Journal: Nucleic Acids Research

Article Title: Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells

doi: 10.1093/nar/gkv1019

Figure Lengend Snippet: 53BP1 and RAP80 altered foci accumulation in FA pathway-deficient cells. ( A ) Representative images of RAP80 (red) and 53BP1 (green) foci in nuclei (DAPI stained, blue) of FANCC-mutated (PD331 FANCC −/− ) and -corrected (PD331 corr ) cells 24 h after MMC exposure (1 μg/ml/1 h). White line: 2 μm. ( B ) Representative images of RIF1 (red) and 53BP1 (green) foci in nuclei (DAPI stained, blue) of FANCC-mutated (PD331 FANCC −/− ) and -corrected (PD331 corr ) cells 24 hours after MMC exposure (1 μg/ml/1 h). White line: 2 μm. ( C ) 53BP1 and RAP80 foci quantificationin untreatyed cells (0 h) and at different time points (1, 6 and 24 h) following MMC exposure (1 μg/ml/1 h). Histograms represent the mean of three independent experiments. Error bars indicate S.D. The data were analyzed by a Student's t -test; ** indicates P < 0.01. ( D ) Size (arbitrary unit, a.u.) of 53BP1 foci in untreated conditions and 24 h after MMC exposure (1 μg/ml/1 h) in FANCC-mutated (PD331 FANCC −/− ) and -corrected (PD331 corr ) cells. The cell size was determined using the ImageJ software. The values on the histogram represent the mean of three independent experiments; at least 50 cells were scored each time. Error bars indicate S.D. Data were analyzed by a Student's t -test; *** indicates P < 0.001. ( E ) Western blot showing the efficiency of the siRNA against 53BP1 and RAP80, as observed 72 h after transfection, in corrected (PD331 corr) and FA-C (PD331 FANCC −/− ) cells. Vinculin was used as a loading control. siCtrl (control) indicates cells transfected with an untargeted siRNA. ( F ) Representative images of RAP80 (red) and 53BP1 (green) foci in nuclei (DAPI stained, blue) of FANCC-corrected cells (PD331 corr ) after depletion of 53BP1 or RAP80 by siRNA transfection. Cells were treated with MMC (1 μg/ml/1 h) and analyzed 24 h later. White line: 2 μm. siCtrl (control) indicates cells transfected with an untargeted siRNA. ( G ) Quantification of RAP80 foci-positive cells after transfection with an untargeted siRNA (siCtrl, control), si53BP1 or siMDC1 in FANCC-mutated (PD331 FANCC −/− ) and -corrected (PD331 corr ). Cells presenting more than 5 foci were considered positive. Data represent the mean of two independent experiments with similar results. ( H ) Left. Representative images of BRCA1/RAP80 interaction dots detected with the Proximity Ligation Assay in nuclei (DAPI stained, blue) in FANCC-mutated (PD331 FANCC −/− ) and -corrected (PD331 corr ) cells under untreated conditions or 24 h following MMC exposure (1 μg/ml/1 h). White line: 8 μm. Right. BRCA1/RAP80 interaction dots quantification 24 h after MMC exposure (1 μg/ml/1 h). Histograms represent the pooled data from three independent experiments. Quantification was conducted using ImageJ software. ( I ) Left. Representative images of BRCA1/CtIP (left) interaction dots detected with the Proximity Ligation Assay in nuclei (DAPI stained, blue) in FANCC-mutated (PD331 FANCC −/− ) and -corrected (PD331 corr ) cells under untreated conditions or 24 hours following MMC exposure (1 μg/ml/1 h). White line: 8 μm. Right. BRCA1/CtIP interaction dots quantification 24 h after MMC exposure (1 μg/ml/1 h). Histograms represent the pooled data from three independent experiments. Quantification was conducted using ImageJ software. ( J ) Representative images of FANCD2 foci (red), RAP80 (red) or 53BP1 (green) in PD20 corr ( FANCD2 -corrected cells), PD20 ( FANCD2 −/- cells) and PD20 FANCD2 K561R (PD20 cells expressing a K561R, non-ubiquitinable FANCD2) cells. White line: 2 μm. Histograms represent the number of RAP80 (red) and 53BP1 (green) foci in the three different cell lines in the left. Data represent the mean of two independent experiments with similar results.

Article Snippet: Cells grown on glass cover slips were fixed in 4% formaldehyde supplemented with 0.1% Triton X100 for 10 min at room temperature before permeabilization in 0.5% Triton X100 for 5 min. After blocking with 3% BSA in PBS containing 0.05% Tween 20, the cells were stained for 1 h in blocking solution with antibodies against FANCD2 (ab2187, Abcam), PhosphoDNA-PKcs (Ser2056 ab18192, Abcam), MDC1 (ab11169, Abcam), γH2AX (JBW301, Upstate), 53BP1 (MAB3802, Millipore), Mre11 (GTX70212, GeneTex), Rad51 (PC130, Calbiochem), RIF1 (A300–569A, Bethyl), RAP80 (NBP1–87156, Novus) and acetylated lysine (MA1–2021, Thermo).

Techniques: Staining, Software, Western Blot, Transfection, Control, Proximity Ligation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells

doi: 10.1093/nar/gkv1019

Figure Lengend Snippet: H4K16 acetylation is impaired in MMC-treated FA cells due to altered TIP60 relocalization to damaged chromatin. ( A ) Western blot analysis of H4K16 acetylation following siRNA-mediated FANCC downregulation in HeLa cells (left) and in FANCC-corrected (PD331 corr ) or -deficient (PD331 FANCC −/− ) cells (right) under untreated conditions (NT) and 24 h after exposure to MMC (1 μg/ml/1 h). FANCD2 is used as control of the loss-of-function of the FANCcore complex activity in absence of FANCC. Vinculin and H3 are presented as loading control. ( B ) Quantitative analysis from four independent experiments of the levels of H4K16 acetylation observed by WB as in (A). In each experiment, H4K16 acetylation was measured by densitometry and normalized relatively to the total H3 levels. Error bars indicate S.D. Statistical analysis: *** indicates P < 0.001 using a Student's t- test. ( C and D ) Western blot analysis of H4K16 acetylation in siFANCC-, siFANCD2- or siFANCA-transfected HeLa cells under untreated conditions (NT) and 24 h after exposure to MMC (1 μg/ml/1 h). H3 is presented as loading control. ( E ) Western blot analysis of the effect of TSA treatment (5 μM) on the levels of H4K16 acetylation in untreated or MMC-treated FANC-corrected (PD331 corr ) and -deficient (PD331 FANCC −/− ) cells. Actin is presented as loading control. ( F ) Representative images of pDNA-PK (red) and MRE11 (green) foci in nuclei (DAPI stained, blue) of FANCC-corrected ( corr ) or -deficient ( FANCC −/− ) cells. The cells were pre-treated with DMSO or TSA (5 μM) for 5 h before MMC (200 ng/ml) exposure and analyzed 24 h later. White line: 2 μm. ( G ) Quantitative analysis of the data presented in (F). Histograms are the mean of three independent experiments. ( H ) Western blot analysis of the subcellular distribution of TIP60 in untreated and MMC-treated FANCC-proficient (Ctrl or corr ) or -deficient (siFANCC or FANCC −/− ) cells 24 h after exposure to MMC (1 μg/ml/1 h). GAPDH and LaminB1 are used as loading controls. ( I ) Quantitative analysis from four independent experiments of TIP60 relocalization to the chromatin in HeLa cells 48 h after siRNA-mediated FANCC downregulation (left panel) or in FANCC-corrected (PD331 corr ) or deficient (PD331 FANCC −/− ) cells (right panel). Error bars indicate S.D. * indicates P < 0.05 and *** P < 0.001 using a Student's t -test. ( J ) Simplified model summarizing our data.

Article Snippet: Cells grown on glass cover slips were fixed in 4% formaldehyde supplemented with 0.1% Triton X100 for 10 min at room temperature before permeabilization in 0.5% Triton X100 for 5 min. After blocking with 3% BSA in PBS containing 0.05% Tween 20, the cells were stained for 1 h in blocking solution with antibodies against FANCD2 (ab2187, Abcam), PhosphoDNA-PKcs (Ser2056 ab18192, Abcam), MDC1 (ab11169, Abcam), γH2AX (JBW301, Upstate), 53BP1 (MAB3802, Millipore), Mre11 (GTX70212, GeneTex), Rad51 (PC130, Calbiochem), RIF1 (A300–569A, Bethyl), RAP80 (NBP1–87156, Novus) and acetylated lysine (MA1–2021, Thermo).

Techniques: Western Blot, Control, Activity Assay, Transfection, Staining

Journal: Scientific Reports

Article Title: Evaluation of polyclonal antibodies raised in rabbits against dengue NS1 antigen

doi: 10.1038/s41598-026-35952-1

Figure Lengend Snippet: Detection of recombinant dengue NS1 antigen using Rabbit Polyclonal Antibodies against the Dengue NS1 antigen (RPAD). The ELISA plate was coated with rDNS1Ag (100 ng/well). To this, polyclonal antibodies raised in rabbits were added at different dilutions (1:50 to 1:50000 dilutions). The antibody response was detected by the secondary antibody, anti-rabbit-HRP (1:50000; 50 µl/well). Absorbance (OD) was measured at 452 nm. ns - statistically not significant ( P > 0.05). A – Animal; D – concentration of rDNS1Ag.

Article Snippet: For raising polyclonal antibody, a pair of healthy rabbits maintained at the Animal house facility of ICMR-VCRC, Puducherry, were immunized with 10 μg and 20 μg recombinant Dengue virus 2 NS1 antigen (A1-D10 μg and A2-D20 μg, respectively) (DG R&D Systems, Inc., Canada, Catalogue No. 9439) emulsified in Freund’s incomplete adjuvant.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Evaluation of polyclonal antibodies raised in rabbits against dengue NS1 antigen

doi: 10.1038/s41598-026-35952-1

Figure Lengend Snippet: The RPAD is sensitive to detect all the four serotypes of dengue virus. The ELISA plate was coated with rDNS1Ag (100 ng/well) of different serotypes of DENV (1–4). To this, polyclonal antibody, RPAD (1:5000) was added. The binding of polyclonal antibodies was detected by anti-rabbit-HRP. Absorbance (OD) was measured at 452 nm in an ELISA reader. D1 to D4 –NS1 antigen of Dengue virus serotypes, D2 (R&D) – NS1 antigen used for immunizing the rabbits. ns- statistically not significant ( P > 0.05). A – Animal; D – concentration of rDNS1Ag.

Article Snippet: For raising polyclonal antibody, a pair of healthy rabbits maintained at the Animal house facility of ICMR-VCRC, Puducherry, were immunized with 10 μg and 20 μg recombinant Dengue virus 2 NS1 antigen (A1-D10 μg and A2-D20 μg, respectively) (DG R&D Systems, Inc., Canada, Catalogue No. 9439) emulsified in Freund’s incomplete adjuvant.

Techniques: Virus, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Evaluation of polyclonal antibodies raised in rabbits against dengue NS1 antigen

doi: 10.1038/s41598-026-35952-1

Figure Lengend Snippet: Evaluation of cross-reactivity of RPAD with the NS1 antigen of other Flaviviruses. The ELISA plate was coated with NS1 Ag of flavivirus (100 ng/well). To this, polyclonal antibody, RPAD (1:5000) was added. The binding of RPAD was detected by anti-rabbit-HRP. JE-Japanese Encephalitis Virus; TBEV Tick-borne Encephalitis Virus; WNV-West-Nile Virus; YFV-Yellow Fever Virus, D2 (R&D) – NS1 antigen used for immunizing the rabbits. ns- statistically not significant ( P > 0.05). A – Animal; D – concentration of rDNS1Ag.

Article Snippet: For raising polyclonal antibody, a pair of healthy rabbits maintained at the Animal house facility of ICMR-VCRC, Puducherry, were immunized with 10 μg and 20 μg recombinant Dengue virus 2 NS1 antigen (A1-D10 μg and A2-D20 μg, respectively) (DG R&D Systems, Inc., Canada, Catalogue No. 9439) emulsified in Freund’s incomplete adjuvant.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Virus, Concentration Assay