Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

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Article Snippet: Following incubation with serum dilutions (1:50), plates were washed and incubated with horseradish peroxidase-conjugated goat anti-human IgG (1:2000; Invitrogen #A18805) for 1 hour.

Techniques: Recombinant, Software

Journal: Epigenetics & Chromatin

Article Title: PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

doi: 10.1186/s13072-023-00501-x

Figure Lengend Snippet: Increased levels of ac4C RNAs after UV irradiation are identical in the G1, S, and G2 phases of the cell cycle. The density of ac4C RNA was detected in A non-irradiated HeLa Fucci cells and B UVA-irradiated HeLa Fucci cells, stably expressing RFP-tagged cdt1 (red) in the G1 phase and GFP-tagged geminin (green) in the G2 phase of the cell cycle. The S phase is characterized by RFP-cdt1 and GFP-geminin positivity. Scale bars show 5 µm. C Quantification of the fluorescence intensity of ac4C RNA is shown in panels A and B

Article Snippet: After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer, at 4 °C (dilution 1:2000 or 1:1000).

Techniques: Irradiation, Stable Transfection, Expressing, Fluorescence

Journal: Epigenetics & Chromatin

Article Title: PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

doi: 10.1186/s13072-023-00501-x

Figure Lengend Snippet: Increased levels of ac4C RNAs after UV-irradiation in early and late telophase of mitotic cells. The distribution profile of ac4C RNA (green fluorescence) was studied in mitotic cells of A non-irradiated MEF cells and B UV-irradiated MEFs. The following mitotic phases were distinguished: prophase, prometaphase, metaphase, early anaphase, late anaphase, early telophase, and late telophase. DNA was stained by DAPI (blue), and α-tubulin is shown in red fluorescence. Scale bars show 15 µm

Article Snippet: After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer, at 4 °C (dilution 1:2000 or 1:1000).

Techniques: Irradiation, Fluorescence, Staining

Journal: Epigenetics & Chromatin

Article Title: PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

doi: 10.1186/s13072-023-00501-x

Figure Lengend Snippet: Recruitment of ac4C RNAs to UVA- and UVC-damaged chromatin. A Local microirradiation by 355-nm laser line showed that ac4C RNA recognizes UVA-microirradiated chromatin immediately after laser irradiation. In the later stages of DDR, 11–45 min post-irradiation, the ac4C RNA signal at DNA lesions was reduced. Scale bars are 5 µm. B , C Dot blot analysis of ac4C RNA documents levels of ac4C in total RNA isolated from non-irradiated, UVA-, and UVC-irradiated MEF cells. B shows the level of ac4C in RNAs (#ab252215, Abcam) studied 5 min and 30 min after UVC irradiation, and panel C documents the density of ac4C in RNAs (#ab252215, Abcam) analyzed 15 min after UVA and UVC irradiation. Negative controls (samples not incubated with the primary antibody) are shown. After fractionalization, it was observed that both large RNAs and small RNAs were notably acetylated on N4-cytidine when the cells were exposed to UVC light for 5 min, while 15 min post-irradiation, the highest level of ac4C was on small RNAs. Quantification of the density of dot spots is shown for both panels B and C . D Representative anti-ac4C dot blot (#ab252215, Abcam) performed on N4-acetylcytidine (NA05753, Biosynth). Chemical deacetylation was induced by hydroxylamine (50 mM, pH7, 65 °C, 1 h) . The specificity of N4-acetylcytidine (NA05753, Biosynth) was verified by antibody against m 6 A in RNA (#202 111, SYSY Antibodies). E Mass spectrometry data on ac4C in total and large RNAs studied in control, non-treated cells, and cells exposed to UVA, UVC light, and γ-rays. Cells were also treated with actinomycin D (ActD). E shows the mean ± standard deviations (SD) for three biological replicates (n = 3). Asterisks (*) show p ≤ 0.05, calculated using the two-tailed Student's t-test, indicating statistically significant differences in the level of ac4C RNA

Article Snippet: After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer, at 4 °C (dilution 1:2000 or 1:1000).

Techniques: Irradiation, Dot Blot, Isolation, Incubation, Mass Spectrometry, Control, Two Tailed Test

Journal: Epigenetics & Chromatin

Article Title: PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

doi: 10.1186/s13072-023-00501-x

Figure Lengend Snippet: A high density of ac4C RNA is inside nucleoli on non-irradiated cells, and ac4C RNA colocalizes with fibrillarin. A , B Ac4C RNAs colocalize with fibrillarin-positive regions of nucleoli in non-irradiated cells. The same protein-RNA colocalization was observed in MEFs treated with actinomycin D, an inhibitor of RNA pol I. In these cells, actinomycin D treatment caused a crescent-like morphology of nucleoli, visualized by antibodies against fibrillarin. Analysis was also performed in UVA-microirradiated cells. Scale bars are 5 µm

Article Snippet: After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer, at 4 °C (dilution 1:2000 or 1:1000).

Techniques: Irradiation

Journal: Epigenetics & Chromatin

Article Title: PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

doi: 10.1186/s13072-023-00501-x

Figure Lengend Snippet: UVC light caused an accumulation of ac4C RNA into well-visible foci in a later stage of DDR. A In non-irradiated control cells, relatively high ac4C RNA positivity was observed in nucleoli (detected using immunostaining by the use of an antibody against fibrillarin). UVC irradiation increased the level of ac4C RNA in the whole nucleoplasm (the most marked changes were 5–20 min post-irradiation). MEFs analyzed 20–120 min after UVC irradiation were characterized by ac4C RNA reorganization into well-visible and ac4C RNA-dense tiny foci. Scale bars showed 5 µm. B Quantification shows the fluorescent intensity (FI) of ac4C RNA in the nucleoplasm (green) compared with fibrillarin-positive regions of nucleoli (red) and DAPI-stained DNA (blue). Quantification by LAS X software was performed across the green lines, shown in panel A. C Box plot graphs display the absolute intensity of ac4C RNA in the nucleoplasm (nucleus), ****p ≤ 0.0001 (ANOVA One-Way test). D Box plot graphs show the total intensity of fluorescently-stained ac4C RNA in nucleoli, ****p ≤ 0.0001, **p ≤ 0.01. E Box plot graphs depict the ratio of the fluorescent intensity of ac4C RNA occupying nucleoli and the nucleoplasm (whole nucleus), ****p < 0.0001

Article Snippet: After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer, at 4 °C (dilution 1:2000 or 1:1000).

Techniques: Irradiation, Control, Immunostaining, Staining, Software

Journal: Epigenetics & Chromatin

Article Title: PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

doi: 10.1186/s13072-023-00501-x

Figure Lengend Snippet: Recruitment of ac4C RNA to UVA-damaged chromatin is PARP-dependent. A UVA irradiated MEFs as a whole-cell population, and B microirradiated MEFs by the use of a 355-nm UVA laser. Local laser microirradiation showed that ac4C RNA did not accumulate to DNA lesions when the cells were treated with the PARP inhibitor. DNA damage was detected by antibodies against ATM and γH2AX. Scale bars are 5 µm. C anti-ac4C dot blot a (#ab252215, Abcam) in samples irradiated by UVC, treated by PARP inhibitor and treated with both PARP inhibitor and UVC irradiation. Panel b shows the quantification of the dot blot from ( a )

Article Snippet: After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer, at 4 °C (dilution 1:2000 or 1:1000).

Techniques: Irradiation, Dot Blot

Journal: Epigenetics & Chromatin

Article Title: PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

doi: 10.1186/s13072-023-00501-x

Figure Lengend Snippet: NAT10-independent recruitment of ac4C RNA to UVA-damaged chromatin. A Nuclear distribution of NAT10 (red) and ac4C RNA (green) in physiological conditions. MCF7 cells were studied instead of MEFs as antibodies are only available against the human epitope. B Local laser microirradiation showed that NAT10 acetyltransferase (red) does not recruit to DNA lesions (positive on ac4C RNA; green) induced in MCF7 cells. Scale bars are 5 µm. C Western blots showed no changes in NAT10 levels in cells exposed to UVA and UVC light. An effect of UV- as well as γ-radiation, was confirmed by an increased γH2AX level. D shows western blot results on the level of NAT10 and γH2AX in NAT10 (wt) and NAT10 (dn) cells. Western blot data were normalized to the level of α-tubulin, and proteins were loaded following the identical total protein levels. E A representative anti-ac4C dot blot (#A18806, Abclonal) was performed to study total, long, and small RNA in NAT10 (wt) and NAT10 (dn) cells. F Quantification of dot blot results from E is shown in panel F. Asterisks (*) indicate a statistically increased level of ac4C in RNA. G The level of ac4C RNA (green) and NAT10 (red) in non-irradiated control and UVA- or UVC-irradiated whole populations of a NAT10 (wt) and b NAT10 (dn) HeLa cells. H Box plot graphs display the absolute intensity of ac4C RNA in the nucleoplasm (nucleus), ***p ≤ 0.001 (ANOVA One-Way test). I Box plot graphs show the total intensity of fluorescently stained ac4C RNA in nucleoli, ***p ≤ 0.001, **p ≤ 0.01. J Box plot graphs depict the ratio of the fluorescent intensity of ac4C RNA occupying nucleoli and the nucleoplasm (whole nucleus), ***p < 0.001, **p ≤ 0.01. K The level of ac4C RNA (green) and NAT10 (red) in microirradiated a NAT10 (wt) and b NAT10 (dn) HeLa cells

Article Snippet: After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer, at 4 °C (dilution 1:2000 or 1:1000).

Techniques: Western Blot, Dot Blot, Irradiation, Control, Staining

Journal: Epigenetics & Chromatin

Article Title: PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

doi: 10.1186/s13072-023-00501-x

Figure Lengend Snippet: NHEJ independent recruitment of ac4C RNA to microirradiated chromatin. A 3D-projection of 60 confocal sections shows the high density of ac4C RNA (red) in microirradiated chromatin and in the proximity of nucleoli (visualized by GFP-tagged UBF1/2) ). Analysis was performed using immortalized wild type iMEFs. The 3D projection and density of ac4C RNA and GFP-UBFs were studied using LEICA AF software. B ac4C RNA was recruited to microirradiated chromatin in 53BP1 double null (dn) MEFs, in a similar density as shown in panel (A) for the wt MEFs. The scale bars show 5 µm. C ac4C RNA significantly accumulated at microirradiated chromatin of RIF1 (wt) and RIF1 double null (dn) HCT116 cells. The scale bar shows 2 µm

Article Snippet: After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer, at 4 °C (dilution 1:2000 or 1:1000).

Techniques: Software