Journal: Scientific Reports

Article Title: Polycomb repressive complex 2 facilitates the nuclear export of the influenza viral genome through the interaction with M1

doi: 10.1038/srep33608

Figure Lengend Snippet: ( a ) Remodeling of nuclear compartments by influenza virus infection. At 6 h post infection, mock-infected (upper panel) or infected (lower panel) HeLa cells were subjected to indirect immunofluorescence assays with anti-EZH2, anti-Pol II, anti-HP1β, anti-RAD21, anti-HDAC1, anti-Brg1, anti-Topo II, and anti-CTCF antibodies, respectively (green). Nuclei were counter-stained with DAPI (blue). Punctate foci of EZH2 are indicated by arrowheads. ( b ) Knockdown efficiency of EZH2. HeLa cells were transfected with either control siRNA or siRNA against EZH2. At 60 h post transfection, 1 × 10 5 cells (lane 1), 5 × 10 4 cells (lane 2), 2.5 × 10 4 cells (lane 3), 1.25 × 10 4 cells (lane 4) of control cells, and 1 × 10 5 cells of EZH2 KD cells (lane 5) were subjected western blotting with anti-EZH2 antibody. The original blot is presented in . ( c ) The amounts of PRC2 complex components in EZH2 KD cells. Control or EZH2 KD cells were subjected to western blotting with anti-EZH2, anti-SUZ12, anti-EED, and anti-β-actin antibodies. The original blots are presented in . ( d ) Production of infectious virions. At 60 h post transfection, control and EZH2 KD cells were infected with influenza virus at MOI of 0.3. The culture supernatants were collected at 24, 28, 32, and 36 h post infection, and subjected to plaque assays. The average titers and standard deviations determined from three independent experiments are shown. ( e ) Expression levels of vRNA and mRNA in EZH2 KD cells. Total RNAs prepared from control and EZH2 KD cells at 6 h post infection were subjected to the quantitative real-time PCR. The amounts of vRNA and mRNA of segment 5 in control cells were indicated as 100%, respectively. The average expression levels and standard deviations determined from three independent experiments are shown. ( f ) Expression levels of M1 and NS2 in control and EZH2 KD cells. At 6 h post infection, control and EZH2 KD cells were subjected to SDS-PAGE, and carried out western blotting with anti-EZH2, anti-M1, anti-NS2, and anti-β-actin antibodies. The original blots are presented in .

Article Snippet: Rabbit polyclonal antibodies against EZH2 (Abcam), EED (Proteintech), SUZ12 (Proteintech), HDAC1 (Upstate), pol II (Santa Cruz), RAD21 (Abcam), and mouse monoclonal antibody against Brg1 (Santa Cruz), and rat polyclonal antibody against HP1β (Abcam) were purchased.

Techniques: Virus, Infection, Immunofluorescence, Staining, Knockdown, Transfection, Control, Western Blot, Expressing, Real-time Polymerase Chain Reaction, SDS Page

Journal: bioRxiv

Article Title: MeCP2 NID interaction with RNA: Implications for Rett Syndrome-Relevant Protein Regulation

doi: 10.1101/2025.11.19.689340

Figure Lengend Snippet: In the presence of RNA, binding to certain proteins, such as TBLR1 and Sin3a, is limited, however there are other unknown protein complexes to which MeCP2 interacts through, perhaps, scaffolding RNAs that may bind at the RBD, at some other region of the protein, or both. When RNA is degraded, some large complexes disassociate from MeCP2, as indicated from sucrose density ultracentrifugation, but MeCP2’s total protein interactome increases through interactions at the RBD, such as to TBLR1, as shown by IP data. When UV crosslinking is applied to create a covalent link between RNA bound to MeCP2, RNase treatment does not result in the increased interactome or TBLR1 binding as before, but instead obstructs TBLR1 binding, allowing other proteins, like Sin3a, to bind MeCP2 at a domain outside the RBD.

Article Snippet: Primary antibodies used were: MeCP2 (M9317, Sigma®, St. Louis, MO, USA), H4 (rabbit serum produced in-house), Sin3a (ProteinTech 14638-1-AP), TBLR1 (Bethyl, A300-408A), HDAC1 (Santa Cruz Biotechnology, sc-81598).

Techniques: RNA Binding Assay, Scaffolding, Binding Assay

Journal: Cell death & disease

Article Title: The novel ZEB1-upregulated protein PRTG induced by Helicobacter pylori infection promotes gastric carcinogenesis through the cGMP/PKG signaling pathway.

doi: 10.1038/s41419-021-03440-1

Figure Lengend Snippet: Fig. 5 PRTG activates the downstream cGMP/PKG signaling pathway in gastric cancer cells. A Expression of top 500 genes positively associated with PRTG expression in gastric patients from TCGA database. B Gene ontology term enrichment analysis for top 8 biological process controlled by differentially expressed genes in gastric patients. C and D The expression of cGMP/PKG signaling pathway related proteins in PRTG- overexpressing AGS cells were detected by qRT-PCR (C) and western blot (D). E Supernatant cGMP levels in PRTG overexpressing AGS cells were detected by ELISA. F PRTG silencing AGS cells were infected with H. pylori for 48 h, and then the expression of cGMP/PKG signaling pathway related proteins were detected by western blot. G AGS cells were transiently co-transfected with ZEB1 overexpressing plasmid and PRTG siRNA. 48 h later, the expression of cGMP/PKG signaling pathway related proteins were detected by western blot. H–K PRTG overexpressing AGS cells were treated with sGC inhibitor (NS-2028, 10 μM) or PKG inhibitor (KT5823, 1 μM) for 24 h. cGMP levels in supernatant and phosphorylated VASP expression were detected by ELISA and western blot, respectively. Data were presented as mean ± SD from the three independent replicates. ***P < 0.0001.

Article Snippet: The following primary antibodies were used: anti-PRTG antibody (CF501394, OriGene), ZEB1(21544-1-AP, Proteintech), E-Cadherin (13-1700, Invitrogen), N-Cadherin (33-3900, Invitrogen), Vimentin (ab92547, abcam), Snail (26183-1-AP, Proteintech), sGC (ab189176, abcam), PKG1 (3248, CST), PKG2 (55138-1-AP, Proteintech), PDE5A (ab28761, abcam), VASP (13472-1-AP, Proteintech), PhosphoVASPSer239 (ab194747, abcam), GAPDH (sc-47724, Santacruz), Caspase-3 (19677-1-AP, Proteintech), p21 (10355-1-AP, Proteintech), Bid (10988-1-AP, Proteintech), BCL2 (12789-1-AP, Proteintech), PhosphoH2AXSer139 (2577, CST), BIRC3 (24304-1-AP, Proteintech), HDAC1 (sc-81598, Santa Cruz), Ubiquitin (10201-2-AP, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Infection, Transfection, Plasmid Preparation

Journal: Cell death & disease

Article Title: The novel ZEB1-upregulated protein PRTG induced by Helicobacter pylori infection promotes gastric carcinogenesis through the cGMP/PKG signaling pathway.

doi: 10.1038/s41419-021-03440-1

Figure Lengend Snippet: Fig. 2 PRTG plays oncogenic activities in gastric cancer progression. A EdU assay after PRTG stable overexpression or transient knockdown in AGS cells. B Cell apoptosis was detected in PRTG overexpressing or silencing AGS cells after treated with chemotherapy drugs (paclitaxel, 20 nM; CDDP, 5 μM) for 48 h. C Statistical analysis of apoptosis in AGS cells (n = 3, related to Fig. 2B). D Western blot was used to detect the effect of PRTG on the expression of apoptosis-related proteins in paclitaxel-treated AGS cells of C. E Transwell assay was used to detect the invasion and migration ability of PRTG overexpressing or silencing AGS cells. F AGS cells stably expressing empty vector (pLVX) or pLVX-PRTG were injected into the nude mice (n = 12) and tumor volumes were monitored. G Tumor volumes of 3 mice were presented 25 days post inoculation. H Tumor volumes and weights (n = 12) were measured 25 days post inoculation. I The expression of Ki67, E-cadherin and N-cadherin were detected by IHC. Data were presented as mean ± SD from the three independent replicates. **P < 0.01; ***P < 0.0001. CDDP, cisplatin; pLVX, empty control pLVX lentivirus.

Article Snippet: The following primary antibodies were used: anti-PRTG antibody (CF501394, OriGene), ZEB1(21544-1-AP, Proteintech), E-Cadherin (13-1700, Invitrogen), N-Cadherin (33-3900, Invitrogen), Vimentin (ab92547, abcam), Snail (26183-1-AP, Proteintech), sGC (ab189176, abcam), PKG1 (3248, CST), PKG2 (55138-1-AP, Proteintech), PDE5A (ab28761, abcam), VASP (13472-1-AP, Proteintech), PhosphoVASPSer239 (ab194747, abcam), GAPDH (sc-47724, Santacruz), Caspase-3 (19677-1-AP, Proteintech), p21 (10355-1-AP, Proteintech), Bid (10988-1-AP, Proteintech), BCL2 (12789-1-AP, Proteintech), PhosphoH2AXSer139 (2577, CST), BIRC3 (24304-1-AP, Proteintech), HDAC1 (sc-81598, Santa Cruz), Ubiquitin (10201-2-AP, Proteintech).

Techniques: EdU Assay, Over Expression, Knockdown, Western Blot, Expressing, Transwell Assay, Migration, Stable Transfection, Plasmid Preparation, Injection, Control

Journal: Cell death & disease

Article Title: The novel ZEB1-upregulated protein PRTG induced by Helicobacter pylori infection promotes gastric carcinogenesis through the cGMP/PKG signaling pathway.

doi: 10.1038/s41419-021-03440-1

Figure Lengend Snippet: Fig. 4 H. pylori promotes ZEB1 stabilization and recruitment to PRTG promoter. A qRT-PCR was used to detect the silencing efficiency of siRNA targeted to corresponded transcription factor in AGS cells. Relative expression meant the expression of siRNA targeted transcription factor refer to the expression level of cells transfected with control siRNA. B The expression of PRTG in AGS cells transfected with indicated siRNA targeted to corresponded transcription factor was detected by qRT-PCR. C The expression of ZEB1 and PRTG in ZEB1 silencing AGS cells were detected by western blot. Liposome represented transfection regent control. D qRT-PCR and western blot were used to the expression of PRTG and ZEB1 in AGS cells transiently transfected with pcDNA3.1 empty vector or pcDNA3.1-ZEB1. E Predicted binding sites of the transcription factor ZEB1 to PRTG promoter (site 1 and site 2). P1 and P4 represented distal regions far from ZEB1 predicted binding sites in PRTG promoter. F The binding site of ZEB1 to PRTG promoter was identified by ChIP-qPCR. G AGS cells were co-transfected with the ZEB1 expression plasmid and PRTG promoter luciferase reporter constructs. Extract luciferase activities were determined 24 h after transfection. H The expression of ZEB1 in AGS cells at different time points post H. pylori infection was detected by western blot. I The effect of H. pylori on the stability of ZEB1 protein in AGS cells treated with CHX (5 μg/mL) for the indicated times was detected by western blot. J AGS cells were infected with H. pylori and treated with MG132 (10 μM) and CHX for 6 h. whole-cell lysates of uninfected and H. pylori-infected cells were immunoprecipitated (IP) with anti-ZEB1 antibody and then immunoblotted for detection of ubiquitinated ZEB1 (Ub-ZEB1). K AGS cells were treated as indicated and ZEB1 protein expression in cellular fractions were detected by western blot. L ChIP-qPCR analysis of the effect of H. pylori infection on the binding of ZEB1 to the PRTG promoter. M The luciferase reporter assay was used to detect the effect of H. pylori infection on the binding of ZEB1 to the PRTG promoter. N Western blot was used to detect the effect of ZEB1 silencing on the H. pylori-induced PRTG expression. Data were presented as mean ± SD from the three independent replicates. ***P < 0.0001. CHX, cycloheximide; siNTC, non-target control siRNA.

Article Snippet: The following primary antibodies were used: anti-PRTG antibody (CF501394, OriGene), ZEB1(21544-1-AP, Proteintech), E-Cadherin (13-1700, Invitrogen), N-Cadherin (33-3900, Invitrogen), Vimentin (ab92547, abcam), Snail (26183-1-AP, Proteintech), sGC (ab189176, abcam), PKG1 (3248, CST), PKG2 (55138-1-AP, Proteintech), PDE5A (ab28761, abcam), VASP (13472-1-AP, Proteintech), PhosphoVASPSer239 (ab194747, abcam), GAPDH (sc-47724, Santacruz), Caspase-3 (19677-1-AP, Proteintech), p21 (10355-1-AP, Proteintech), Bid (10988-1-AP, Proteintech), BCL2 (12789-1-AP, Proteintech), PhosphoH2AXSer139 (2577, CST), BIRC3 (24304-1-AP, Proteintech), HDAC1 (sc-81598, Santa Cruz), Ubiquitin (10201-2-AP, Proteintech).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Western Blot, Plasmid Preparation, Binding Assay, ChIP-qPCR, Luciferase, Construct, Infection, Immunoprecipitation, Reporter Assay

Journal: Cell death & disease

Article Title: The novel ZEB1-upregulated protein PRTG induced by Helicobacter pylori infection promotes gastric carcinogenesis through the cGMP/PKG signaling pathway.

doi: 10.1038/s41419-021-03440-1

Figure Lengend Snippet: Fig. 5 PRTG activates the downstream cGMP/PKG signaling pathway in gastric cancer cells. A Expression of top 500 genes positively associated with PRTG expression in gastric patients from TCGA database. B Gene ontology term enrichment analysis for top 8 biological process controlled by differentially expressed genes in gastric patients. C and D The expression of cGMP/PKG signaling pathway related proteins in PRTG- overexpressing AGS cells were detected by qRT-PCR (C) and western blot (D). E Supernatant cGMP levels in PRTG overexpressing AGS cells were detected by ELISA. F PRTG silencing AGS cells were infected with H. pylori for 48 h, and then the expression of cGMP/PKG signaling pathway related proteins were detected by western blot. G AGS cells were transiently co-transfected with ZEB1 overexpressing plasmid and PRTG siRNA. 48 h later, the expression of cGMP/PKG signaling pathway related proteins were detected by western blot. H–K PRTG overexpressing AGS cells were treated with sGC inhibitor (NS-2028, 10 μM) or PKG inhibitor (KT5823, 1 μM) for 24 h. cGMP levels in supernatant and phosphorylated VASP expression were detected by ELISA and western blot, respectively. Data were presented as mean ± SD from the three independent replicates. ***P < 0.0001.

Article Snippet: The following primary antibodies were used: anti-PRTG antibody (CF501394, OriGene), ZEB1(21544-1-AP, Proteintech), E-Cadherin (13-1700, Invitrogen), N-Cadherin (33-3900, Invitrogen), Vimentin (ab92547, abcam), Snail (26183-1-AP, Proteintech), sGC (ab189176, abcam), PKG1 (3248, CST), PKG2 (55138-1-AP, Proteintech), PDE5A (ab28761, abcam), VASP (13472-1-AP, Proteintech), PhosphoVASPSer239 (ab194747, abcam), GAPDH (sc-47724, Santacruz), Caspase-3 (19677-1-AP, Proteintech), p21 (10355-1-AP, Proteintech), Bid (10988-1-AP, Proteintech), BCL2 (12789-1-AP, Proteintech), PhosphoH2AXSer139 (2577, CST), BIRC3 (24304-1-AP, Proteintech), HDAC1 (sc-81598, Santa Cruz), Ubiquitin (10201-2-AP, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Infection, Transfection, Plasmid Preparation

Journal: Cell death & disease

Article Title: The novel ZEB1-upregulated protein PRTG induced by Helicobacter pylori infection promotes gastric carcinogenesis through the cGMP/PKG signaling pathway.

doi: 10.1038/s41419-021-03440-1

Figure Lengend Snippet: Fig. 7 Schematic diagram of the ZEB1/PRTG/cGMP/PKG regulatory mechanism in H. pylori-infected gastric cells. H. pylori promotes ZEB1 stabilization and recruitment to PRTG promoter, and which resulted the upregulation of PRTG expression. Then, PRTG activated the subsequent cGMP/PKG signaling pathway to perform oncogenic activities. PKG inhibitor KT5823 antagonizes the effect of PRTG-induced gastric cancer progression and enhance the effect of chemotherapy.

Article Snippet: The following primary antibodies were used: anti-PRTG antibody (CF501394, OriGene), ZEB1(21544-1-AP, Proteintech), E-Cadherin (13-1700, Invitrogen), N-Cadherin (33-3900, Invitrogen), Vimentin (ab92547, abcam), Snail (26183-1-AP, Proteintech), sGC (ab189176, abcam), PKG1 (3248, CST), PKG2 (55138-1-AP, Proteintech), PDE5A (ab28761, abcam), VASP (13472-1-AP, Proteintech), PhosphoVASPSer239 (ab194747, abcam), GAPDH (sc-47724, Santacruz), Caspase-3 (19677-1-AP, Proteintech), p21 (10355-1-AP, Proteintech), Bid (10988-1-AP, Proteintech), BCL2 (12789-1-AP, Proteintech), PhosphoH2AXSer139 (2577, CST), BIRC3 (24304-1-AP, Proteintech), HDAC1 (sc-81598, Santa Cruz), Ubiquitin (10201-2-AP, Proteintech).

Techniques: Infection, Expressing