Fig 2: hSSB2 depletion delays XPC recruitment, impairs RPA recruitment to UV damage sites and increases sensitivity to UVB exposure. (a) Representative images of HeLa cells probed for CPD (green, left panels) and RPA32 (red, middle panels) without UV exposure or 1 h and 3 h after UVC irradiation with a 5 µm filter. Cells were irradiated 48 h after negative control (si Control) and hSSB2 (si hSSB2) siRNAs transfection with UVC covered with 5 µm filter. DNA is stained with DAPI (blue, right panels). Average RPA32 fluorescent staining intensity at the CPD sites (± sem, n = 3) has been reported in graph (b). (c) Representative images of HeLa cells probed for CPD (green, left panels) and XPC (red, middle panels) without UV exposure or 15 min and 1 h after UVC irradiation. DNA is stained with DAPI (blue, right panels). Average XPC fluorescent staining intensity at the CPD sites (± sem) has been reported in graph (d). Scale bar represents 5 µm. (e,f) Measure of the average live cells (± sem, n = 3) 48 h after UVB exposure of HeLa cells transfected with negative control (si Control) or hSSB2 (si hSSB2) siRNAs (e) and U-2OS cells (f). Analysis was carried out with the Cytell imaging system 20 min after Hoechst and Propidium Iodide staining (****P-value < 0.0001 in a Student test).

Fig 3: SerRS is involved in the hypoxia response to induce VEGFA expression through phosphorylation by ATM and ATR kinases at S101 and S241 residues.(A, B) qRT-PCR analysis of VEGFA expression in HEK293 cells transfected with sh-SerRS, sh-GlyRS, or nonspecific sh-Control under hypoxia or normoxia conditions. VEGFA levels (A) and relative induction of VEGFA under hypoxia (B) were plotted as means ± SEM (n = 4, biological replicates, Student t test, **p < 0.01, ****p < 0.0001). (C) Sequence alignment of SerRS proteins from vertebrates and non-vertebrates flanking serine 101 and serine 241 (highlighted in red) sites numbered according to the human sequence. The conserved ATM/ATR substrate motif residues are underlined. (D) 32P-labeling to confirm phosphorylation of SerRS, but not GlyRS, in vitro using recombinant tRNA synthetases. Double-stranded DNA oligonucleotides are used to mimic DNA damage to activate ATM/ATR from the nuclear extract of HEK293 cells. (E) Western blot analysis to confirm recombinant SerRS, but not GlyRS and TyrRS, is phosphorylated by ATM/ATR by using a specific antibody against phosphor-ATM/ATR substrate (p-S*Q). The purified His6-tagged aaRS proteins are recognized by anti-His6-tag antibody. Activation of ATM and ATR was confirmed by autophosphorylation of ATM (at S1981) and phosphorylation of RPA32 (at S33), respectively. (F, G) IP and western blot analysis to confirm hypoxia-induced SerRS phosphorylation in HEK293 (F) and HUVEC (G) cells. IP was performed with anti-SerRS antibody from mouse and rabbit for HEK293 (F) and HUVEC (G) cells, respectively. Phosphorylated SerRS was detected with the antibody against phosphor-ATM/ATR substrate (p-S*Q) from rabbit. Activation of ATM and ATR was confirmed by autophosphorylation of ATM (at S1981) and phosphorylation of CHK1 (at S345) and P53 (at S15). (H) Hypoxia-induced phosphorylation of SerRS in HEK293 cells is reduced in SerRSAA compared with SerRSWT. (I) Hypoxia-induced phosphorylation of SerRS was decreased when ATM and ATR were knocked down separately or together by siRNAs (si-ATM, si-ATR). At least 2 biological replicates were performed for western blot experiments with consistent results. Representative images were shown. See S1 Data for quantitative data and statistical analysis. See S2 Data for original, uncropped images supporting blots and gel results. aaRS, aminoacyl-tRNA synthetase; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia mutated and RAD3-related; GlyRS, glycyl-tRNA synthetase; HUVEC, human umbilical vein endothelial cell; IP, immunoprecipitation; qRT-PCR, real-time quantitative reverse transcription PCR; SerRS, seryl-tRNA synthetase; SerRSAA, S101A/S241A; SerRSWT, wild-type SerRS; sh-Control, control shRNA; sh-GlyRS, shRNAs targeting GlyRS; sh-SerRS, shRNAs targeting SerRS; siRNA, small interfering RNA; TyrRS, tyrosyl-tRNA synthetase; VEGFA, vascular endothelial growth factor A.

Fig 4: Interrogation of potential targets for therapeutic intervention in mPTCL AGSEA of HALLMARK gene signature was performed on whole mPTCL‐infiltrated versus wildtype spleens and purified TCRvβ8+ mPTCL versus wildtype CD4+ T‐cells (n = 6). Eighteen of the top 20 positive enriched signatures that were identified in whole spleen and purified cells are shown. Red circles depict significant enrichment (FDR q‐value < 0.05). NES—normalized enrichment score. FDR Q‐values can be found in Dataset EV5.BWestern blot for phosphorylated and total ZAP70, AKT, S6, and GSK3β in lysates from three control (C) C57bl/6J and three mPTCL (T) spleens. Murine DLBCL cell line A20 is a positive control for constitutively active AKT signaling.C, D(C) Kaplan–Meyer curve showing time to welfare endpoint (survival) in mice implanted IV with 104 mPTCL cells and dosed with PI3Kδ inhibitor PI‐3065 at 75 mg/kg PO QD (n = 10) (log‐rank test P > 0.9999) or (D), the pan‐AKT inhibitor capivasertib (AZD5363) dosed at 130 mg/kg PO BID 4 days on/3 days off (4/3; n = 9) versus vehicle control (n = 10) (log‐rank test P = 0.0498 in favor of the control arm). Results in C and D are representative of three independent studies of PI3K pathway inhibitors.EGSEA of REACTOME terms in the comparison of transcriptome from whole spleens and purified cells as in (A). A selection of 18 of the top 35 pathways are shown (all with FDR P‐value < 0.05). FDR Q‐values can be found in Dataset EV5.FHeatmap of log2 normalized expression of significantly upregulated genes (P‐adjusted < 0.05) in “REACTOME Activation of ATR in response to replication stress” gene signature from panel E.GImmunostain for DNA damage marker γH2AX in wildtype and mPTCL spleens. mPTCL data are representative of six mice. Arrowhead indicates positive nuclear foci.HWestern blot for γH2AX, total H2AX, CHK1, and RPA32 in control C57bl/6J and mPTCL spleens. GAPDH is a loading control. Three representative samples per group are shown in the blot (left), and quantification was performed on n = 6 at right. T‐test P = 0.0798 (CHK1; not statistically significant, ns), P = 8.69E‐05 (RPA32), P = 2.11E‐03 (γH2AX). ***P < 0.001.

Fig 5: PIK3CA blockade in molecular processes regulated by PIK3CA mutation.A Immunoblotting shows the phosphorylation of AKT and S6 in lysates from WT and C35 cells treated with alpelisib at 1 μM or 10 μM for 24 h. β-actin was used as a loading control. The data presented are representative of two independent experiments. B WT and PIK3CA-mutant DHSA-1426 cells were treated with increasing concentrations of alpelisib for 72 h. Cell viability was measured using the CellTiter 96™ AQueous One Solution Cell Proliferation Assay. Data were compiled from two independent experiments. Error bars indicate mean ± SD. The IC50 was calculated using non-linear regression method (left). The boxplot shows the IC50 data from each individual experiment (black point) and their mean value (horizontal black line, right). C The alpelisib sensitivity data across human breast invasive carcinoma (BRCA) cell lines were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC) database (version 2 and release 8.4). Each dot represents an individual cell line for BRCA cell lines and a biological replicate of the experiment for DHSA-1426 and COSB cell lines. The horizontal black bars indicate the median values. D Immunoblotting analysis shows γH2AX and RPA32 status in WT and PIK3CA-mutant DHSA-1426 cells treated with alpelisib and ZSTK474. Bar plots indicate the signal intensity of γH2AX normalized with RPA32 expression. ELISA analysis shows secreted GM-CSF (E), CXCL8 (F), IL-6 (G), and MCP-1 (H) from cell culture supernatants after treatment with 10 μM of the indicated drugs. Statistical significance was determined by two-tailed unpaired t-tests; ns: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.