Journal: Nature communications
Article Title: Nuclear retention of unspliced HIV-1 RNA as a reversible post-transcriptional block in latency.
doi: 10.1038/s41467-025-57290-y
Figure Lengend Snippet: Fig. 4 | Nuclear retention of US HIV-1 RNA in PBMCs from 22 ART-treated PWH. a PBMCs from healthy donors were left untreated or were treated with PHA [1.66 µg/ml] and after 24 h collected for immunoblotting with the use of anti- MATR3, anti-MTR4, anti-αtubulin and anti-GAPDH antibodies. αtubulin and GAPDH are loading controls. b–e Freshly isolated PBMCs from 16 ART-treated PWH were subjected to nucleocytoplasmic fractionation for subsequent nucleic acid isolation and RT-qPCR quantification of (b) US-short HIV-1 RNA, (c) gapdh mRNA, and (d) US- long HIV-1 RNA. Percentages of each of the measured RNAs in the cytoplasmic fraction are shown in (e). f–k Ex vivo cultures of CD8+-depleted PBMCs from 6 ART- treated PWH were mock-treated, treated with SAHA [0.5 µM], disulfiram [0.5 µM], romidepsin [17.5 nM] or PHA [1.66 µg/ml] as a positive control in the presence of ARV [280 nM ritonavir, 180 nM azidothymidine, 200 nM raltegravir, 100 nM efa- virenz]. Three days post-treatment cells were subjected to nucleocytoplasmic fractionation for subsequent nucleic acid isolation and RT-qPCR quantification of (f, g) US-short HIV-1 RNA, (h, i) gapdh mRNA, and (j, k) US-long HIV-1 RNA. Per- centages of each of the measured RNAs in the cytoplasmic fraction are shown in (g, i, k). b–k Medians are indicated as horizontal lines, and for (e, g, i, k) inter- quartile ranges are indicated as well. The RNA copy numbers were normalized to the cell numbers measured by the beta-actin qPCR assay. Open symbols in (b, d, f, j) indicate undetectable measurements of US RNA that were assigned the values
Article Snippet: Antibodies for Western blot: against MATR3 were purchased from Bethyl (1:2000, #A300-591A, rabbit) or Sigma (1:1000, MABN1587, mouse); MTR4 (1:500, #A300-614A) were purchased from Bethyl; PSF (1:4000; #P2860) and FLAG (1:1000; #F1804) were purchased from Sigma; EXOSC-10 (1:1000; #ab50558) were purchased from Abcam; GFP (1:1000, #2555), GAPDH (1:4000, #2118) and H3 histone (1:5000; #9715) were purchased from Cell Signaling Technology; α-tubulin (1:200; #sc12462-R) were purchased from Santa Cruz.
Techniques: Western Blot, Isolation, Fractionation, Quantitative RT-PCR, Ex Vivo, Positive Control
Bethyl is a verified supplier 
![Fig. 4 | Nuclear retention of US HIV-1 RNA in PBMCs from 22 ART-treated PWH. a PBMCs from healthy donors were left untreated or were treated with PHA [1.66 µg/ml] and after 24 h collected for immunoblotting with the use of anti- MATR3, <t>anti-MTR4,</t> anti-αtubulin and anti-GAPDH antibodies. αtubulin and GAPDH are loading controls. b–e Freshly isolated PBMCs from 16 ART-treated PWH were subjected to nucleocytoplasmic fractionation for subsequent nucleic acid isolation and RT-qPCR quantification of (b) US-short HIV-1 RNA, (c) gapdh mRNA, and (d) US- long HIV-1 RNA. Percentages of each of the measured RNAs in the cytoplasmic fraction are shown in (e). f–k Ex vivo cultures of CD8+-depleted PBMCs from 6 ART- treated PWH were mock-treated, treated with SAHA [0.5 µM], disulfiram [0.5 µM], romidepsin [17.5 nM] or PHA [1.66 µg/ml] as a positive control in the presence of ARV [280 nM ritonavir, 180 nM azidothymidine, 200 nM raltegravir, 100 nM efa- virenz]. Three days post-treatment cells were subjected to nucleocytoplasmic fractionation for subsequent nucleic acid isolation and RT-qPCR quantification of (f, g) US-short HIV-1 RNA, (h, i) gapdh mRNA, and (j, k) US-long HIV-1 RNA. Per- centages of each of the measured RNAs in the cytoplasmic fraction are shown in (g, i, k). b–k Medians are indicated as horizontal lines, and for (e, g, i, k) inter- quartile ranges are indicated as well. The RNA copy numbers were normalized to the cell numbers measured by the beta-actin qPCR assay. Open symbols in (b, d, f, j) indicate undetectable measurements of US RNA that were assigned the values](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_1667/pm40021667/pm40021667__page8_image1.jpg)