Fig 1: Autoregulation of XBP1. hSAECs were RSV infected (MOI = 1.0) for 24 h in the presence of DMSO, KIRA8 (KIRA), or ceapin-A7 (A7) at 10 μM. A and B: Q-RT-PCR of total XBP1 (XBP1-Total) and unspliced XBP1 (XBP1u). Shown is fold change of mRNA relative to mock infection (DMSO) in n = 3 independent experiments. Recruitment of XBP1 (C) and RNA Pol II (D) to XBP1 promoter. XChIP was conducted using XBP1 or RNA Pol II-specific antibodies (XBP1, Cat. No. ab37152 at Abcam; RNA Pol II, Cat. No. ab26721 at Abcam). The XChIP-enriched genomic DNA was analyzed by Q-gPCR using XBP1 promoter-specific primers (Table 2). Data are calculated as fold change relative to uninfected hSAECs (DMSO) and plotted as means ± ranges plus all data points of duplicate independent immunoprecipitates. Note the RNA Pol II dependence on IRE1α-XBP1 signaling. **P < 0.01, ANOVA. hSAECs, human small airway epithelial cells; MOI, multiplicity of infection; Q-RT-PCR, quantitative reverse transcription polymerase chain reaction; RSV, respiratory syncytial virus; XBP1, X-box binding protein 1.
Fig 2: BRD4 navigates the translocation of the RPAP2-Pol II complex to SEs and drives ZBTB16 transcription. a Co-IP experiment showing the binding of RPAP2 and RPB1. b Immunofluorescence showing the effects of RPAP2 knockdown, leptomycin B pretreatment prior to RPAP2 knockdown and JQ1 treatment on the subcellular distribution of RPAP2 and RPB1 in MSCs. c–f Western blot analysis of protein fractions showing the distribution of RPB1, BRD4 and RPAP2 in the cytoplasm, nucleus and chromatin. Tubulin, lamin A/C and histone 3 were the internal controls for proteins in the cytoplasm, nucleus and chromatin, respectively (c). Scatter plots showing the respective abundances of RPB1 (d), BRD4 (e) and RPAP2 (f) in different protein extracts. g ChIP-seq signal traces showing POL II binding to ZBTB16 in NC or OB group MSCs. h Location of ChIP‒qPCR primers for ZBTB16. i ChIP‒qPCR analysis showing POL II occupancy on ZBTB16 in NC and OB group MSCs. j ChIP‒qPCR analysis showing the effects of JQ1 treatment, leptomycin B and leptomycin B pretreatment prior to RPAP2 knockdown on POL II occupancy on ZBTB16 in MSCs. The statistical data are represented as the means ± SEMs, n = 9, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001
Fig 3: RuvBL1 complex formation during infection accompanies decreased RuvBL1-E1A and -STAT2 binding affinity and decreased transcription factor recruitment to ISG promoters. (A) HAdV E1As bind to RuvBL1 and RuvBL2. A549 cells were infected with indicated viral strains for 24 hours, lysed, and indicated proteins were immunoprecipitated. Precipitates were resolved on SDS gel, transferred to the PVDF membrane, and blotted with indicated antibodies. M: mock infected, C2: HAdV-C2, B7: HAdV-B7, B14: HAdV-B14. (B) HAdV-B7 and HAdV-B14 induce complex formation of RuvBL1 in infected cells. A549 cells were transfected with HA-tagged RuvBL1 expression vector and infected with indicated viruses for 24 hours. Eight hours before harvest, indicated samples were treated with IFN α-2A. Cells were cross-linked with formaldehyde, harvested, and lysed, and HA-RuvBL1 was precipitated using 12CA5 anti-HA mouse antibody. Precipitates were resolved on precast, 4%–12% gradient SDS gel, transferred to the PVDF membrane, and blotted with the indicated antibodies (RuvBL1 was blotted with a rat anti-HA antibody different from that used to precipitate it); HMW, high molecular weight. (C) HAdV infection disrupts RuvBL1/RNA Pol II interaction. A549 cells were infected with indicated viral strains for 24 hours, lysed, and indicated proteins were immunoprecipitated. Precipitates were then resolved on SDS gel, transferred to the PVDF membrane, and blotted with the indicated antibodies. RPB1: RNA Polymerase II. (D) HAdV-B7 and B14 disrupt STAT2/RuvBL1 interaction. A549 cells were transfected with HA-tagged RuvBL1 expression vector and infected with the indicated HAdV strains 24 hours later. Sixteen hours after infection, IFN α-2A was applied. After 8 more hours, cells were harvested and lysed, and HA-tagged RuvBL1 was precipitated via 12CA5 anti-HA antibody. Samples were resolved on SDS gel, transferred to PVDF membrane, and blotted with the indicated antibodies. (E) Recruitment of STAT2 and RNA polymerase II to ISG promoters is dramatically reduced in HAdV-B7 and HAdV-B14 compared to HAdV-C2. A549 cells were treated with IFN α-2A 20 hours after infection with the indicated HAdVs, and 4 hours later (24 hours after infection), cells were cross-linked, harvested, and indicated proteins were immunoprecipitated. Cross-linking was reversed, DNA was isolated, and relative levels were analyzed via qPCR. Statistical analysis was performed via unpaired student’s t-test. One asterisk signifies a P-value of <0.05, two represent <0.01, and three indicate <0.001; n = 2. (F) Ubiquitination of STAT2 is reduced in HAdV-B7 and B14-infected cells. HT1080 cells were transfected with the HA-ubiquitin expression vector and subsequently infected with the indicated viruses for 24 hours at an MOI of 10. Cells were lysed and immunoprecipitations were carried out for STAT2 in RIPA buffer as described. Immunoprecipitates were then resolved on a 4%–12% gradient SDS gel and blotted for the indicated proteins. PENTON was used to indicate infection, note that higher levels of PENTON in C2 do not reflect higher protein expression in this virus, but rather the sensitivity of the anti-PENTON antibody for C2, for which it was raised, is much higher than for B7 and B14.
Fig 4: Nuclear ANLN directly interacts with Pol II large subunit.A Representative immunofluorescence staining images of ANLN (pink) and DNA (blue) in different cell cycles. Scale bar, 5 μm. B Western blotting was performed to examine the different subcellular fractions of the indicated cells. Lamin B1 and histone H3 were used as marker proteins of the chromatin. GAPDH was used as a marker of the cytoplasm. C BrUTP was transfected into HeLa cells using liposomes for 15 min, and then cultured in fresh medium for another 15 min. Immunofluorescence staining was performed using ANLN (green) and BrdU (red) antibodies. Scale bar, 5 μm. D The enrichment analysis of ANLN interacting proteins identified by mass spectrometry was performed using Metascape database. Based on the selected terms, accumulative hypergeometric P-values and enrichment factors were calculated and used for filtering. Remaining significant terms were hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships. The 0.3 kappa score was applied as the threshold to cast the tree into term clusters. E Immunoprecipitation was performed using ANLN antibody, and the indicated antibodies were subsequently used for the western blotting. F The indicated plasmids were transfected into HEK293T cells for 48 h, and then the cells were lysed and subjected to immunoprecipitation with HA magnetic beads. G, H The interaction between recombinant POLR2A fragments and ANLN full-length protein was examined using an in vitro pulldown assay. I Representative PLA images for the interaction between endogenous ANLN and Pol II in KYSE510 cells. Scale bar, 5 µm. The data are representative of at least three independent experiments with similar results. Source data are provided as a Source Data file.
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