Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Immunofluorescence microscopy quantification of NFκB translocation, phosphorylated SP1 residue Ser101, phosphorylated cJun residues Ser63 and Ser73, and phosphorylated RNA polymerase II residues Ser2 and Ser5 in differentiated THP1 cells infected with Vpr WT or control viruses in the presence or absence of ATM, ATR, or DNA-PK inhibition ( n = 50 cells). Cells were infected for 24 hours, treated with vehicle or the indicated inhibitor for 24 hours, and then subjected to immunofluorescence microscopy. Analyses were performed using a one-way ANOVA; ns, not significant; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . (B) Representative immunofluorescence microscopy images of R-loop abundance in primary MDM and HeLa cells infected with indicated viruses 48 hours post-infection ( n = 50 cells). Analyses performed using a student t test; *** p < 0.001. (C–E) Immunofluorescence microscopy quantification of R-loop abundance in HeLa cells infected with indicated viruses 48 hours post-infection. Samples were left untreated (C) or treated with RNaseH (D, left), triptolide (D, right), or with the indicated DDR inhibitors (E), respectively ( n = 50 cells). For RNaseH treatment, cells were infected for 48 hours prior to methanol fixation and addition of recombinant RNaseH to deplete RNA associated with R-loops. For inhibitor treatments, HeLa cells were infected for 24 hours, treated with the indicated inhibitor for 24 hours, and then subjected to immunofluorescence microscopy. Analyses were performed using a one-way ANOVA; ns, not significant; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . (F) Immunofluorescence microscopy quantification of DDR activation (left) and R-loop abundance (right) in HeLa cells infected with indicated viruses and transfected with RNaseH constructs ( n = 50 cells). Cells were infected for 24 hours before transfection of control or RNaseH-expressing plasmids for 24 hours prior to being prepared for immunofluorescence microscopy. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001; * p < 0.05. The data underlying this Figure can be found in . (G) Flow cytometric histograms of HeLa cells infected with control or Vpr WT LTR-mCh viruses for 24 hours prior to transient expression of RNaseH for 24 hours and quantification of LTR-mCh MFI in transfected cells via flow cytometry ( n = 3 experiments). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ).

Article Snippet: For inhibitor experiments, cells were treated 24-hours post-infection with either 10 nM ATM inhibitor (Fisher, #AZD1390), 10 μM ATR inhibitor (Fisher, #NU6027), 3 mM caffeine, 16 μM DNA-PK inhibitor #1 (MedChemExpress, #NU7026), or 16 μM DNA-PK inhibitor #2 (MedChemExpress #NU7441) for 24 hours.

Techniques: Immunofluorescence, Microscopy, Translocation Assay, Residue, Infection, Control, Inhibition, Recombinant, Activation Assay, Transfection, Construct, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: A. Specificity profile of TFU72 and AZD7648 against kinases of the PI3K and PIKK families. Data is shown as half maximal inhibitory concentration (IC 50 ) against (cellular IC 50 for DNA-PKcs, biochemical IC 50 for other kinases) different kinases. B-C. Heatmap showing the top kinases inhibited by TFU72 and AZD7648 (both at 1 µM) following testing on a human kinase panel. The data is shown in descending order sorted for kinases inhibited at 50% or above relative to a target-specific inhibitor for TFU72 ( B ) or AZD7648 ( C ). D-E. Allelic HDR editing frequencies at AAVS1 locus determined by NGS analysis at D5 post RNP and ssODN editing with different concentrations of TFU72/AZD7648 as indicated ( D ). The compounds were removed either at 24 hours post editing (1 wash) or left in the cells for 5 days (0 wash). Viability of the gene edited cells as measured by CellTiter-Glo® 2.0 Assay is shown as percentage relative to the untreated sample (gene editing without compound) ( E ) (n=2). F. Allelic distribution of HDR, INDEL and WT frequencies in K562 cells edited across different gene loci (FANCF, HEK4, HBB) with or without TFU72 treatment based on NGS analysis. U denotes untreated (editing without compound), T denotes editing with TFU72 (1 µM) (n=2).

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: iPSC, HSPC and T cells were edited at CCR5 locus using RNP and AAV6 gene editing for knock-in of short sequence (two stop codons) with different concentrations of AZD7648 and TFU72 as indicated. HDR, NHEJ, MMEJ and WT allelic frequencies were calculated at D3 post editing using Sanger Sequencing and ICE analysis (n=3). A, C, E . Scatter plots show the allelic HDR frequency in iPSC ( A ), HSPC ( C ) and T cells ( E ). B, D, F . Bar graphs show the distribution of allelic HDR, NHEJ, MMEJ and WT frequencies in iPSC ( B ), HSPC ( D ) and T cells ( F ). GT denotes gene targeting (HDR) with RNP, AAV6 editing. A and T denote AZD7648 and TFU72, respectively.

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Knock-In, Sequencing

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: iPSC ( A ), HSPC ( B ) and T cells ( C ) were edited individually at CCR5, HBB and STING1 loci with two different gRNAs at each loci using RNP and AAV6 gene editing for the knock-in of short sequence with AZD7648 and TFU72 at the indicated concentrations. Bar graphs show allelic HDR frequencies (% targeted alleles) measured at D3 post editing using Sanger Sequencing and ICE analysis (n=3). GT denotes gene targeting (HDR) with RNP, AAV6 editing. AAV6 HDR template for CCR5 locus was designed to knock-in two stop codons at the target site. At the HBB locus, the AAV6 HDR template was designed to knock-in silent mutations and correction of the sickle cell disease mutation (E6V). For the STING1 locus, the AAV6 HDR template was designed to knock-in a point mutation (V155M) along with silent mutations at the gRNA target site.

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Knock-In, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: A-C . iPSC ( A ), HSPC ( B ) and T cells ( C ) were edited at CCR5, HBB and STING1 loci individually using RNP and AAV6 gene editing for the knock-in of multi-kb sequence with AZD7648 and TFU72 at the indicated concentrations and at different AAV6 doses, multiplicity of infection (MOI): 500, 1000, 2500, 5000. Bar graphs show allelic HDR frequencies (% targeted alleles) measured at D3 post editing using ddPCR analysis (n=3). GT denotes gene targeting (HDR) with RNP, AAV6 editing. AAV6 HDR templates for CCR5 and HBB loci were designed to knock-in a 2.2-kb sequence consisting of UBC promoter driven GFP followed by a bGH polyA signal sequence. For the STING1 locus, the AAV6 HDR template was designed to knock-in a 1.4-kb sequence consisting of PGK promoter driven GFP followed by a sv40 polyA signal sequence.

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Knock-In, Sequencing, Infection

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: HSPCs were edited at HBB locus using RNP, AAV6 (Hifi or WT Cas9) with AZD7648 (A, 0.5 µM) or TFU72 (T, 0.5 µM) or no treatment (U). Mock electroporated cells (M) were used as a negative control. GT denotes gene targeting (HDR) with RNP, AAV6. A . At D3 post editing, a previously characterized off-target site at Chr9 (OT1) was PCR amplified from genomic DNA and sequenced by NGS. Bar graphs show INDEL frequency at the OT1 site as determined by the CRISPResso2 tool analysis of the NGS data (n=2). WT Cas9 edited samples were assessed for the INDELs using Sanger Sequencing and ICE analysis. B . Translocation between on-target (HBB) site and OT1 off-target site was assessed by ddPCR analysis and data is shown as percentage of translocation which is the sum of 4 different translocation outcomes (n=2). C . HSPCs edited at HBB loci with Hifi Cas9 were assessed for the frequency of large deletions at the on-target site using Nanopore sequencing of a 10-kb PCR amplicon. Bar graph shows the frequency of reads with deletions of the sizes 50-1000, 1000-3000, 3000-5000 and above 5000 bp (n=3). D-E. HSPCs edited at HBB locus using RNP, AAV6 with different incubation times of AAV6 (U), AZD7648+AAV6 (A) and TFU72+AAV6 (T) were assessed for the off-target INDELs at the OT1 site as described above. D. Bar graphs show the INDEL frequency at the OT1 site at D3 post editing in samples with 12h, 24h and 72h incubation times (n=1). E. Bar graphs show the frequency of reads with deletions of the sizes 50-1000, 1000-3000, 3000-5000 and above 5000 bp as determined by Nanopore sequencing described above (n=1).

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Negative Control, Amplification, Sequencing, Translocation Assay, Nanopore Sequencing, Incubation

Journal: Cell reports

Article Title: DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

doi: 10.1016/j.celrep.2025.116796

Figure Lengend Snippet: (A) Subcellular fractionation of E6.1 Jurkat T cells shows increased DNA-PKcs phosphorylation at S2056 after 2 min of 5 μg/mL αCD3/CD28 TCR stimulation, which is reduced by the DNA-PKcs inhibitor NU7441 (5 μM). (B and C) (B) ImageJ quantification reveals a 3.5-fold increase in pDNA-PKcs band intensity in whole-cell extract and (C) a 4.8-fold increase in pDNA-PKcs band intensity in cytosolic extract, with one dot representing one experiment. Data are represented as mean ± SEM. (D) LSCM imaging at 63× magnification of E6.1 Jurkat T cells reveals that two minutes of αCD3/CD28 TCR stimulation (5 μg/mL) increases protein expression (green) in the cytosol and at the plasma membrane alongside F-Actin (red). (E) Quantification of pDNA-PKcs by mean fluorescence intensity (MFI) is shown for whole-cell pDNA-PKcs and the ratio of pDNA-PKcs outside the nucleus. Data are represented as mean ± SEM. (F) Among PIKK family members, DNA-PKcs, but not ATM or ATR, is activated in the cytosol following TCR stimulation with 5 μg/mL αCD3/CD28 or 100 nM doxorubicin (DNA damage-inducing reagent) for 2 min in E6.1 Jurkat T cells. (G) ImageJ quantification reveals a 3-fold increase in pDNA-PKcs band intensity following both TCR stimulation (CD3/28) and DNA damage (doxorubicin), with one dot representing one experiment. Data are represented as mean ± SEM. (H) LSCM imaging at 63× magnification of E6.1 Jurkat T cells reveals that two minutes of αCD3/CD28 TCR stimulation increases only pDNA-PKcs cytosolic presence, but not pATM or pATR. (I and J) (I) Quantified MFI values and (J) the cytosolic-to-whole-cell ratio of phosphorylated PIKKs, with each dot representing a single cell. Data are represented as mean ± SEM. Scale bars, 5 μm. Representative western blots and microscopy images from n = 3 independent experiments, with all cells within the field of view quantified on ICC. Statistical significance determined using one-factor ANOVA plus Tukey’s multiple comparisons (α = 0.05, * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.0001).

Article Snippet: NU7441 (KU-57788) DNA-PK inhibitor , Selleck-Chem , Cat#S2638.

Techniques: Fractionation, Phospho-proteomics, Imaging, Expressing, Clinical Proteomics, Membrane, Fluorescence, Single Cell, Western Blot, Microscopy

Journal: Cell reports

Article Title: DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

doi: 10.1016/j.celrep.2025.116796

Figure Lengend Snippet: (A and B) (A) LSCM imaging at 63× magnification and (B) histogram MFI analysis of 2-min αCD3/CD28 TCR-stimulated Jurkat T cells identify areas of colocalization where pDNA-PKcs and LAT peaks overlap (*) and areas where peaks do not overlap (arrow). (C) LSCM imaging at 63× magnification of SEE-pulsed Raji B cells (blue) cocultured and conjugated with E6.1 Jurkat T cells shows pDNA-PKcs (green) colocalized with LAT (red) at the immune synapse (arrow). (D) LSCM immune synapses were quantified by MFI of pDNA-PKcs and LAT by drawing a quantifying line along the interface of Raji B cell and Jurkat T cell, each dot representing an area of immune synapse. Data are represented as mean ± SEM. (E) Co-immunoprecipitation (coIP) in Jurkat T cells shows that DNA-PKcs interacts with LAT following TCR stimulation. (F) TCR stimulation increases LAT pull-down by DNA-PKcs 7.5-fold, which is reduced by DNA-PKcs inhibitor NU7441 to 2.5-fold when quantified on ImageJ with one dot representing one coIP experiment. Data are represented as mean ± SEM. (G) LSCM imaging at 63× magnification of E6.1 Jurkat T cells demonstrates LAT (red) localization at the plasma membrane with pDNA-PKcs (green) upon TCR stimulation, which decreases with NU7441 (5 μM). Arrows identify areas of colocalization. (H) LSCM quantification reveals significant increases in MFI for pDNA-PKcs and LAT after TCR stimulation, along with colocalization events, which decrease upon DNA-PKcs inhibition with NU7441. Each dot represents a single quantified cell. Data are represented as mean ± SEM. (I and J) shRNA-mediated knockdown of DNA-PKcs (>70% reduction) reduces total DNA-PKcs expression on western blot. Data are represented as mean ± SEM. (K) LSCM imaging at 63× magnification shows that shRNA inhibition of DNA-PKcs attenuates LAT (red) localization at the plasma membrane after 2 min of 5 μg/mL αCD3/CD28 TCR stimulation. (L) Quantification of LSCM with each dot representing one shRNA-transfected cell. Data are represented as mean ± SEM. Scale bars, 5 μm. Representative images and western blots from n = 3 independent experiments with all cells quantified within the field of view on ICC. Statistical significance determined using one-factor ANOVA plus Tukey’s multiple comparisons (α = 0.05, ** p < 0.01, ** p < 0.005, and **** p < 0.0001).

Article Snippet: NU7441 (KU-57788) DNA-PK inhibitor , Selleck-Chem , Cat#S2638.

Techniques: Imaging, Immunoprecipitation, Clinical Proteomics, Membrane, Inhibition, shRNA, Knockdown, Expressing, Western Blot, Transfection

Journal: Cell reports

Article Title: DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

doi: 10.1016/j.celrep.2025.116796

Figure Lengend Snippet: E6.1 Jurkat T cells were stimulated with αCD3/CD28 for 15 min and then lysed with Golgi fractionation buffer. (A) Total and phosphorylated DNA-PKcs (pDNA-PKcs) are present in both the cis - and trans -Golgi fractions. Upon TCR stimulation, the secretory fraction exhibits an increase in pDNA-PKcs expression in the presence of LAT. (B) LSCM imaging at 63× magnification shows that LAT expression at the plasma membrane is attenuated by both DNA-PKcs inhibition (NU7441, 5 μM) and inhibition of secretory vesicle blebbing (brefeldin, 10 μg/mL). (C) Quantification of LSCM, with each dot representing one area of the plasma membrane. Data are represented as mean ± SEM. (D) LSCM imaging at 63× magnification shows that inhibition of DNA-PKcs with NU7441 (5 μM) prevents early TCR signaling markers like pLck (p-Y394) and CD3ζ from localizing to the plasma membrane in Jurkat T cells. (E) Quantification of LSCM with each dot representing one cell (LAT and CD3ζ) or an area of the plasma membrane (pLck). Data are represented as mean ± SEM. Scale bars, 5 μm. Representative images and blots from n = 3 independent experiments with all cells within the field of view quantified on ICC. One-factor ANOVA plus Tukey’s multiple comparisons was used to determine statistical significance (α = 0.05, **** p < 0.0001).

Article Snippet: NU7441 (KU-57788) DNA-PK inhibitor , Selleck-Chem , Cat#S2638.

Techniques: Fractionation, Expressing, Imaging, Clinical Proteomics, Membrane, Inhibition

Journal: Cell reports

Article Title: DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

doi: 10.1016/j.celrep.2025.116796

Figure Lengend Snippet: (A–D) (A and B) MC38 adenocarcinoma cells or (C and D) B16 melanoma cells were injected into the rear flank of Cre + CD8-PKcs fl/fl and Cre + CD8-PKcs wt/wt mice. (A) Cre + CD8-PKcs fl/fl mice exhibited decreased median survival time (MST: 23 days) compared to Cre + CD8-PKcs wt/wt mice (MST: 36 days) (* p < 0.05, log rank analysis). (C) Cre + CD8-PKcs fl/fl mice exhibited decreased median survival (MST: 17.5 days) compared to Cre + CD8-PKcs wt/wt mice (MST: 22 days) (*** p < 0.0005, log rank analysis). (B) MC38 and (D) B16 tumor volume in Cre + CD8-PKcs fl/fl mice had an accelerated rate of growth compared to Cre + CD8-PKcs wt/wt mice (**** p < 0.0001, nonlinear regression, error bars represent SEM, n = 3 and 8 mice per group, respectively). Data are represented as mean ± SEM. MC38 adenocarcinoma cells were injected into the rear flank of Cre + CD4-PKcs wt/wt and Cre + CD4-PKcs fl/fl mice. (E) Cre + CD4-PKcs fl/fl mice exhibited similar MST (24 days) compared to Cre + CD4-PKcs wt/wt mice (MST: 27 days) ( p = 0.74, log rank analysis). (F) Tumor volume in Cre + CD4-PKcs fl/fl mice had a similar rate of growth compared to Cre + CD4-PKcs wt/wt mice (* p < 0.05, nonlinear regression, error bars represent SEM, n = 6/group). Data are represented as mean ± SEM. Naive BALB/c tail skin allografts were transplanted onto the backs of syngeneic BALB/c ( n = 3), or fully allogeneic Cre + -PKcs wt/wt ( n = 5), Cre + -PKcs wt/wt + NU7441 ( n = 4), Cre + CD4-PKcs fl/fl ( n = 7), or Cre + CD8-PKcs fl/fl ( n = 10) recipient mice. (G) Representative examples of skin graft healing on day 0 (day of surgery), 11 days post-transplant, and 18 days post-transplant. (H) Graft necrosis was visually measured daily, with the DNA-PKcs inhibitor NU7441 at 10 mg/kg slowing the graft necrosis progression. Data are represented as mean ± SEM. Cre + CD8-PKcs fl/fl recipient mice had no necrosis until integration, where minor necrosis (<20%) was apparent in 2 of 10 mice; whereas, 8 of 10 mice did not show any necrosis with integration. (I) Fully allogeneic Cre + -PKcs wt/wt mice (MST: 13 days), median survival of allograft was significantly increased in Cre + -PKcs wt/wt mice treated with NU7441 (MST: 23 days, p < 0.0001) and Cre + CD8-PKcs fl/fl recipient mice (MST: >30 days, p < 0.0001) and marginally increased in CD4-PKcs −/− mice (MST: 18 days, p < 0.05), compared by log rank analysis. (J and K) (J) Syngeneic BALB/c and (K) allogeneic Cre + CD8-PKcs fl/fl recipient mice had successful graft integration and graft hair growth by day 30.

Article Snippet: NU7441 (KU-57788) DNA-PK inhibitor , Selleck-Chem , Cat#S2638.

Techniques: Injection