Fig 2: Abrogation of ATM phosphorylation on BRCA1-S1152 reduces SKP2 recruitment and ATM-NBS1 interaction upon DNA damage(A) Coimmunoprecipitation shows reduced interaction between BRCA1-S1198A and SKP2 in HEK293 cells.(B) Recruitment of SKP2 upon microirradiation. SKP2 laser stripes (green) and γH2AX (red) at 10, 30, and 60 min time points are shown. Scale bar, 10 μm. Bar graph below is quantification of three independent experiments. For 10 min (n = 326 for Brca1WT and n = 351 for Brca1S1152A/S1152A, p = 0.007656); 30 min (n = 336 for Brca1WT and n = 427 for Brca1S1152A/S1152A, p = 0.009519); 60 min (n = 336 for Brca1WT and n = 380 for Brca1S1152A/S1152A, p = 0.009258).(C) Immunoprecipitation of endogenous NBS1 shows reduced K63-linked ubiquitination of NBS1 in Brca1S1152A/S1152A MEF cells, but not in Brca1S971A/S971A MEF cells, in response to DNA damage.(D) Coimmunoprecipitation shows reduced interaction between NBS1 and ATM upon DNA damage in Brca1S1152A/S1152A MEF cells but not in Brca1S971A/S971A MEF cells.

Fig 3: NBS1 K388 lactylation promotes DNA-damaging treatment resistance via HR repair.a-d, Cytotoxic effects of cisplatin on AGS-P and AGS-NBS1-K388R cells were examined by cell viability (a), western blot (b), flow cytometry (c), and colony formation assay (d). e-f, Cytotoxic effects of IR on AGS-P and AGS-NBS1-K388R cells were examined by cell viability (e), western bolt (f). g-h, The protective effects of lactate on cisplatin resistance were examined in AGS-P and AGS-NBS1-K388R cells by western blot (g) and flow cytometry (h). i-j, The protective effects of lactate on IR resistance were examined in AGS-P and AGS-NBS1-K388R cells by cell viability (i) and flow cytometry (j). k-l, The protective effects of LDHA on cisplatin resistance were examined in AGS-P and AGS-NBS1-K388R cells by flow cytometry (k) and cell viability (l). m, n, Immunoblotting of γ-H2AX in both AGS-P and AGS-NBS1-K388R cells after cisplatin (2.5 μM) (m) or IR (10 Gy) (n) treatment for indicated time. o-p, Immunostaining with anti-BRCA1 (o) and anti-RAD51 (p) was performed at 60 min after IR (2 Gy). Scale bars, 15 μm. Data are presented as mean ± SD. n = 3 biologically independent samples for a, c, d, e, h, i, j, k, l. P value was determined by t-test (two-sided) for c, d, h, j, k. For gel source data, see Supplementary Fig. 1. Source Data

Fig 4: Abrogation of ATM phosphorylation on BRCA1-S1152 affects assembly of NBS1 but not on the recruitment of MRE11/CtIP/53BP1 upon DNA damageRecruitment of MRE11 (A), CtIP (B), 53BP1 (C), and NBS1 (D) after microirradiation in BRCA1 WT and S1152A MEF cells. Laser stripes of indicated proteins (red) and γH2AX (green) from S/G2 cells at 10, 30, and 60 min time points. Scale bar, 10 μm. Bar graph shows quantification of laser strips. Average from 3 to 4 independent experiments. For MRE11: 10 min (n = 452 for Brca1WT and n = 437 for Brca1S1152A/S1152A, p = 0.1176); 30 min (n = 405 for Brca1WT and n = 433 for Brca1S1152A/S1152A, p = 0.0695); 60 min (n = 511 for Brca1WT and n = 676 for Brca1S1152A/S1152A, p = 0.1299). These data are from 3 independent experiments. For 53BP1: 10 min (n = 534 for Brca1WT and n = 687 for Brca1S1152A/S1152A, p = 0.007104); 30 min (n = 436 for Brca1WT and n = 407 for Brca1S1152A/S1152A, p = 0.170465); 60 min (n = 543 for Brca1WT and n = 386 for Brca1S1152A/S1152A, p = 0.267004). The data are from 4 independent experiments. For CtIP: 10 min (n = 147 for Brca1WT and n = 170 for Brca1S1152A/S1152A, p = 0.024461); 30 min (n = 115 for Brca1WT and n = 215 for Brca1S1152A/S1152A, p = 0.251565); 60 min (n = 103 for Brca1WT and n = 214 for Brca1S1152A/S1152A, p = 0.178625). The data are from 4 independent experiments. For NBS1, 10 min (n = 125 for Brca1WT and n = 102 for Brca1S1152A/S1152A, p = 0.002956746); 30 min (n = 203 for Brca1WT and n = 174 for Brca1S1152A/S1152A, p = 0.000222756); 60 min (n = 159 for Brca1WT and n = 165 for Brca1S1152A/S1152A, p = 0.002122252).(E) Interaction of BRCA1 with RAD51 and CtIP is comparable among Flag-tagged human BRCA1 (WT, S1189A, and S988A) as shown in coimmunoprecipitation assay in HEK293 cells. However, interaction between NBS1 and Flag-BRCA1-S1189A is reduced after IR.

Fig 5: The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μm. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110β, p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD (n = 3). ∗P < 0.05; ∗∗P < 0.01.