ht 29 cells  (ATCC)

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, Colony survival of control (sgROSA) and ABRAXAS KO HEK293T cells treated with the indicated doses of CPT alone or in combination with 200 nM ATMi. Relative colony survival (%) in individual genotypes and treatment conditions are shown. Data represent mean ± SD of n =3 independent experiments. b, Line graph showing relative colony survival of control (sgHPRT1), ABRAXAS KO, ATM KO and ATM-ABRAXAS double KO (DKO) HT-29 cells exposed to the indicated doses of CPT. Data represent mean ± SD of n =3 independent experiments. c, Representative images of RPA immunofluorescence in control (sgHPRT1) and ABRAXAS KO cells treated with 50 nM CPT +/- 250 nM ATMi for 1 h. Cells were labeled with EdU (10 µM) to identify S-phase cells. Scale bar is shown. d, Scatter plot showing significantly reduced RPA foci in CPT-treated EdU positive S-phase cells upon ATMi. ABRAXAS KO significantly restored RPA foci under these conditions. Data represent mean ± SEM derived from n ≥ 200 EdU positive nuclei examined over two independent experiments; p values are indicated, unpaired two-tailed t test. e, Schematic showing experimental scheme of native IdU assay. Cells were treated with 50 nM CPT +/- 250 nM ATMi as indicated. IdU was added 20 minutes after CPT addition to label newly synthesized DNA encountering CPT-induced lesions. Scatter plot showing quantification of native IdU experiment. Nascent ssDNA (IdU foci) was significantly reduced in either ATM inhibited or ATM KO cells. ABRAXAS KO significantly restored ssDNA under these conditions. Data represent mean ± SEM derived from n ≥ 180 cells examined over two independent experiments; p values are indicated, two-tailed Mann–Whitney test. f, Scatter plot showing reduced BRCA1 foci in S404A/S406A ABRAXAS expressing cells as compared to WT. WT and S404A/S406A ABRAXAS expressing HT-29 cells were treated with CPT (1 µM) alone or in combination with ATMi (250 nM) for 1 h. ATMi was added 10 min before CPT treatment. Experiments were performed four times with similar results. Data represent mean ± SEM derived from n ≥ 150 cells examined over two independent experiments; p values are indicated, unpaired two-tailed t test. g, ABRAXAS S404A/S406A mutations restore end resection in ATM-inhibited cells. Scatter plot represents quantification of RPA foci in S404A/S406A cells. Experiments were performed as described in (f) and repeated three times with similar results. Data represent mean ± SEM derived from n ≥ 110 cells examined over two repeats; p values are indicated, unpaired two-tailed t test. h, Clonogenic survival assay showing CPT+ATMi resistance in HT-29 cells expressing S404A/S406A ABRAXAS. Data are mean ± SD from n =3 independent experiments.

Article Snippet: HT-29 and HEK293T cell lines were purchased from ATCC.

Techniques: Control, Immunofluorescence, Labeling, Derivative Assay, Two Tailed Test, Synthesized, MANN-WHITNEY, Expressing, Clonogenic Cell Survival Assay

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 blotting showing diminished CPT-induced phospho (S824)-KAP1 (p-KAP1) levels upon AZD0156 treatment, confirming potency of the ATM inhibitor. Control (sgROSA) and ABRAXAS KO HEK293T cells were treated with CPT (1 µM) +/- AZD0156 (250 nM) for 40 min and analyzed by Western blotting using the indicated antibodies. GAPDH serves as loading control. b, Colony survival assay showing CPT+ATMi resistance upon ABRAXAS KO in HEK293T cells. Experiments were repeated n =3 times with similar results. c, Western blotting validating ATM and ABRAXAS DKO in HT-29 cells. GAPDH serves as loading control. d, Colony survival assay showing CPT resistance in ATM and ABRAXAS DKO HT-29 cells. Experiments were repeated n =3 times with similar results. e, Colony survival assay showing olaparib+ATMi resistance in ABRAXAS KO HEK293T cells. Experiments were repeated n =3 times with similar results.

Article Snippet: HT-29 and HEK293T cell lines were purchased from ATCC.

Techniques: Western Blot, Control, Clonogenic Cell Survival Assay

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, Clonogenic survival assays showing CPT+ATMi resistance in HT-29 cells expressing ABRAXAS S404A/S406A mutant. b, Western blots showing ABRAXAS levels in ABRAXAS KO HEK293T cells complemented with WT or S404A/S406A ABRAXAS. GAPDH serves as loading control. c, Clonogenic survival assay showing CPT+ATMi resistance in ABRAXAS S404A/S406A expressing HEK293T cells.

Article Snippet: HT-29 and HEK293T cell lines were purchased from ATCC.

Techniques: Expressing, Mutagenesis, Western Blot, Control, Clonogenic Cell Survival Assay

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, Experimental scheme of the iPOND-MS in control (sgROSA) and ABRAXAS KO cells treated with CPT (1 µM) + ATMi (250 nM). ATMi was added 5 min before CPT treatment and present throughout the experiment. (b and c), iPOND purified samples (5% beads) were analyzed by silver staining (b) and Western blotting (c). Enrichment of replisome component PCNA in iPOND purified samples but not in control no click indicates specificity of the iPOND purification. d, Scatter plot showing iPOND-MS data ( n =5). X axis shows log2 fold change (FC) of proteins in ABRAXAS KO cells over sgROSA. Y axis shows average log2 intensity of proteins across sgROSA and ABRAXAS KO cells. e, Gene-annotation clusters network showing top 10 significantly enriched GO terms for the upregulated proteins in ABRAXAS KO cells. f, Experimental scheme of BrdU-coupled chromatin accessibility assay in HEK293T cells treated with CPT (1 µM) +/- ATMi (250 nM). g, Representative gel images showing increased MNase accessibility at BrdU-labeled damaged forks in ABRAXAS KO HEK293T cells. Equal number (∼30 X10 6 ) of control (sgROSA) and ABRAXAS KO cells were treated with CPT (1 µM) + ATMi (250 nM) and labeled with BrdU. BrdU-labeled nuclei were digested with MNase (250 gel units) for the indicated time followed by chromatin isolation. Equal amount of digested chromatin was run on 1.2% agarose gel (left panel), stained with Ethidium bromide (EtBr) and subsequently transferred to nylon membrane by Southern blotting. MNase accessibility at BrdU labeled damaged chromatin was determined by Western blotting using an anti-BrdU antibody (right panel). h, Bar graph showing increased BrdU-labeled mononucleosome fractions in ABRAXAS KO cells as compared to control. Relative percentage of BrdU-labeled mononucleosome fractions normalized to total amount of BrdU-labeled DNA per lane is shown. Data represent mean ± SEM of n =3 independent experiments; p values are indicated, unpaired two-tailed t test. (i and j), ABRAXAS complementation in ABRAXAS KO cells reduced MNase accessibility at BrdU-labeled damaged chromatin. Nuclei isolated from ABRAXAS KO and ABRAXAS-complemented HEK2923T cells were treated with CPT (1 µM) + ATMi (250 nM) and were subjected to MNase digestion for 10 min followed by Southern-Western blotting ( i ). Bar graph showing decreased BrdU-labeled mononucleosome fractions upon ABRAXAS complementation ( j ). Data represent mean ± SEM of n =3 independent experiments; p values are indicated, unpaired two-tailed t test.

Article Snippet: HT-29 and HEK293T cell lines were purchased from ATCC.

Techniques: Control, Purification, Silver Staining, Western Blot, Labeling, Isolation, Agarose Gel Electrophoresis, Staining, Membrane, Southern Blot, Two Tailed Test