EMBO/soda-nel huggingface dataset:
NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres (2021)
Figure 7

Figure 7. hTR overexpression induces a DNA-PK and hnRNPA1-dependent depletion of RPA from ALT telomeres.

A. Representative images for co-localization between RPA32 (green) and single stranded C-rich telomeric sequences (ss-Telo-C, red FISH probe), in G2/M-synchronized cells. White arrows indicate co-localization events.

B. Quantification of images in (A). A total of at least 55 nuclei were scored from three independent experiments. Each dot represents the number of events per nucleus.

C. Quantification of images in (A) as the percentage of single stranded C-rich telomeric sequences associated with RPA32 to total single stranded C-rich telomeric sequences A total of at least 55 nuclei were scored from three independent experiments.

D. Representative images for co-localization between RPA32 or p-RPA32S33 (green) and telomeres (red FISH probe), in the indicated cell lines treated with siLuc or sihnRNPA1 for 48 h. White arrows indicate co-localization events.

E. Quantification of images in (D). A total of 85-135 nuclei were scored from three independent experiments. Each dot represents the number of events per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons.

F. Representative images for co-localization between RPA32 (green) and telomeres (red FISH probe) in the indicated cell lines treated with either DMSO or 10 μM NU7026 DNA-PK-inhibitor (DNA-PK-i) for 48 h. White arrows indicate co-localization events. Quantifications are shown on the right. A total of 53-84 nuclei were scored from three independent experiments. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons.

Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.
NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres (2021)
Figure 8

Figure 8. NHP2 regulates 53BP1 recruitment at damaged telomeres and IR-induced DSBs.

A. hTR and NHP2 mRNA expression in U2OS-hTR cells 72 h post-transfection with siLuc or siNHP2, normalized to ACTB mRNA and to SI14/TEL+ cells (n=2).

B. Western blot showing NHP2 protein levels in the indicated cells treated with siLuc or siNHP2, 72 h post-transfection. β-Actin served as loading control.

C. Representative images for co-localization between p-RPA32S33 (green) and telomeres (red FISH probe) in U2OS-hTR cells treated with control siLuc or siNHP2, 72 h post-transfection. White arrows indicate co-localization events. Quantification of images is shown on the right. Each dot indicates the number of co-localization events per nucleus (n=2). Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution.

D. Representative images for TIF quantification: co-localization between 53BP1 (green) and telomeres (red FISH probe), measured 72 h post-transfection with siLuc or siNHP2 in cells further treated with either DMSO or Doxorubicin (DOXO, 1.5 μM) for 2 h. White arrows indicate TIF. Quantification of TIF is shown on the right. A total of at least 100 nuclei were scored from two independent experiments. Each dot represents the number of TIF per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons.

E. Western blot analyses in U2OS and U2OS-hTR cells treated with the indicated siRNAs, 72 h post-transfection to detect total protein levels using the indicated antibodies. β-Actin and Vinculin served as loading controls.

F. Representative images for 53BP1 foci formation in U2OS and U2OS-hTR cells, 72 h post-transfection with siLuc or siNHP2, exposed to 1 Gy irradiation and fixed at various time points post-irradiation as indicated.

G. Quantification of images shown in (F). Each dot represents the number of 53BP1 foci per nucleus. A total of 114-140 nuclei were scored from two independent experiments. Statistical analyses were performed using two-way ANOVA Mixed effect model.

Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.
Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA damaging
chemotherapeutic agents (2019)
Figure 1

Figure 1. Chemotherapeutic drugs and irradiation deplete BRD7. (A) Western blot analysis of BRD7 and γH2AX protein levels in breast cancer cell lines: MCF7 parental or doxorubicin-resistant MCF7/ADR (ADR, 0.5μg/ml) and irradiation-resistant MCF7/IR (IR, 10Gy), MDA-MB-231 parental or doxorubicin-resistant MDA-MB-231/ADR (ADR, 0.5μg/ml) or cisplatin-resistant MDA-MB-231/DDP (1μg/ml), non-small lung cancer cell lines: A549 parental or cisplatin-resistant A549/DDP (1μg/ml). (B) MCF7, MCF7/ADR and MCF7/IR, MDA-MB-231 and MDA-MB-231/ADR cells were harvested and mRNA levels of BRD7 were determined by real-time PCR (n=3). Values are mean ± SEM and Error bars indicate SEM. (C and D) Western blot analysis of BRD7 protein levels in MDA-MB-468 and MDA-MB-231 cell after treatment with ADR (5 μM) or camptothecin (CPT) (1 μM) for different intervals (n=3). (E) Representative images of endogenous BRD7 (green) and γH2AX foci (red) in paraformaldehyde-fixed MDA-MB-468 cells after treatment with CPT (1 μM) for different intervals. Visualized by immunofluorescence using anti-BRD7 and Alexa Fluor 555 anti-γH2AX antibodies. DNA staining with DAPI; Scale bars, 2 μm. (F) Quantification of average fluorescence intensity of BRD7 of cells in (E). Error bars indicate SEM; n>100.
Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA damaging
chemotherapeutic agents (2019)
Figure 2

Figure 2. BRD7 interacts with PARP1. (A) Silver staining of the BRD7 complex separated by SDS-PAGE. HEK293T cells stably expressing SFB-tagged BRD7 were used for tandem affinity purification (TAP) of protein complexes. BRD7-interacting proteins, including PARP1 and PIK3R2, are indicated. (B) Table summarizes proteins identified by mass spectrometry analysis. (C and D) HEK293T cells transiently transfected with Flag-PARP1 and Myc-BRD7 for 24 h were lysed with RIPA buffer. Followed by immunoprecipitation (IP) using antibodies to either Myc (C) or Flag (D) conjugated to agarose followed by Western blot with the indicated antibodies (n=3). (E and F) HeLa and MDA-MB-231 cells were lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti-IgG, or BRD7 or PARP1 antibodies, and analysed by Western blot (n=3). (G) MDA-MB-231 cells were treated first with Olaparib (10 μM) for 6 h and lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti-IgG or PARP1 antibodies, and analysed by Western blot (n=3). (H and I) Association of endogenous BRD7 with PARP1 in HeLa cells was performed by co-immunoprecipitation using anti-BRD7 or anti-PARP1 antibody. HeLa cell were treated with CPT (1 μM, 1 h), followed by IP using indicated antibodies, and Western blot was performed. γH2AX was used as a marker of DNA damage induced by CPT (n=3).
Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA damaging
chemotherapeutic agents (2019)
Figure 3

Figure 3. BRD7 is ADP-ribosylated by PARP1 in vitro and in vivo. (A) HeLa cells were untreated or treated with CPT (1 μM) for 1 h followed by lysing with RIPA buffer and lysates were then immunoprecipitated using anti-IgG or anti-PAR antibodies and immunoblotted with the indicated antibodies (n=3). (B) HeLa cells were untreated or treated with CPT (1 μM) for 1 h and cellular lysates were immunoprecipitated using anti-IgG or anti-BRD7 antibodies and immunoblotted were performed using the indicated antibodies (n=3). (C) HeLa and 293T cells transfected with Myc-BRD7 plasmid for 24 h were lysed with RIPA buffer. Lysates were then immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies. Ribosylation levels of exogenous BRD7 were detected using anti-PAR antibody (n=3). (D) HeLa cells transfected with Myc-BRD7 plasmid. After 24 hours, cells were treated with either CPT (1 μM) or ADR (5 μM) combined with MG132 (10 μM) for indicated times. Cellular lysates were immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies (n=3). (E) HeLa PARP1 wild-type and PARP1 knockout cells were transfected with Myc-BRD7 for 24 h, lysates were subjected to immunoprecipitation using anti-Myc agarose and analysed by Western blot (n=3). (F) HeLa were transfected with BRD7 wild-type and various BRD7 mutant plasmids for 24 h, lysed with RIPA, followed by anti-Myc IP and Western blot with indicated antibody (n=3). (G) Ribosylation of BRD7 by PARP1 in vitro. Recombinant BRD7 was subjected to in vitro ribosylation either in absence or presence of biotin-labeled NAD+. Recombinant proteins were detected by indicated antibodies, and ribosylated proteins were determined with anti-biotin antibody (n=3). (H) PAR-binding motif of BRD7 is required for its ribosylation by PARP1. Recombinant Myc-BRD7-WT and Myc-BRD7-mutant were subjected to in vitro ribosylation assay and analysed by Western blot as indicated (n=3).
Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA damaging
chemotherapeutic agents (2019)
Figure 4

Figure 4. PARP1 regulates BRD7 protein stability through ubiquitination-dependent pathway. (A) HeLa cells were transfected with Flag-PARP1 and Myc-BRD7 for 24 h, then treated with MG132 (10 μM) for additional 4 h, and proteins were detected by Western blot with indicated antibodies (n=3). (B) HeLa cells were transfected with either scrambled or PARP1 siRNAs for 48 h, protein levels were detected by Western blot with the indicated antibodies (n=3). (C) Expression of BRD7, p21 and PARP1 measured in PARP1 wild-type and knockout HeLa cells using PARP1 sgRNA by Western blot (n=3). (D) MDA-MB-231 cell were treated with different concentration of PARG inhibitor PDD0017273 for 48 h, cell lysates were analysed by Western blot (n=3). (E) MDA-MB-231 cell were treated with 10 μM of PDD0017273 for indicated times, cell lysates were analysed by Western blot (n=3). (F and G) Wild-type and PARP1 knockout HeLa cells were incubated with 10 μg/ml cycloheximide (CHX) for the indicated times. Lysates were harvested and analysed by Western blot. Quantification of BRD7 protein are shown in F (n=3) and results represent mean ± SEM. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h of Control or sgPARP1 cells, respectively. (H) PARP1 wild-type and knockout HeLa cells were transfected with HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h, cells were lysed with RIPA buffer; followed by anti-BRD7 IP and analysed by Western blot with the indicated antibodies (n=3). (I) SFB-BRD7 stably overexpressing HeLa cells transfected with scramble or PARP1 siRNA for 24 h were transfected with vector or HA-ubiquitin (Lys48 or Lys63 only) for 24 h, and MG132 (10 μM) was added for an additional 4 h, lysed with RIPA, subjected to IP using S tag beads followed by Western blot (n=3). (J) HeLa cells were co-transfected either wild-type or BRD7 mutant with Flag-PARP1 for 24 h and analysed by Western blot (n=3). (K) HeLa cells were co-transfected with either wild-type or BRD7 mutant plus HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h and lysed with RIPA buffer, followed by anti-Myc agarose IP and analysed by Western blot with the indicated antibodies (n=3).
Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA damaging
chemotherapeutic agents (2019)
Figure 5

Figure 5. E3 ligase RNF146 promotes BRD7 ubiquitination and degradation. (A) HeLa and MDA-MB-231 cells were transfected with either scrambled or RNF146 siRNAs (48 h). Protein levels were detected by Western blot. PTEN, which is a known RNF146-interacting protein, was included as a positive control (n=3). (B) HeLa cells were transfected with either scrambled or RNF146 siRNAs for 48 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for the indicated periods of time. Lysates were harvested and analysed by Western blot (n=3). (C) Quantification of BRD7 protein levels from B, n=3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h. (D) Control and RNF146 stably overexpressing HeLa cells were transfected with Myc-BRD7-WT for 24 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for indicated periods of time. Lysates were subjected to Western blot analysis (n=3). (E) Quantification of BRD7 protein levels from D, n=3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h. (F) HeLa cells transfected with either RNF146 or control siRNA for 24 h followed by transfection of HA-ubiquitin for another 24 h; MG132 (10 μM) was added for 4 h and lysed with RIPA, followed by anti-BRD7 IP and analysed by Western blot with the indicated antibodies (n=3). (G) HeLa cells stably expressing RNF146 were transfected with HA-ubiquitin for 24 h; MG132 (10 μM) was added for an additional 4 h, followed by anti-BRD7 IP and Western blot (n=3).
Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA damaging
chemotherapeutic agents (2019)
Figure 6

Figure 6. E3 ligase RNF146 interacted with BRD7 in a PARP1-dependent manner. (A and B) HeLa cells transfected with the indicated plasmids; after 24 h, cells were lysed with RIPA buffer followed by immunoprecipitation (IP) using either anti-Myc or anti-Flag agarose and Western blot with indicated antibody (n=3). (C) HeLa and MDA-MB-231 cells were lysed with RIPA buffer, and lysates were subjected to IP using either anti-IgG, anti-BRD7 or anti-RNF146 antibodies followed by analysis with Western blot (n=3). (D) Association of endogenous BRD7 with RNF146 in HeLa cells was performed by co-IP using anti-RNF146 antibody. Cell lysates were incubated with protein-G agarose beads conjugated with indicated antibodies, and Western blot was performed (n=3). (E) HeLa cells with wild-type or PARP1 knockout were transfected with Myc-BRD7-WT for 24 h, their lysates were subjected to IP using anti-Myc agarose and analysed by Western blot with indicated antibodies (n=3). (F) HeLa cells stably expressing Flag-RNF146 were transfected with either Myc-BRD7-WT or Myc-BRD7-mutant for 24 h, lysates were subjected to IP using anti-Flag agarose and analysed by Western blot with indicated antibodies (n=3).
Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA damaging
chemotherapeutic agents (2019)
Figure 7

Figure 7. Inhibiting PARylation of BRD7 enhanced sensitivity of cancer cells to DNA damaging agents. (A) MDA-MB-231 cells were transfected twice with indicated siRNAs, the absorbance values of indicated cell lines was measured at different time points by CCK8 assay (n=3). Data from three independent experiments are expressed as mean ± SEM. Error bars indicate SEM. (B) Cell lysates treated in A were analysed by western blot with indicated antibodies (n=3). (C) MDA-MB-231 cells were treated with Olaparib (10 μM) combined with ADR (0.5 μM), CPT (0.5 μM) or VP16 (10 μM) for 72 h, and CCK8 activity of the cells was detected (n=3). Results represent mean ± SEM of three experiments and Error bars indicate SEM. N.S., not significant; **P < 0.01, Student's t test. (D) MDA-MB-231 cells were treated with Olaparib (10 μM) combined with ADR (0.5 μM) for 72 h, and their lysates were subjected to IP using anti-BRD7 antibodies and analysed by Western blot with indicated antibodies (n=3). (E) MDA-MB-231 cells were transfected twice with BRD7 siRNAs, and these cells were subjected to CCK8 assay after treatment with Olaparib (10 μM) combined with either ADR (0.5 μM) or VP16 (10 μM) for 72 h (n=3). Results represent mean ± SEM of three experiments and Error bars indicate SEM. N.S., not significant; **P < 0.01, Student's t test. (F) Resistance of BRD7-depleted cells to Olaparib was reversed by the expression of wild-type BRD7 but not mutant BRD7. shRNA-resistant BRD7 wild-type and BRD7 mutant plasmids were transduced into BRD7-depleted MDA-MB-231 cells, and cell viability was determined as indicated above (n=3). Results represent mean ± SEM of three experiments and Error bars indicate SEM. N.S., not significant; **P < 0.01, Student's t test.
A new patient derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan (2019)
Figure 1

Fig. 1.

Functional glycosylation of α-dystroglycan and characterization of dystroglycanopathy patient-specific iPSCs.

(A) Current model of the core M3 functional glycan structure on α-dystroglycan and enzymes involved in its synthesis. ECM ligands, such as laminins, bind to the Xyl-GlucA disaccharide repeats (IIH6 epitope). Man, Mannose; GlcNAc, N-Acetylglucosamine; GalNAc, N-Acetylgalactosamine; Rbo5P, ribitol-5-phosphate; Xyl, xylose; GlcA, glucuronic acid.

(B) Representative images of immunostaining demonstrate that FKRP-iPSCs express specific pluripotency-associated markers, including NANOG, OCT4, Tra-1-60 and SSEA4.

(C) FKRP-iPSCs have a normal karyotype.

(D) In vitro differentiation of FKRP-iPSCs to cells representing ectoderm (β-III Tubulin, Tuj1), mesoderm (SMA, smooth muscle actin) and endoderm (AFP, α-fetoprotein).

Data information: Scale bars, 50 μm.
A new patient derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan (2019)
Figure 2

Fig. 2.

Targeted gene correction of FKRPA455D-iPSCs by CRISPR/Cas9-mediated genome editing.

(A) Cas9 protein and the specific sgRNA targeting the human FKRP locus. The FKRP c.1364C>A (p.A455D) mutation is 43 bases upstream of the sgRNA target sequences. Red arrowheads indicate putative cleavage site. PAM, protospacer-adjacent motif.

(B) A schematic diagram shows the genome editing strategy based on CRISPR/Cas9-stimulated homologous recombination, followed by positive selection with puromycin and negative selection with FIAU. Homology left and right arms on the targeting donor vector are indicated as black boxes, flanking the piggyBac (PGK-puro∆tk) selection cassette, which is under the control of PGK promoter. PCR genotyping primers are shown as blue arrows. Note that the TTAA sequences are designed to accommodation the selection cassette excision sites, yet code the same amino acids.

(C) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones (5D17, 5D23 and 3B17), compared with their parental FKRPA455D-iPSCs. Selection cassette excision sites are identified in the corrected-iPSC lines.

(D) CRISPR/Cas9 corrected-iPSC lines show a normal karyotype.
A new patient derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan (2019)
Figure 3

Fig. 3.

Characterization of NSCs and cortical neurons derived from FKRP- and CRISPR/Cas9 corrected-iPSCs.

(A and B) Representative images of NSCs derived from FKRP- and corrected-iPSC lines expressing SOX1, SOX2 and nestin.

(C and D) Quantification of percentage of SOX1+ (C) and SOX2+ (D) cells in culture. The efficiency of neural induction is more than 99% in FKRP- and corrected-iPSC (5D17, 5D23 and 3B17) lines. Data are mean ± s.d. n = 4 technical replicates.

(E and F) FKRP- and corrected NSC lines can be further differentiated to cortical neural progenitor cells, expressing PAX6, OTX2 and vimentin.

(G-I) Quantification of percentage of PAX6+ (G) and OTX2+ (H) cells in culture. About 91-98% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express PAX6 (G). About 93-96% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express OTX2 (H). Of the OTX2+ population, about 60-67% cells are also Ki67+ cycling progenitors (I). Data are mean ± s.d. n = 4 technical replicates.

(J and K) Glutamatergic projection neurons derived from FKRP and corrected (5D17, 5D23 and 3B17) progenitor cells. The vast majority of neurons contain vGlut1+ punctae in their neurites (labelled by Tuj1). Right panels are enlarged images from the insets of left panels.

Data information: Scale bars, 50 μm.
A new patient derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan (2019)
Figure 4

Fig. 4.

Functional glycosylation of α-dystroglycan in cortical neurons derived from CRISPR/Cas9-corrected iPSCs.

(A) Molecular weight of glycosylated α-dystroglycan (IIH6 epitope) in WT-iPSC derived cortical neurons is similar to that in the mouse brain (~120 kDa) and lower than that in the mouse muscle (~156kDa). IIH6 reactivity was almost not detected in cortical neurons derived from FKRP-iPSCs. Note that the β-Actin antibody does not cross-react with the muscle sample.

(B) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC-derived cortical neurons. Intensities of IIH6 reactivity are normalized to β-Actin expression, indicating mean ± s.d. (n = 3 biological replicates; t-test; ** p<0.01)

(C) Representative laminin overlays show that targeted gene correction restores laminin-binding activity in iPSC-derived cortical neurons. Intensities of laminin-binding activity are normalized to β-dystroglycan, indicating mean ± s.d. (n = 3 biological replicates; t-test; * p<0.05).