Journal: Cell reports

Article Title: De novo assembly of RNA m 6 A modification factors into viral genome-associated nuclear bodies drives HCMV RNA accumulation

doi: 10.1016/j.celrep.2025.115826

Figure Lengend Snippet: (A) NHDFs treated with DMSO or cycloheximide (CHX) for 1 h were infected with HCMV Ad169-GFP, fixed at 6 hpi, probed simultaneously with WTAP and YTHDC1 antibodies, and visualized by confocal microscopy. Nuclei were identified by DAPI staining (blue in merged image). Scale bar, 20 μm. (B) Quantitation of WTAP and YTHDC1 NBs detected within the same domain in HCMV-infected cells treated with either DMSO or CHX using ImageJ macros ( n = 3, 100 cells per replicate) and shown as a percentage of NBs (±SD). (C) Relative abundance of WTAP and YTHDC1 in the same NBs measured by the MFI of indirect immunofluorescent signals within NBs ( n = 3, 100 cells per replicate), ±SD. (D) NHDFs infected with TB40/E were treated with water or α-amanitin for 0–6 hpi. Following fixation, WTAP or YTHDC1 were visualized by epifluorescence microscopy. Scale bar, 20 μm. (E) As in (D), except after 6 h, cultures were washed with PBS and treated with media + water or α-amanitin for an additional 3 h before fixing. Scale bar, 10 μm. (F) As in (D), except that water or α-amanitin was only added from 6 to 9 hpi before fixing. Scale bar, 10 μm. (G) NHDFs infected with HCMV Ad169-GFP were fixed at 6 hpi, probed simultaneously with antibodies recognizing WTAP and RNAPII phosphorylated at serine 5 (RNAPII S5P), and visualized by confocal microscopy. Nuclei were identified by DAPI staining. Scale bar, 10 μm. (H) IF signal intensity corresponding to WTAP and RNAPII S5P was plot profiled across two sections (indicated by numbered white lines in merged image in G) to determine the extent of co-localization.

Article Snippet: Cells were washed three times (5 min ea.) with PBS, incubated with Alexa Fluor (488, 555, or 647)-conjugated secondary antibody (Thermo Fisher Scientific #A11029, #A21429, #A21424, # PIA32733 , # PIA32787 , #A11008) in blocking solution (1 h, RT), washed three times with PBS, and covered with PBS containing a 1:5000 dilution of DAPI (Fisher Scientific #D1306) for 5 min, followed by a final wash with PBS and then mounted with mounting medium (Fisher Scientific # P36984 ).

Techniques: Infection, Confocal Microscopy, Staining, Quantitation Assay, Epifluorescence Microscopy

Journal: Cell reports

Article Title: De novo assembly of RNA m 6 A modification factors into viral genome-associated nuclear bodies drives HCMV RNA accumulation

doi: 10.1016/j.celrep.2025.115826

Figure Lengend Snippet: (A and B) NHDFs infected with HCMV TB40/E in the presence of CHX were fixed at 6 hpi. YTHDC1 was visualized by indirect IF, and HCMV IE1/2 mRNA (A) or HCMV RNA5.0 (B) was visualized using smFISH probes. Scale bar, 10 μm. (C and D) As in (A), except infected cells were identified using indirect IF for HCMV IE1/2 proteins, and the cellular GAPDH (C) or IFNB1 (D) mRNA was visualized using smFISH probes. (E) HCMV TB40/E-infected cells were permeabilized at 6 hpi and subsequently treated with PBS or RNase A before fixation and YTHDC1 or WTAP visualization. DAPI stained nuclei are included in the merged images. Scale bar, 20 μm.

Article Snippet: Cells were washed three times (5 min ea.) with PBS, incubated with Alexa Fluor (488, 555, or 647)-conjugated secondary antibody (Thermo Fisher Scientific #A11029, #A21429, #A21424, # PIA32733 , # PIA32787 , #A11008) in blocking solution (1 h, RT), washed three times with PBS, and covered with PBS containing a 1:5000 dilution of DAPI (Fisher Scientific #D1306) for 5 min, followed by a final wash with PBS and then mounted with mounting medium (Fisher Scientific # P36984 ).

Techniques: Infection, Staining