Journal: bioRxiv

Article Title: Construction and characterization of coronavirus nonstructural protein 3-host protein interaction networks unravel an important role of cleavage and polyadenylation specificity factor 6 in regulation of viral RNA replication

doi: 10.1101/2025.02.27.640555

Figure Lengend Snippet: (A) Interaction of IBV and SARS-CoV-2 nsp3 with a selected host protein in cells overexpressing the proteins. HEK293T cells were co-transfected with plasmids encoding HiBiT-tagged IBV or SARS-CoV-2 nsp3, together with a FLAG-tagged host protein (DDX5, DDX39B, DHX9, eIF4A3, SRRT, and UBA1), followed by IP with anti-FLAG beads. The total cell lysates (input) and immunoprecipitated samples were analyzed by immunoblot with anti-FLAG and HiBiT, respectively. The molecular weights of the proteins are indicated on the left and the names of the corresponding proteins are shown on the right. (B) Interaction of endogenous CPSF6 in cells overexpressing either IBV or SARS-CoV-2 nsp3. HEK293T cells were transfected with plasmid encoding FLAG-tagged IBV nsp3 or HA-tagged SARS-CoV-2 nsp3, followed by IP with anti-Flag or HA beads. The total cell lysates (input) and immunoprecipitated samples were analyzed by immunoblot with anti-FLAG and HiBiT, respectively. The molecular weights of the proteins are indicated on the left and the names of the corresponding proteins are shown on the right. (C) Regulation of the transcription of seven selected genes by IBV infection of H1299 and Vero cells. Cells were infected with IBV at a multiplicity of infection (MOI) of approximately 2, and harvested at indicated time points for RNA extraction. Equal amounts of total RNA were reverse-transcribed, and the level of IBV gRNA as well as mRNA levels of DDX5, DDX39B, DHX9, CPSF6, elF4A3, SRRT, and UBA1 were determined by qPCR. Significance levels are presented by the P value (ns, no significance, *, P < 0.05;**, P < 0.01; ***, P < 0.001).

Article Snippet: The relative expression levels of IBV and OC43 gRNA and sgRNA were measured by quantitative real-time PCR (RT-qPCR) using the Talent qPCR PreMix SYBR Green kit (Tiangen).

Techniques: Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Infection, RNA Extraction, Reverse Transcription

Journal: bioRxiv

Article Title: Construction and characterization of coronavirus nonstructural protein 3-host protein interaction networks unravel an important role of cleavage and polyadenylation specificity factor 6 in regulation of viral RNA replication

doi: 10.1101/2025.02.27.640555

Figure Lengend Snippet: (A) Western blot analysis of the effect of CPSF6-knockdown on the replication of IBV. H1299 cells were transfected with siEGFP and siCPSF6 before being infected with IBV at an MOI of 2. Cell lysates were harvested at the indicated time points and subjected to Western blot analysis using the indicated antibodies. Beta-actin was included as the loading control. Protein ladders in kilodaltons are indicated on the left. Significance levels are presented by the P value (*, P<0.05) (B) RT-qPCR analysis of the effect of CPSF6-knockdown on the replication of IBV. Total RNAs were extracted from cells infected and harvested as described in panel (A). Equal amounts of total RNA were reverse-transcribed, and the level of IBV gRNA, -gRNA and sgRNAs were determined by qPCR. Significance levels are presented by the P value (*, P < 0.05;**, P < 0.01; ***, P < 0.001). (C) HiBiT luminescence assessment of the effect of CPSF6-knockdown on the replication of IBV. Cells were infected as described above in panel (A), and the culture supernatants were collected at the specified time point for HiBiT luminescence. Significance levels are presented by the P value (***, P < 0.001) (D) Western blot analysis of the effect of CPSF6-knockdown on the replication of HCoV-OC43. H1299 cells were transfected with siEGFP and siCPSF6 before being infected with HCoV-OC43 at an MOI of 2. Cell lysates were harvested at the indicated time points and subjected to Western blot analysis using the indicated antibodies. Beta-actin was included as the loading control. Sizes of protein ladders in kilodaltons are indicated on the left. Significance levels are presented by the P value (*, P<0.05) (E) RT-qPCR analysis of the effect of CPSF6-knockdown on the replication of HCoV-OC43. Cells were treated as described above in panel (D), and harvested at indicated time points for RNA extraction. Equal amounts of total RNA were reverse-transcribed. The level of OC43 gRNA and sgRNAs were determined by qPCR. Significance levels are presented by the P value (*, P < 0.05;**, P < 0.01; ***, P < 0.001)

Article Snippet: The relative expression levels of IBV and OC43 gRNA and sgRNA were measured by quantitative real-time PCR (RT-qPCR) using the Talent qPCR PreMix SYBR Green kit (Tiangen).

Techniques: Western Blot, Knockdown, Transfection, Infection, Control, Quantitative RT-PCR, Reverse Transcription, RNA Extraction

Journal: Neuron

Article Title: CONSERVED HIGHER ORDER CHROMATIN REGULATES NMDA RECEPTOR GENE EXPRESSION AND COGNITION

doi: 10.1016/j.neuron.2014.10.032

Figure Lengend Snippet: (A) 500kb of linear genome (mm9, chromosome 6:135,623,529-136,123,529), encompassing Grin2b, including TSS (green), proximal intronic sequences targeted by SETDB1 (yellow), and two loop loopings (red) (1) Grin2bTSS+378kb and (2) Grin2bTSS+471kb, with sequences homologue to human GRIN2BTSS+348kb and GRIN2BTSS+449kb (Fig. S1) (B) (Top) Browser tracks for histone marks H3K4me3 and H3K27ac in adult mouse cerebral cortex(Dixon et al., 2012). (C) Fold-change (CK-Setdb1 transgenic (Tg) /wildtype littermate (Wt) of 3C PCR from adult Tg and Wt cortex (see also Fig. S2). Notice increased physical interactions of (yellow) TSS-bound +15 to +40kb intronic sequence, and significant decrease in (red box no. 1) Grin2bTSS+378kb and (red box no. 2) Grin2bTSS+471kb. Two-way ANOVA, 3C interaction × genotype F(7,32)=54.905(p<0.001). Newman-Keuls post-hoc *, **; P< 0.05, 0.01. (D) Quantification of Grin2b (left) RNA and (right) protein in adult (6–8 week) CK-Setdb1 Tg and wildtype (Wt) littermate control cortex. mean ± S.D, N=5 (RNA) and N=3 (immunoblot)/group. (E) Activity-dependent regulation of Grin2b higher order chromatin in hippocampal neurons. (top, left to right) Grin2b RNA, 3C quantification of Grin2bTSS+378kb and Grin2bTSS+471kb and SETDB1 occupancy across four regulatory sequences at Grin2b locus. Note significant increase at SETDB1 target site after 15 hours of picrotoxin (PTX) or vehicle control (DMSO). N=3–6 experiments/group, data shown as mean ± S.D..(F) (left) Grin2b-TALE-VP64-GFP specifically targets mouse Grin2b loop no. 2 (Grin2bTSS+471kb), 471 kb downstream of TSS. Images show Neuro2A cells and cultured hippocampal neurons expressing GFP-tagged TALE-VP64. RT-PCR for Grin2b and Gapdh control showing specific expression in cultured neurons. (right) anti-TALE-VP64 ChIP in N2A and NG108 cells and primary cortical neurons, expressed (y-axis) as fold change compared to non-transfected condition (N=3/group; (mean ± S.E.M., * Two-way ANOVA cell type × TALE-VP64 binding F(2,12)=4.04, P<0.05, Bonferroni posthoc P < 0.05).RT-PCR Grin2b, Grin2a and Setdb1 RNA levels in neurons transfected with TALE-VP64-EGFP (TALE), compared to mock-transfected neurons (Con). Notice specific TALE-VP64 mediated increase in Grin2b RNA (N = 6 per group, mean ± S.E.M, *P < 0.05, t-test). See also Figures S1 and S2.

Article Snippet: TALE-based designer transcription factor Two Transcription Activator-Like Element DNA-binding proteins (TALE), fused to four tandem copies of the Herpes simplex Viral protein 16, amino acids 437–447, DALDDFDLDML connected with glycine-serine linkers, were targeted to intergenic sequences in the mouse genome, 471 kb downstream of the Grin2b TSS using the TALE tool box kit (Addgene).

Techniques: Transgenic Assay, Sequencing, Control, Western Blot, Activity Assay, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Binding Assay