Interaction of SMARCB1 and UL44 in HCMV-infected fibroblast cells and with recombinant proteins.

<p>(<b>A</b>) Co-immunoprecipitation of endogenous SMARCB1 and UL44 in HCMV-infected fibroblast cells. Equal numbers of mock infected and HCMV infected fibroblast cells were lysed at 72 hpi, and the extracts were immunoprecipitated with anti-SMARCB1. Co-immunoprecipitated proteins were resolved electrophoretically and subjected to immunoblot analysis with anti-UL44 and anti-SMARCB1. Lane 1: Mock lysate immunoprecipitated with anti-SMARCB1. Lane 2: HCMV lysate immunoprecipitated with anti-SMARCB1. Lane 3: HCMV lysate immunoprecipitated with no antibody. Lanes 4 and 5, SMARCB1 and UL44 input in extracts (7 µg, 1% of the total in Lane 4 and 35 µg, 5% of the total in Lane 5). IgGHc: IgG heavy chain. (<b>B</b>) <i>In vitro</i> pull-down assay of GST-SMARCB1 and His₆-UL44. Purified GST-SMARCB1 or GST incubated with purified His₆-UL44 immobilized on magnetic His-tag Dynabeads. Samples were analyzed by SDS-PAGE and Coomassie blue staining. Lane 1: GST-SMARCB1+Dynabeads His-tag. Lane 2: His₆-UL44+Dynabeads His-tag. Lane 3: GST-SMARCB1+His₆-UL44+Dynabeads His-tag. Lane 4: His₆-UL44 (2 µg, 10% of input). Lane 5: GST-SMARCB1 (2 µg, 10% of input). Lane 6: GST+His₆-UL44+Dynabeads His-tag. Lane 7: GST (2 µg, 10% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lane 5). (^……) indicates that samples were run on the same gel.</p

SMARCB1 and UL114 interact <i>in vitro</i>.

<p>(<b>A</b>) <i>In vitro</i> binding analysis of HA-tagged Δ379-SMARCB1 and c-myc-tagged UL114 in ³⁵S-labeled proteins using the TNT coupled transcription/translation system. The proteins were transcribed and translated <i>in vitro</i> with ³⁵S-methionine in the translation mixture to generate radioactive labeled products from vectors pACT2-Δ379-SMARCB1 (HA-epitope) and pGBKT7-UL114 (c-myc epitope). The translated Δ379-SMARCB1-HA and UL114-c-myc were immunoprecipitated with either anti-HA or anti-c-myc-antibodies and eluted from the Protein G beads. Samples were subjected to 8% SDS-PAGE and PhosphoImaging. Lane 1: UL114-c-myc+c-myc antibody. Lane 2: Δ379-SMARCB1-HA+HA-antibody. Lane 3: Δ379-SMARCB1-HA+UL114-c-myc+HA-antibody. Lane 4: Δ379-SMARCB1-HA+UL114-c-myc+c-myc antibody. Lane 5: UL114-c-myc+HA-antibody. Lane 6: Δ379-SMARCB1-HA+c-myc-antibody. (^.......) indicates that samples were run on the same gel and (——) indicates that samples were run on a different gel. (<b>B</b>) and (<b>C</b>) GST pull-down assays to detect the interaction of SMARCB1 and UL114. (<b>B</b>) Purified GST-SMARCB1 or crude extract of <i>E. coli</i> cells over-expressing GST were incubated with purified UL114. The GST pull-down products were immunoblotted with anti-SMARCB1, anti-UL114 and anti-GST. Lane 1: GST-extract+UL114. Lane 2: GST-SMARCB1+UL114. Lane 3: purified GST-SMARCB1 (2 µg, 10% of input). Lane 4: Purified UL114 (1 µg, 5% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lanes 2 and 3). (<b>C</b>) Purified GST-SMARCB1 or crude extract of <i>E. coli</i> cells over-expressing GST were incubated with lysates of HCMV-infected cells. The GST pull-down products were immunoblotted with anti-SMARCB1, anti-UL114, anti-GST and anti-UL57. Lane 1: GST-extract+HCMV lysate. Lane 2: GST-SMARCB1+HCMV lysate. Lane 3: purified GST-SMARCB1 (2 µg, 10% of input). Lane 4: HCMV lysate (30 µg, 1% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lanes 2 and 3). The asterisks (*) on Lanes 1 and 4 indicates unspecific bands by the use of anti-SMARCB1.</p

Recruitment of SWI/SNF chromatin remodeling factors to nuclear virus DNA replication foci.

<p>(<b>A</b>) SMARCB1 co-localizes with UL44 in HCMV-infected fibroblast cells harvested at 24, 48, and 72 hpi. (<b>B</b>) Co-localization of SMARCB1 and other essential factors of the SWI/SNF complex; BRG-1, BAF155, BAF170, in HCMV infected fibroblast cells harvested at 48 hpi. The cells were fixed and subjected to double-staining for UL44 (mouse Mab-UL44) and SMARCB1 (rabbit Pab-SMARCB1) and SMARCB1 (rabbit Pab-SMARCB1) and either BRG-1 (mouse Mab-BRG-1), BAF155 (mouse Mab BAF155), BAF170 (mouse Mab BAF170) for immunofluorescence microscopy. Secondary antibodies were used for staining UL44, BRG-1, BAF155, BAF170 in green (anti-mouse 488) and SMARCB1 in red (anti-rabbit 594), and the cells were further visualized by confocal microscopy. Co-localization was visualized by a merge of the two microscopic determinations, and counterstaining of the nuclei was achieved by the use of DAPI.</p

SMARCB1, UL114 and UL44 are associated with the chromatin and the nuclear matrix in HCMV infected fibroblast cells.

<p>Mock- and HCMV-infected fibroblast cells harvested at indicated time points were subjected to sub-nuclear fractionation to obtain whole chromatin fraction and core nuclear matrix. Proteins from equal cell equivalents from each fraction were analyzed by western blotting with the indicated antibodies.</p

Increased expression of the SWI/SNF core subunits in HCMV infected cells.

<p>(<b>A</b>) The expression of UL114, SMARCB1 and UL44 in nuclear extracts (20 µg) from mock and HCMV-infected fibroblast cells was analyzed at immediate –early (12 hpi), early (24 hpi) and late (48 and 72 hpi) times of infection by western blot. The western blots were analyzed by Thyphoon scanning. GAPDH was used as a loading control. (<b>B</b>) The expression of BRG1, BAF155 and BAF 170 in nuclear extracts (40 µg) from mock and HCMV-infected fibroblast cells was analyzed at late (72 hpi) time of infection by western blot. The western blots were analyzed by Thyphoon scanning. GAPDH was used as a loading control.</p

DataSheet_1_Depletion of the m1A writer TRMT6/TRMT61A reduces proliferation and resistance against cellular stress in bladder cancer.docx

BackgroundBladder cancer (BLCA) is a common and deadly disease that results in a reduced quality of life for the patients and a significant economic burden on society. A better understanding of tumorigenesis is needed to improve clinical outcomes. Recent evidence places the RNA modification m1A and its regulatory proteins TRMT6/TRMT61A and ALKBH3 in BLCA pathogenesis.MethodsTRMT6/TRMT61A, ALKBH1, and ALKBH3 expression was examined in human BLCA cell lines and a normal urinary tract epithelium cell line through qRT-PCR and western blot analysis. Prestoblue Cell Viability Reagent, wound-healing assay, and live-cell imaging-based cell displacement analysis, were conducted to assess proliferation, migration, and displacement of this BLCA cell line panel. Cell survival was assessed after inducing cellular stress and activating the unfolded protein response (UPR) with tunicamycin. Moreover, siRNA-mediated gene silencing in two BLCA cell lines (5637 and HT1197) was conducted to investigate the biological roles of TRMT6/TRMT61A.ResultsHeterogeneous morphology, proliferation, displacement, tunicamycin sensitivity, and expression levels of m1A regulators were observed among the panel of cell lines examined. In general, TRMT61A expression was increased in BLCA cell lines when compared to SV-HUC-1. Depletion of TRMT6/TRMT61A reduced proliferation capacity in both 5637 and HT1197 cell lines. The average cell displacement of 5637 was also reduced upon TRMT6/TRMT61A depletion. Interestingly, TRMT6/TRMT61A depletion decreased mRNA expression of targets associated with the ATF6-branch of the UPR in 5637 but not in HT1197. Moreover, cell survival after induction of cellular stress was compromised after TRMT6/TRMT61A knockdown in 5637 but not in HT1197 cells.ConclusionThe findings suggest that TRMT6/TRMT61A plays an oncogenic role in BLCA and is involved in desensitizing BLCA cells against cellular stress. Further investigation into the regulation of TRMT6/TRMT61A expression and its impact on cellular stress tolerance may provide insights for future BLCA treatment.</p