quantification of the MRE11-EdU SIRF foci normalized to average EdU-EdU signal in individual experimental conditions ( d ). Data represent mean ± SEM derived from n ≥ 140 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. (e and f ) Representative images of RPA immunofluorescence in the indicated genotypes following treatment with CPT (1 µM) +/- ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. γH2AX marks cells with CPT-induced replication damage. Scale bar is shown ( e ). Scatter plot showing quantification of the RPA foci ( f ). Data represent mean ± SEM derived from n ≥ 90 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. (g and h), Representative images of RPA immunofluorescence in the indicated genotypes treated with combined CPT (1 µM) and ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. Scale bar is shown ( g ). Scatter plot showing quantification of the RPA immunofluorescence ( h ). Data represent mean ± SEM derived from n ≥ 100 cells examined over two biological repeats; p values are indicated, unpaired two-tailed t test. i, Colony survival assay showing EXO1 KO resensitizes ABRAXAS KO cells to combined CPT and ATMi treatment. Data represent mean ± SD derived from n =3 independent experiments.

Article Snippet: Images with 100X magnification were acquired using a 100X oil objective lens of Nikon Eclipse Ti2 microscope.

Techniques: Binding Assay, Control, Labeling, Derivative Assay, Two Tailed Test, Immunofluorescence, Clonogenic Cell Survival Assay