EMBO/soda-nel huggingface dataset:
SIRT7 promotes genome integrity and modulates non-homologous end joining DNA repair (2016)
Figure 4

Figure 4 - SIRT7 protects cells from endogenous and induced DNA damage.
A Quantitation of neutral comet assays, using passage 3 WT and SirT7-/- primary MEFs, showing (left) the amount (%) of DNA in the tail and (right) the tail moment (see Fig S3A for representative images).
B Western blot showing ATM and KAP1 phosphorylation and total protein levels in WT and SirT7-/- primary fibroblasts after IR (8Gy). ATM inhibitor (ATMi) KU-55933 was added 30 min prior to irradiation where indicated. One representative blot from 4 independent experiments is shown.
C-F IF analysis of WT and SirT7-/- primary fibroblasts showing γH2AX dynamics after DNA damage induction. Cells were untreated (NoIR) or treated with 1Gy of X-rays (IR) and fixed at the indicated times post insult. Cells were pulsed with EdU (green) 30 min prior to fixation, then stained for γH2AX (red) and counterstained with DAPI (blue) (n>30 cells per group/mice; 3 mice per genotype). (C) Representative images of untreated S-phase (NoIR, EdU positive) WT and SirT7-/- nuclei (scale bar 5µm). (D) Quantitation of the number of γH2AX foci per nucleus at the indicated time points post IR (1Gy), and indicated cell cycle phases. (E) Quantitation of experiment described in (C) showing mean number of γH2AX foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after IR in G2 (mean ± SEM from 3 independent experiments). Total γH2AX foci and γH2AX foci overlapping with or at the periphery of heterochromatic regions were enumerated. Nuclei and pericentric heterochromatin (chromocenters) were segmented by DAPI staining. Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number. (F) Representative images of WT and SirT7-/- primary fibroblasts in G2 phase showing γH2AX (red) and DAPI (blue) at the indicated period of time after IR. (Right) 3D rendering of the IF segmentation depicting nuclei (pale blue), chromocenters (darker blue), and γH2AX (yellow denotes foci associated with pericentric heterochromatin, otherwise foci are red). Scale bar 2 µm.
G FACS quantitation of WT and SirT7-/- MEF cells in S-phase after insult with 10 mM hydroxyurea (HU) for 24 hours. Cells were fixed and stained with 7AAD and cell cycle was monitored by FACS (mean ± SEM; 5 samples per genotype).
H, I DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7-/- MEFs. (H) Representative images from cells labeled for 20 min with IdU (green) followed by 20 min of CldU (red). (I) Quantitation of fork velocity (fiber length/labeling time; mean ± SEM; 3 samples per genotype per condition).
*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.
SIRT7 promotes genome integrity and modulates non-homologous end joining DNA repair (2016)
Figure 5

Figure 5 - Impaired NHEJ DNA repair pathway in SirT7-/- cells.
A-E IF analysis of WT and SirT7-/- primary fibroblasts after IR (1Gy) and 1 hour chase. Cells were pulsed with EdU 30 min prior to fixation, stained with antibodies against γH2AX and 53BP1 and then counterstained with DAPI (scale bar 5µm). (A) Representative images from each cell cycle stage showing merge (top-left) including EdU (green), 53BP1 (top-right, magenta), γH2AX (bottom-left, red), and 3D rendering of reconstructed Z-stacks with 53BP1 foci modeled as spheres (bottom-right; pale-blue: nucleus; magenta spheres: 53BP1 foci). (B) Quantitation of the number of 53BP1 foci per nucleus, and (C) mean volume of 53BP1 foci (n>30 cells per group/mice; 3 mice per genotype). (D-E) Same as in (B-C), upon overexpression of SIRT7 WT or catalytically inactive point mutant SIRT7-H188Y in WT and SirT7-/- primary fibroblasts.
F Detection of endogenous chromatin-bound and nucleoplasmic 53BP1 protein from WT and SirT7-/- MEFs before and after IR (8Gy) by western blot. Histone H3 was used as loading control, and tubulin as control for fractionation.
G Same as (F), using 293T-REX cells treated with doxycycline or vehicle to induce SIRT7-HA expression.
H ChIP-on break assay. (Top) Schematic of I-SceI substrate construct introduced into HT1080 cells, which contains a single I-SceI site located within a puromycin resistance cassette. Shown along the top are the coordinates for amplicons probed by Q-PCR. (Bottom) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells. Q-PCR measurements were normalized to input DNA and non-I-SceI treated samples (No Cut) (mean ± SEM; one of two independent experiments shown). (Bottom Right) western blot demonstrating efficient knockdown of SirT7, with Histone H3 as a loading control.
I, J NHEJ repair assay using GFP expression-based reporter system in SirT7-depleted HT1080 cells (SirT7 Knockdown) versus control (Scramble Control). (I) Representative FACS dot plots. (J) Quantitation of (I); mean ± SEM of 3 independent clones per condition. Values are normalized to control (Scramble Control + I-SceI) mean.
K, L Class-switch recombination in splenic B cells from WT and SirT7-/- mice stimulated with lipopolysaccharides (LPS) and interleukin 4 (IL4). (K) The switching from IgM to IgG1 (left) and to IgG3 (right) was measured by FACS. (L) Quantitation of (K) showing mean ± SEM of 3-4 samples per genotype. One representative experiment from two is shown.
*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.
SIRT7 promotes genome integrity and modulates non-homologous end joining DNA repair (2016)
Figure 6

Figure 6 - SIRT7 modulates levels of H3K18Ac at DSB.
A, B H3K18Ac IF in WT and SirT7-/- fibroblasts. Cells were pulsed with EdU (green), stained with antibody against H3K18Ac (magenta) and then counterstained with DAPI (blue). (A) Representative IF images of late S-phase nuclei (EdU positive; scale bar 5µm). (B) Quantitation of (A) throughout the cell cycle.
C Same as in (B), in S-phase cells at the indicated times after IR (1Gy) (mean ± SEM of >30 cells per cell cycle, time point, and genotype).
D Laser-induced DNA damage in HT1080 cells expressing HA-tagged SirT7 stained with antibodies against γH2AX (red) and HA (green), then counterstained with DAPI (blue) 30 min post damage (scale bar 3 µm).
E ChIP assays of SIRT7 enrichment at the indicated loci as described in Figure 5H (mean ± SEM; one of two independent experiments is shown).
F Recruitment kinetics of GFP-tagged SirT7 after laser-induced microirradiation. (Top) Representative cell at the indicated times after induction of DNA damage (white circle; scale bar 2µm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU-55933 ATM inhibitor (ATMi), 10M Olaparib PARP inhibitor (PARPi), or DMSO. KU-55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage (mean ± SEM; sample size: SIRT7-GFP, n=34; SIRT7-GFP + ATMi, n=36; SIRT7-GFP + PARPi=18). (Bottom) Same as (middle) except quantitation of SIRT7-GFP relative intensity within the nucleolus. Data was acquired at five second intervals over a span of five min.
G ChIP assays of H3K18Ac enrichment at the indicated loci as described in Figure 5H. H3K18Ac enrichment adjacent to the induced DSB in control and SIRT7 depleted cells (bottom left), or in cells overexpressing SIRT7 in the presence or absence of PARP inhibitors (PARPi) (bottom right) (mean ± SEM; one of two independent experiments is shown).
*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.
SIRT7 promotes genome integrity and modulates non-homologous end joining DNA repair (2016)
Figure 7

Figure 7 - H3K18 deacetylation is required for efficient 53BP1 recruitment to chromatin.
A-H NHEJ activity in HT1080 or NIH3T3 cells stably overexpressing H3-WT, H3-K18Q or H3-K18R (naive, acetylated or deacetylated H3K18 residues, respectively).
A Random integration assay in HT1080 cells using a linearized pSMCV vector containing a puromycin resistance cassette (mean number of puromycin resistant colonies normalized by plating efficiency ± SEM; One representative transfection from 3 is shown).
B NHEJ repair assay using GFP expression-based reporter system described in Fig 5I (mean ± SEM of 3 independent experiments. See Fig EV5B for representative FACS dot plots).
C-D IF staining of NIH3T3 cells after IR (1Gy) and 1 hour chase. Cells were pulsed with EdU 30 min prior to fixation, stained with antibodies against γH2AX and 53BP1, and counterstained with DAPI. Representative images can be found in Fig EV5C. (C) Quantitation of the number of 53BP1 foci per nucleus, and (D) mean volume of 53BP1 foci (mean ± SEM of >30 cells per condition and cell cycle stage).
E Western blot analysis of 53BP1 in chromatin and non-chromatin fractions from NIH3T3 cells expressing H3-WT, H3-K18Q or H3-K18R vectors and exposed to IR (10Gy). One representative blot is shown from 3 independent experiments. Shown is 53BP1, GAPDH for fractionation control, and H3 for loading control.
F-H Same as in (C-D) except using antibodies against γH2AX (red) and RIF1 (magenta). (F) Representative images from cells in S-phase (EdU positive; scale bar 5µm). (G) Quantitation of the number of RIF1 foci per nucleus, and (H) mean volume of RIF1 foci (mean ± SEM of >30 cells per condition and cell cycle stage).
I 53BP1 enrichment at the indicated loci as described in Figure 5H in Scramble control or SirT7 knockdown cells expressing the indicated H3 constructs (mean ± SEM; one of two independent experiments).
J Western blot analysis of chromatin fractions from primary WT and SirT7-/- MEFs expressing the indicated H3 constructs and exposed to IR (10Gy). Shown are levels of 53BP1, SIRT7, H3-Myc, GAPDH for fractionation control, and H3 for loading control.
K Model for the role of SIRT7 in NHEJ DNA repair: SIRT7 is recruited to DNA damage sites, where it regulates the extent of histone H3K18 acetylation, which in turn is required for efficient 53BP1 focus formation and overall NHEJ-mediated DNA repair.
*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.
Fibrotic enzymes modulate wound-induced skin tumorigenesis (2021)
Figure 1

Figure 1: Dermal CAFs from wound-induced tumours exhibit a fibrotic gene signature. a. Schematic representation of experimental set-up for genome-wide fibroblast expression profiling.

b. Heatmap depicting mean expression of the top 20 upregulated genes of fibroblast isolated from wound-induced tumours (Pap) compared to fibroblasts from wild-type (WT) and inflamed (InvEE) back skin (n=3 per condition). PRSS35 is indicated in red as a top upregulated gene in CAFs.

c. To visualize DEGs between subsets of fibroblasts sorted from homeostatic (WT), inflamed (InvEE) or tumour-associated (Pap) skin, each gene was plotted in a hexagonal triwise diagram in which the position of a point represents the relative increased expression in one or two populations, whereas the distance from the origin represents the magnitude of expression. Genes that are 32-fold or more upregulated are plotted on the outer grid line. Grey dots in the centre of the triwise plot represent genes that are not differentially expressed. Genes associated with a fibrotic response are depicted in red.

d. Hexagonal triwise plots of genes belonging to the respective gene-ontology (GO) term, which were among the highest upregulated GO-terms. Dots in red represent genes that are differentially expressed between the three conditions.
Fibrotic enzymes modulate wound-induced skin tumorigenesis (2021)
Figure 3

Figure 3: PRSS35 is selectively upregulated in wound-induced tumours.

a. Log2 fold change of probe intensities of PRSS35 gene expression in different subsets of dermal fibroblasts observed in Affymetrix GeneChip® Mouse 430 2.0 array (n=3 per condition).

b. Hexagonal triwise plot depicting the PRSS35 gene expression level.

c. Relative mRNA levels of PRSS35 in lysates from normal skin (WT, n=4 mice), inflamed skin (InvEE, n=4 mice), wounds (d14pw, n=4 mice) and wound-induced papillomas (Pap, n=5 mice). (* p=0.0159; Mann-Whitney test). Data represent means of three technical replicates ± S.E.M.

d. Wound healing dynamics of wild-type (WT, n=10) and PRSS35-/- (n=11) mice after full-thickness wounding with an 8 mm punch biopsy. Wound size expressed as percentage of initial wound size (ns; Wilcoxon matched-pairs signed rank test ). Data represent means ± S.E.M.

e. Representative pictures of back skin of WT and PRSS35-/- mice at different days post-wounding (dpw). Scale bar: 1 cm.

f. H&E-stained skin sections of WT and PRSS35-/- wounds at different days post-wounding. Scale bars: 100 µm.
Fibrotic enzymes modulate wound-induced skin tumorigenesis (2021)
Figure 4

Figure 4: Mice lacking PRSS35 are more sensitive to wound- and chemically-induced carcinogenesis.

a. Incidence of wound-induced papilloma formation in InvEE PRSS35+/+ (WT; n=8) and InvEE PRSS35-/- (n=10) mice at site of wounding. (* p=0.0218; Gehan-Breslow-Wilcoxon test).

b. H&E-stained skin sections of wound-induced papillomas of InvEE PRSS35+/+ and InvEE PRSS35-/- mice. Scale bars: 100 µm.

c, d. Incidence of papilloma formation (ns: non-significant; Gehan-Breslow-Wilcoxon test) (c) and number of tumours per mouse (d) in wild-type (WT, n=17) and PRSS35-/- (n=22) mice treated with DMBA-TPA (*p=0.0171; Wilcoxon matched-paired rank test). Data represent means ± S.E.M.

e. Representative pictures of the back skin of WT and PRSS35-/- mice undergoing the DMBA-TPA treatment at week 15 and week 20 after the start of TPA treatment. White arrows indicate tumours. Scale bar: 1 cm.

f. Tumour size at week 20 after the start of TPA treatment. The biggest tumour of each mouse was measured (WT; n=8; KO; n=14; ns: Mann Whitney test). Data represent means ± S.E.M.
Fibrotic enzymes modulate wound-induced skin tumorigenesis (2021)
Figure 5

Figure 5: PRSS35 expression by primary fibroblasts is inhibited by TGF-β stimulation, and in vivo ablation of PRSS35 leads to an altered collagen deposition in skin wounds and tumours .

a. Relative mRNA expression levels of PRSS35 in primary fibroblast cultures isolated from WT and PRSS35-/- mice, untreated or after 24 hours stimulation with 10 ng/mL TGF-β1 (n>8 biological replicates per condition) (**** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means of three technical replicates ± S.E.M.

b. Relative mRNA expression levels of the indicated ECM genes in primary fibroblast cultures (col1a1: collagen type 1 alpha 1; αSMA, alpha smooth muscle actin; Cyr61: Cysteine-rich angiogenic inducer 61 (=CCN1)) in primary fibroblast cultures isolated from WT and PRSS35-/- mice, untreated or after 24 hours stimulation with 10 ng/mL TGFβ1 (n≥7 biological replicates per condition) (** p<0.01, *** p<0.001, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M.

c Relative mRNA expression levels of the indicated cytokines/growth factors in primary fibroblast cultures (n≥8 per condition) (VEGF-α, vascular endothelial growth factor-alpha; FGF2: basic fibroblast growth factor; FGF7: keratinocyte growth factor) (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M.

d. Picrosirius red staining of normal skin, day 14 post-wounding (d14pw) skin or wound-induced papillomas from WT and PRSS35 KO mice. Red colouring indicates thick fibres, green colouring indicates thin fibres. Scale bars: 200 µm.

e. Quantification of picrosirius red staining as total collagen density (upper panel) and ratio of red versus green picrosirius staining (lower panel). Using QuPath Bioimage analysis software pixels colouring red or green were determined per area. (n≥4 technical replicates per condition) (* p<0.05, ** p<0.01, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M.
Fibrotic enzymes modulate wound-induced skin tumorigenesis (2021)
Figure 6

Figure 6: PRSS35 is selectively upregulated in high-grade human squamous cell carcinomas.

a. Human skin sections were stained with a PRSS35-specific antibody (red) and counterstained with Dapi (n=5 per condition). Boxed areas are magnifications as shown in the lower panel and dotted line represents the epidermal-dermal boundary. Scale bars: 100 μm.

b. Quantification of PRSS35 positive signal by Volocity analysis (n≥4 per condition; ** p<0.01; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M.
A phosphatase-centric mechanism drives stress signaling response (2021)
Figure 2

Figure 2. Deletion of Cdc55 affects phosphorylome at the onset of hyperosmotic stress. (A) Scatter plot displaying log2 SILAC ratios of individual phosphorylation sites in the experiments setup SR on the y-axis and setup cdc55Δ on the x-axis. Black dots represent Hog1-dependent sites (ratio in setup SR ≥ 2, and affected by Hog1as-inhibition (ratio ≤ 0.5)) (Romanov et al., 2017). (B) Histogram displaying the SILAC ratios (log2) of stress-induced phosphorylation sites in setup cdc55Δ (upper panel) and setup rts1Δ (lower panel) (Hollenstein et al., 2020), stratified by their Hog1-dependence (grey area versus thick black line). (C) Schematic illustration of suggested model of Cdc55 regulation in the hyperosmotic stress response, with grey arrows signifying hypothetical relationships. (D) Line plot monitoring growth rate (y-axis) of indicated strains (colored lines) under different osmolarity conditions (x-axis). (E) Histogram displaying the SILAC ratios (log2) of stress-induced phosphorylation sites that are induced in setup cdc55Δ in two separate experiments. The blue histogram shows the distribution of fold-changes when IGO1 and IGO2 are deleted and cells are exposed to hyperosmotic stress (setup SR igo1∆igo2∆). The orange histogram shows the distribution of fold-changes after deletion of CDC55 and upon exposure to hyperosmotic stress (setup SR cdc55∆). The same set of sites was compared between the two experiments. (F) Scatter plot displaying log2 SILAC ratios of phosphorylation sites in the experiments setup SR on the x-axis and setup SR igo1∆igo2∆ on the y-axis. Phosphorylation sites that do not exhibit Cdc55-dependence are shown in grey, whereas Cdc55-dependent phosphorylation sites are highlighted in orange. We found an enrichment for Cdc55-dependent sites in the SR- and Igo1/Igo2-dependent set (Set1) (Fisher Exact test, p-value = 3.54 x 10-31, odds-ratio = 21.40). (G) Waffle plot showing the impact of different signaling mechanisms on the stress phosphoproteome. Each box corresponds to 1% of the entire set. Orange boxes represent the percentage of phosphosites regulated by PP2ACdc55, whereas grey boxes represent phosphosites regulated by Hog1 and downstream of the MAPK. Light purple boxes indicate the phosphosites that are neither regulated by Hog1 nor PP2ACdc55. Dependency was defined as log2 SILAC ratio below -1 or above 1. Rck2-dependency was extracted from (Hollenstein et al., 2020).
A phosphatase-centric mechanism drives stress signaling response (2021)
Figure 4

Figure 4. Inhibition of PP2ACdc55 by Igo1/2 is required for an efficient transcriptional response to hyperosmotic stress. (A) Schematic illustration of iAID-conditioned Cdc55 depletion system: Cdc55 is fused with an AID (Auxin-inducible degron) tag rendering the protein sensitive to auxin (Morawska and Ulrich, 2013; Nishimura et al., 2009). Expression of AID-Cdc55 is controlled by a TetOff promoter and is repressed in the presence of doxycycline. Upon addition of auxin Cdc55 is rapidly depleted. (B) Western-blot illustrating the depletion of AID-Cdc55 at various time points after addition of auxin (upper panel); Pgk2 serves as a negative control (lower panel). (C) Scatter plot of log2 fold-changes of overlapping phosphorylation sites from setup cdc55Δ (x-axis) and the Cdc55-depletion system (y-axis). Stress-independent sites are shown in grey, stress-induced sites are shown in black. The dotted lines mark two-fold thresholds. (D) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) under different strain conditions (i.e. presence/absence of IGO1/2 and induction of iAID-Cdc55 depletion). Measured time points after stress application are depicted on the x-axis, target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion at 30 minutes post-stress. (E) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress. Target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion. dep.: Cdc55 depleted, n.d.: Cdc55 non-depleted.
A phosphatase-centric mechanism drives stress signaling response (2021)
Figure 5

Figure 5. Transcriptional activity of Gis1 and Rph1 is activated by inhibition of PP2ACdc55. (A) Venn diagram of 14 transcription factors (as recovered from the YEASTRACT database (Teixeira et al., 2006, 2018)) with at least one phosphorylation site changing two-fold in setup SR (grey) or SR igo1∆igo2∆ (blue). For the six overlapping transcription factors the log2 SILAC ratios of phosphorylation sites are shown (y-axis) in the corresponding setups (color code as above). The first two box plots illustrate the distribution of log2 SILAC ratios of all phosphorylation sites quantified in each setup ('all'). Gis1 and Rph1 were found to be significantly different in their distributions (p-value < 0.01, t-test relative to "all"). (B) M-Track proximity assay of Cdc55 fused to the protA-H3-HA prey-tag and Gis1, respectively Rph1, tagged with the HKMT bait-tag expressed in a rim15∆ strain background. Background signal intensity was defined using a strain expressing only Cdc55-protA-H3-HA and no tagged bait protein (noHKMT). Positive control: Nup2-HKMT, CDC55-protA-H3HA. me3: antibody recognizing me3K9H3; HA: 12CA5 antibody (C) Mobility shift assays monitoring phosphorylation-induced mobility changes of Gis1 (two upper rows) and Rph1 (two lower rows) upon hyperosmotic stress in wild type, igo1∆igo2∆ and Cdc55-depleted cells (right-hand side). (D) Mobility shift assay of Gis1 and Rph1 across several time points after Cdc55 depletion (without exposure to hyperosmotic stress). Cdc28: loading control (shown in Figure EV5C). (E) Western blot phosphorylation shift assays of wild type Gis1-myc (upper panel) and Rph1-Flag (lower panel), and mutated forms Gis1-5A-myc (upper panel) and Rph1-5A-Flag (lower panel). The measurement was done using Phos-tag (Kinoshita et al., 2006) and at the indicated time points after exposure to hyperosmotic stress (0.5 M NaCl) using Pgk1 protein levels as a negative control. The location of the mutated residues is shown in the respective schematics. Red boxes: demethylase associated domains (JmjN and JmjC), blue box: zinc finger domain. (F) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress across indicated strains expressing either wild type or point-mutated forms of Gis1 and Rph1 in a gis1∆rph1∆ deletion background, with the empty vector as control.