EMBO/soda-nel huggingface dataset:
Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors (2022)
Figure 3

Figure 3: Flagellar assembly intermediates in H. pylori.

A-D) slices through electron cryotomograms of H. pylori (left panels) indicating the presence of various flagellar intermediates. Middle panels show central slices through STAs. Right panels show schematic representations. Question marks in the schematic in (C) indicate that it is unclear whether the densities surrounding the upper part of the C-ring are the same as the novel cytoplasmic rings seen in the MS-complex. Empty panels indicate that there were not enough examples to produce a STA.

E & F) cryotomogram slices, STAs, and schematics of H. pylori fliP* cells showing the MS-complex (E) and the C-complex (F). The two novel cytoplasmic rings are highlighted with light and dark green arrows in the STA panel. White arrows in (A-F) point to the particles.

G & H) central slices through the STAs of the MS-complexes of the motile H. pylori (G)and H. pylori FliP* mutant (H) with the diameters of their various constituent rings indicated.

Data information: Scale bars are 50 nm for cryotomogram slices and 20 nm for STAs.
HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS (2007)
figf1

a–e, Scanning electron microscopy (SEM) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. a, Normal eyes in DTS7 flies reared at 22 °C. b, Rough eyes in DTS7 flies reared at 28 °C. c, d, Degeneration was suppressed by expression of Drosophila HDAC6 (dHDAC6; c) or human HDAC6 (hHDAC6; d), but not a catalytically dead mutant of hHDAC6 (cat. mut.; e). f–j, SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. f, Normal eyes in AR52 flies reared without (-) DHT. g, Rough eyes in AR52 flies reared with (+) DHT. Degeneration was suppressed by expression of Drosophila HDAC6 (h) or human HDAC6 (i), but not a catalytically dead mutant of human HDAC6 (j). k–p, Detection of UPS reporter in imaginal eye discs from third-instar larvae by confocal microscopy. High level fluorescence was found in flies expressing GFP (k, positive control), but fluorescence was barely detectable in control flies expressing CL1–GFP (l, negative control). CL1-GFP accumulates in DTS7 flies with temperature-dependent proteasome impairment (compare m to n) and in AR52 flies with ligand-dependent degeneration (compare o to p). The retinal phenotypes of 200 to >1,000 flies of each genotype were examined. Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3, respectively. (DHT, dihydrotestosterone.)
HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS (2007)
figf2

a–e, Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in (f). a, An autophagosome (red arrow) containing cytoplasmic contents in a photoreceptor neuron from an AR52 fly reared on DHT. b, Higher magnification of the autophagosome in a. c, Multiple autophagolysosomes (red arrows) containing dense, amorphous material from an AR52 fly reared on DHT. d, A juxtanuclear multilamellar body (red arrow) from a DTS7 fly reared at 28 °C. e, A multivesicular body (red arrow) from a DTS7 fly reared at 28 °C. f, A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 °C compared to those reared at 22 °C, and in AR52 flies reared on DHT compared to those reared off DHT. Data show mean ± s.d., n = 59–82 neurons in 5 sections per condition. No accumulation of autophagic figures was found in AR12 flies. g–l, SEM images of fly eyes expressing the indicated transgenes. RNAi knockdown (KD) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 °C (compare h, i to g) and AR52 flies reared on DHT (compare k, l to j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2. (N, nucleus; Rh, rhabdomere.)
HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS (2007)
figf3

a, b, d, Western blots from flies expressing the indicated transgenes. a, Steady state levels of AR52 protein are reduced in flies overexpressing Drosophila HDAC6, but are elevated in flies in which atg6 or atg12 has been knocked down. b, Western blots showing the temporal profile of AR52 protein monomer and high molecular weight aggregate levels after a brief pulse of expression. AR52 protein became detectable by 2.5 h after treatment with RU486, reached a peak at 10 h, and then slowly decayed. c, A logarithmic plot of AR52/actin ratios was used to determine the line of best fit by regression analysis (y = Ae-Kx). R2 = 0.9117 (AR52 - DHT), R2 = 0.7808 (AR52 + DHT), R2 = 0.9719 (HDAC6 + (AR52 - DHT)), R2 = 0.9644 (HDAC6 + (AR52 + DHT)). Half-life was determined by the slope of the best fit line with the equation t1/2 = 0.693/K. Half-life of AR52 in vivo was reduced ∼2-fold in flies co-expressing Drosophila HDAC6 and did not differ significantly in the presence (broken lines) or absence (solid lines) of DHT. Plots of the mean AR52/actin ratios are shown in Supplementary Fig. S12. d, Flies co-expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b, but AR protein decayed at an accelerated rate. Exogenous Drosophila HDAC6 was detected by immunoblot against the V5 epitope.
HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS (2007)
figf4

a–l, SEM images of fly eyes expressing the indicated transgenes. The rough eye phenotypes caused by proteasome mutation (a) or by expression of polyQ-expanded AR (b) were both suppressed by rearing flies on the TOR inhibitor rapamycin (c, d). e, f, Rapamycin failed to suppress degeneration in an autophagy-deficient background created by knockdown of atg12, confirming that rescue by rapamycin is autophagy-dependent. g, h, Rapamycin also failed to suppress degeneration when HDAC6 levels were knocked down, demonstrating that suppression via the TOR pathway is HDAC6-dependent. k, j, HDAC6 failed to suppress degeneration in an autophagy-deficient background, confirming that rescue by HDAC6 is dependent on autophagy (compare k, l to i, j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2.
Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation (2014)
figf2

(a) DAPK1-targeting peptide HA-GluN2Bct-CTM and its nonfunctional control peptide HA-GluN2Bct-CTMm. (b) Reciprocal coimmunoprecipitation (IP) followed by immunoblotting revealed that GluN2Bct specifically interacted with cDAPK1 but not wtDAPK1. Flag-tagged inactive wtDAPK1 or constitutively active cDAPK1 was expressed with either HA-GluN2Bct-CTM or HA-GluN2Bct-CTMm at various ratios in HEK cells, and coimmunoprecipitation and/or immunoblotting were performed 24 h thereafter. Anti-HA was used to detect HA-GluN2Bct-CTM and HA-GluN2Bct-CTMm, anti-FLAG to detect wtDAPK1 and cDAPK1. (c) HA-GluN2Bct-CTM specifically and dose-dependently decreased the level of cDAPK1 (n = 4 independent experiments from 4 separate cell cultures and transfections; P 0.001; F(5,18) = 18.27) but not wtDAPK1 (d; n = 3 independent experiments from 3 separate cell cultures and transfections; P = 0.933; F(5,12) = 0.249). HA-GluN2Bct-CTM–mediated cDAPK1 knockdown was significantly reduced by NH4Cl (c; 20 mM; n = 4; ΔΔΔP 0.001 compared to HA-GluN2Bct-CTM:cDAPK1 8:1 group) and by mutational inactivation of CTM (e; HA-GluN2Bct-CTMm; P = 0.785 compared to control (white bar); F(4,35) = 0.432, 8 independent experiments from 8 separate cell cultures and transfections). Levels of cDAPK1 or wtDAPK1 cotransfected with pcDNA3.0 vector (0:1, white bar) represent the control values, arbitrarily set as 1. *P 0.05, **P 0.01, ***P 0.001, compared with the control; n.s., not significant. Bars represent relative means ± s.e.m. Full-length blots are presented in Supplementary Figure 9. Membranes reprobed for β-actin were used as loading controls. One-way ANOVA with Fisher least significant difference test.
Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation (2014)
figf3

(a) Left, NMDA (50 μM; 30 min) activated DAPK1, resulting in a time-dependent decrease in its phosphorylation levels (pDAPK1, n = 4). Right, coimmunoprecipitation (IP) with anti-GluN2B and sequential immunoblotting for DAPK1 and GluN2B showed an NMDA-induced association between DAPK1 and GluN2B (n = 3). (b) Design and production of TAT-GluN2Bct-CTM and TAT-GluN2Bct peptides (left) using an E. coli expression system. Coomassie blue staining after SDS-PAGE assessed their purity (right); left lane, size marker. (c) Bath application of TAT-GluN2Bct-CTM (200 μM; n = 9), but not TAT-GluN2Bct (200 μM; n = 6), knocked down activated DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; one-way ANOVA; P 0.001, F(5,36) = 10.891) and dose- (d, doses in μM; n = 4; P 0.001; F(6,21) = 18.14) and time-dependent (e; P 0.001; F(8,44) = 12.074). (f) A single pretreatment of TAT-GluN2Bct-CTM (sing; 200 μM, 60 min before and during the 30-min NMDA stimulation) produced a transient reduction of DAPK1, returning to baseline within 7 h (n = 4; P = 0.888), and an additional dose of the peptide after NMDA washout resulted in a persistent decrease in DAPK1 up to 7 h (mult; n = 4; ΔΔP = 0.002). One-way ANOVA; P 0.001, F(4,15) = 10.389. (g) Synthetic peptides TAT-GluN2B and TAT-GluN2BCTM. (h) TAT-GluN2BCTM (25 μM; n = 5; P = 0.001), but not control TAT-GluN2B (25 μM; n = 4; P = 0.223), decreased native DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; P = 0.302). One-way ANOVA, P 0.001, F(5,24) = 13.591. Relative levels of DAPK1 were normalized to those in the saline control group and compared to the saline control (white bar; *P 0.05, **P 0.01 and ***P 0.001) or NMDA-treated group (gray bar; ΔP 0.05, ΔΔP 0.01 and ΔΔΔP 0.001). Membranes reprobed for β-actin were used as a loading control. Bars represent relative means ± s.e.m. Sample sizes represent number of individual experiments. Full-length blots are presented in Supplementary Figure 9.
Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation (2014)
figf4

(a) Top, the synthetic cell-penetrating α-synuclein targeting peptide TAT-βsynCTM and its control TAT-βsyn. Middle, immunoblots demonstrate that TAT-βsynCTM (25 μM; n = 5), but not the CTM-lacking control peptide TAT-βsyn (25 μM; n = 5), specifically decreased the targeted endogenous α-synuclein (one-way ANOVA, Tukey post hoc, P 0.001, F(3,16) = 12.435), without affecting the level of unrelated control protein PSD-95 at 4 h (bottom), and this reduction was prevented in the presence of NH4Cl (20 mM; n = 5). Sample sizes represent individual experiments from at least 3 separate primary cultures. (b) Top, PSD-95 targeting peptide TAT-GluN2B9cCTM and control TAT-GluN2B9c. Middle, TAT-GluN2B9cCTM (25 μM; n = 4), but not Tat-GluN2B9c (25 μM; n = 4), effectively degraded endogenous PSD-95 (one-way ANOVA, Tukey post hoc, P 0.001, F(3,12) = 18.154) without perturbing untargeted protein α-synuclein (bottom). NH4Cl rescued PSD-95 degradation. Sample sizes represent individual experiments from at least 2 separate primary cultures. Membrane reprobing for β-actin was used as an additional specificity and loading control. ΔP 0.05, **,ΔΔP 0.01 and ***P 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (arbitrarily set as 1). Full-length blots are presented in Supplementary Figure 9.
Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation (2014)
figf5

(a) Immunoblotting for phosphorylated DAPK1 (pDAPK1) revealed a time-dependent activation (dephosphorylation) of DAPK1 by H2O2 treatment (300 μM; 30 min; n = 4). (b) Bath application of 100 μM TAT-GluN2Bct-CTM (36.54 ± 7.1% of control; n = 8; P = 0.001), but not TAT-GluN2Bct (89.13 ± 10.78%; n = 7; P = 0.311), 60 min before and during H2O2 treatment (300 μM; 30 min) knocked down DAPK1 at 2 h after washout, and this effect was rescued by NH4Cl (20 mM; n = 8; **P = 0.003 to control; P = 0.106 to H2O2-treated). One-way ANOVA, F(4,34) = 11.628, P 0.001. Bars represent DAPK1 levels relative to saline control group. β-actin was used as a loading control. (c) LDH assay revealed that H2O2 treatment (300 μM; 30 min) resulted in a significant increase in neuronal death 12 h after treatment (n = 8; 2.50 ± 0.12; P 0.001 to control), which was rescued by breaking down H2O2 with catalase (100 U in 10 μl phosphate-buffered saline; n = 4; 1.17 ± 0.02; P = 0.001 to H2O2 group). H2O2-induced neurotoxicity was significantly reduced by TAT-GluN2Bct-CTM (50 μM; applied 60 min before and maintained throughout the experiments; n = 9; 1.56 ± 0.08; P = 0.001 to H2O2 group), but not by TAT-GluN2Bct (50 μM; n = 9; 2.63 ± 0.10; P = 0.105 to H2O2 group) or the NMDAR antagonist APV (1 μM; n = 4; 2.16 ± 0.14; P = 0.169 to H2O2 group). NH4Cl abolished the neuroprotective effect of TAT-GluN2Bct-CTM (n = 4; 2.39 ± 0.27; P = 0.538 compared to H2O2 group). One-way ANOVA, P 0.001, F(6,41) = 26.842. ***,ΔΔΔP 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (white bar, arbitrarily set as 1). n represents individual experiments from at least 3 separate primary cultures. Full-length blots are presented in Supplementary Figure 9.
Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation (2014)
figf6

(a) Timeline of tissue collection for analysis of DAPK1 degradation in rats; IHC, immunohistochemistry. (b) 2,3,5-Triphenyltetrazolium chloride staining of a series of transverse brain sections showed reliable damage in the ipsilateral side following unilateral MCAo. (c) Black dashed lines on a section stained as in b represent brain areas removed for immunoblotting. (d) Immunoblots demonstrated specific DAPK1 knockdown in the infarct (Ipsi) but not contralateral (Contra) side following application of TAT-GluN2BCTM (10 mg per kilogram, i.v.; n = 3; t(4) = 14.459, P 0.001) but not TAT-GluN2B (10 mg per kilogram, i.v.; n = 3; t(4) = 0.739, P = 0.501). β-actin was used as a loading control; two-tailed Student's t-test ***P 0.001; n.s., not significant. (e) HE (left) and immunohistochemical DAPK1 (right) staining of adjacent brain sections. Compared with those in saline (top) and TAT-GluN2B–treated (middle) controls, TAT-GluN2BCTM treatment (bottom) selectively reduced infarct area (left) and DAPK1 levels (right) ipsilaterally. Black outlining delineates infarct areas as visualized with HE staining. (f) Left, brain sections stained with Fluorojade B in rats injected with saline (n = 6), TAT-GluN2B (n = 5) or TAT-GluN2BCTM (n = 5) after treatment as shown in a. Right, quantification of cellular damage by counting the number of Fluorojade B–positive cells in each 640 × 640 pixel image at 10× magnification. TAT-GluN2BCTM (10 mg per kilogram) displayed more prominent neuroprotection in the cortex (P 0.001) and striatum (P 0.001) as compared to TAT-GluN2B (10 mg per kilogram). Cortex: H(2) = 41.235; P 0.001; striatum: H(2) = 38.808; P 0.001. Kruskal-Wallis ANOVA on ranks with Dunn's post hoc; bars represent relative means ± s.e.m., ***,ΔΔΔP 0.001. n values represent tissue from 3 animals collected from at least 2 litters. Scale bars 1 mm in b,c,e, 20 μm in f. Full-length blots are presented in Supplementary Figure 9.
Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA-damage repair (2022)
Figure 1

Figure 1. DNA damage triggers an interaction between DPP9 and BRCA2

A. Sequence alignment of BRCA2 homologues from 108 placental mammals (Logo plot) shows conservation of the cleavage site for DPP9 at the BRCA2 N-terminus. The cartoon depicts the full-length BRCA2 and its conserved domains with the corresponding interaction partners.

B. Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non-targeting siRNA (siNT) or silenced with the indicated oligos. 300 ng / mL MMC was added for 24 h. Each dot represents the number of PLA events in a single cell, from two to seven biological replicates (siNT-MMC (n = 6), siNT+MMC (n = 7), siDPP9-MMC (n = 4), siDPP9+MMC (n = 5), siBRCA2-MMC (n = 2), siBRCA2+MMC (n = 3), siFLNA-MMC (n = 5), siFLNA+MMC (n = 6), NgtCntrl-MMC (n = 3), NgtCntrl+MMC (n = 6)). The number of foci are shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, **** = p ≤ 0.0001).

C. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells. Exposure of cells to MMC triggers more PLA events (white). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071 (these images, along with the corresponding controls can be found in Appendix Figure S1A&B).

D. Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S-Phase. Each dot represents the number of PLA events in a single cell, from three biological replicates. Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS (250 ng / mL for 30 min). Cells were sampled prior to NCS treatment, after NCS treatment and with 1.5 h and 3 h of recovery after NCS treatment. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001).

E. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells. Exposure of cells to NCS triggers more PLA events (white), which increase in time. Cells in S-Phase show a greater fold-increase in the number of PLA events in comparison to asynchronous cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Antibodies as in (C).

F. Co-immunoprecipitation assays showing binding of BRCA2 and DPP9-SWT. HEK293 DPP9 KO + DPP9WT cells, were treated with 1µg/mL Dox (24 h) to induce the expression of DPP9-FLAG. DNA damage was induced with 300 nM MMC treatment for 24h. Control cells do not express DPP9 (- Dox). Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting (anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-FLAG RRID:AB_262044).
Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA-damage repair (2022)
Figure 4

Figure 4: DPP9 triggers BRCA2 degradation in response to MMC

A-B. Representative Western Blots and accompanying graph from more than three biological replicates show that MMC (300 nM, 24 h) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells, which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 (100µM), in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 (siDPP9). RAD51 stability is not altered in HeLa DPP9 KD cells. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test. (*= p ≤ 0.03). Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-Vinculin: RRID:AB_477629, anti-Tubulin: RRID:AB_628412, anti-RAD51: RRID:AB_1142428.

C-D. Representative Western Blots and graph summarizing results of more than three biological replicates. CHX chase assays show that the MMC-induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9-SS729A cells overexpressing an inactive DPP9-S mutant. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. Cells that were overexpressing the active variant (DPP9-SWT) show similar levels of MMC-induced BRCA2 degradation. RAD51 stability is not altered by MMC and is similar in all cell lines. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03, **= p ≤ 0.002). Antibodies as described in (A-B).

E. Graphical presentation of the plasmid used for production of the N-terminal truncated mutant BRCA2∆MP3-1000 that was transfected in H-I. The BRCA21-1000 insert was cloned in a similar manner. Both constructs were tagged with a C-terminal FLAG. The ubiquitin moiety is removed in cells by endogenous ubiquitin isopeptidases.

F-G. Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2∆MP3-1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21-1000. Cells were co-transfected with a GFP expressing plasmid, as transfection and loading control. Control cells were transfected with GFP only (U.T). Image originates from one representative experiment. BRCA2-FLAG signals are related to the transfection and loading control GFP. BRCA2 levels in relation to GFP were defined as 100 % at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03). Anti-FLAG: RRID:AB_262044, anti-GFP: RRID:AB_641123.
Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA-damage repair (2022)
Figure 5

Figure 5: DPP9-deficient cells accumulate more DNA Damage and are hypersensitive to genotoxic agents

A. Representative immunofluorescence images of γH2AX signals (white, RRID:AB_309864) in gki MEF DPP9S729A, showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin (NCS), quantification in (B). MEF DPP9S729A and control MEF cells were treated with 250 ng/mL NCS for 30 min and allowed to recover for the indicated time-points. γH2AX signals at time 0, reflect 30 min of NCS and no recovery time. Nuclei are shown in blue (DAPI). Scale bar 10 µm.

B. Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in (A). Signals from more than 1700 cells were quantified per condition per experiment. Mean ± SEM from six biological replicates, each in technical duplicates. Data were analysed by an unpaired two-way ANOVA with Sidak's multiple comparison test (* = p ≤ 0.05, ** = p ≤ 0.01).

C-E. Dose-dependent viability assays showing a higher sensitivity of HeLa DPP9 KD cells (C and D) and DPP9 silenced cells (siDPP9) (E) to MMC (C) and Olaparib (D, E). BRCA2 was silenced for control. Mean ± SEM of three biological replicates. Data were analysed by an unpaired two-way ANOVA with Sidak's multiple comparison test (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001).

F, G. Quantification of colonies formed after γ-radiation of HeLa WT and DPP9 KD (F), and MEF WT and gki MEF DPP9S729A cells (G), showing the mean ± SEM of the survival fraction (SF) from three biological replicates. Data were analysed by an unpaired two-way ANOVA.