Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) Immunoblot analysis of STAT1 and STAT2 protein levels in PARP7 WT and KO MEF cells. The experiment was repeated twice. (B) Co-immunoprecipitation (coIP) analysis showing that STAT1 and STAT2 interact with PARP7. FLAG-PARP7 was ectopically expressed in HEK293T cells. FLAG-PARP7 was immunoprecipitated and then blotted for endogenous STAT1 and STAT2. The experiment was repeated twice. (C and D) Immunoblot analysis of ADP-ribosylated STAT1 (C) and STAT2 (D) in PARP7 WT and KO MEF cells. Cells were lysed in the presence of biotin-NAD + or NAD + . Proteins that were ADP-ribosylated by biotin-NAD + were pulled down with streptavidin beads and then blotted for STAT1 or STAT2. Samples treated with NAD + were used as a negative control. Both experiments were repeated three times. (E) PARP7 WT, but not the H532A mutant, ADP-ribosylates STAT1. GFP-tagged empty vector (EV), WT PARP7, or the H532 mutant were co-transfected with FLAG-STAT1 into HEK293T cells. FLAG-STAT1 was pulled down to detect interaction with PARP7 (blotted with GFP antibody) and ADP-ribosylation (blotted with ADP-ribose antibody). The experiment was repeated twice. See also .

Article Snippet: Recombinant human STAT1 (1 μg, MCE #HY- P71335 ) and STAT2 (1 μg, Sinobiological #S53-54G) were incubated with purified GST-PARP7 (0.5 μg) in ADP-ribosylation reaction buffer containing 50 mM Tris-HCl (pH 8.0), 4 mM MgCl 2 , 0.2 mM DTT, and 100 μM NAD + (Sigma-Aldrich).

Techniques: Western Blot, Immunoprecipitation, TNKS1 Histone Ribosylation Assay, Negative Control, Mutagenesis, Plasmid Preparation, Transfection

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) HEK293T cells were transfected with FLAG-WT or H532A mutant PARP7 overnight. FLAG-PARP7 was detected by immunofluorescence with anti-FLAG (green) antibodies. Endogenous STAT1 was detected by immunofluorescence with Alexa Flour 647. Nuclei were stained with Hoechst stain (blue) (scale bar: 2 μm). Right: quantification of STAT1 and PARP7 co-localization using Pearson’s correlation coefficient. Data are presented as mean ± SD. *** p < 0.001, unpaired t test. (B) HEK293T cells were transfected with FLAG-WT or H532A mutant PARP7 overnight. FLAG-PARP7 was detected by immunofluorescence with anti-FLAG (green) antibodies. Endogenous STAT2 was detected by immunofluorescence with Alexa Flour 647. Nuclei were stained with Hoechst stain (blue) (scale bar: 2 μm.). Right: quantification of STAT2 and PARP7 co-localization using Pearson’s correlation coefficient. Data are presented as mean ± SD. *** p < 0.001, unpaired t test. All experiments were repeated twice. See also .

Article Snippet: Recombinant human STAT1 (1 μg, MCE #HY- P71335 ) and STAT2 (1 μg, Sinobiological #S53-54G) were incubated with purified GST-PARP7 (0.5 μg) in ADP-ribosylation reaction buffer containing 50 mM Tris-HCl (pH 8.0), 4 mM MgCl 2 , 0.2 mM DTT, and 100 μM NAD + (Sigma-Aldrich).

Techniques: Transfection, Mutagenesis, Immunofluorescence, Staining

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) Western blot analysis of STAT1 and STAT2 in PARP7 WT or KO MEF cells after treatment of MG132 at 20 μМ (left) and cycloheximide (CHX) at 50 μМ (right). The experiment was repeated three times. (B) Western blot analysis of STAT1 and STAT2 in PARP7 WT or KO MEF cells after bafilomycin A1 treatment at 5 μМ. The experiment was repeated three times. (C) coIP analysis of p62 and PARP7 WT or H532A catalytic mutant. FLAG-tagged PARP7 WT and H532A mutant were transfected into HEK293T cells, and coIP was performed to detect interactions between p62 and PARP7 WT or H532A mutant. The experiment was repeated three times. (D) Co-localization of hemagglutinin (HA)-tagged p62 with GFP-tagged PARP7 WT or H532A mutant in HEK293T cells. HA-p62 was detected by immunofluorescence with anti-HA (red) antibody. Nuclei were stained with Hoechst stain (scale bar: 5 μm). The experiment was repeated twice. (E) PARP7 promotes STAT1 and STAT2 ubiquitination. HEK293T cells were co-transfected with FLAG-STAT1 (left) or FLAG-STAT2 (right) and GFP-tagged PARP7 WT or H532A mutant. STAT1 or STAT2 was immunoprecipitated by anti-FLAG affinity resins, and the ubiquitination of STAT1 or STAT2 was analyzed by western blot. The experiment repeated twice. (F) PARP7 promotes p62 interaction with STAT1 and STAT2. HEK293T cells were co-transfected with FLAG-STAT1 (left) or FLAG-STAT2 (right) and GFP-tagged PARP7 WT or H532A mutant. STAT1 or STAT2 was immunoprecipitated by anti-FLAG affinity resin and blotted for p62. The experiment was repeated twice. (G) p62 knockdown diminishes the difference in STAT1 and STAT2 levels between PARP7 WT and KO MEF cells. STAT1 and STAT2 protein levels in PARP7 WT and KO MEF cells with or without p62 knockdown were analyzed by western blots. The experiment was repeated twice. (H) Proposed model depicting the negative regulation of STAT1 and STAT2 by PARP7. PARP7 binds and ADP-ribosylates STAT1 and STAT2. The ADP-ribosylation recruits E3 ubiquitin ligases, leading to the ubiquitination of STAT1 and STAT2. The ubiquitinated STAT1 and STAT2 then recruits p62, leading to autophagy-mediated degradation. Created with BioRender.com . See also – .

Article Snippet: Recombinant human STAT1 (1 μg, MCE #HY- P71335 ) and STAT2 (1 μg, Sinobiological #S53-54G) were incubated with purified GST-PARP7 (0.5 μg) in ADP-ribosylation reaction buffer containing 50 mM Tris-HCl (pH 8.0), 4 mM MgCl 2 , 0.2 mM DTT, and 100 μM NAD + (Sigma-Aldrich).

Techniques: Western Blot, Mutagenesis, Transfection, Immunofluorescence, Staining, Ubiquitin Proteomics, Immunoprecipitation, Knockdown

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Molecular docking and dynamic simulations of ROF and JAKs/STATs signaling molecules (A) The binding modes of ROF toward JAK2, JAK3, STAT1, and STAT3. (B–F) The dynamic simulations of JAK2-ROF and JAK3-ROF complexes. The HBond (B), RMSD curves (C), RMSF values (D), Rg values (E), and SASA (F) were analyzed by GROMACS.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: Binding Assay

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Effects of ROF on the JAK2/3-STAT1/3 signaling pathways in Con A-induced hepatitis (A) The phosphorylated levels of JAK2, STAT1, and STAT3 were measured in hepatic tissues using IHC staining ( n = 3; scale bar = 40 μm). (B) The IODs of each protein were detected by using ImageJ. (C) The JAK2/3-STAT1/3 signaling molecules were measured in hepatic tissues with Western blot analysis ( n = 3). The gray value analysis was standardized using the intensity of β-actin and is depicted in bar graphs. The results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs the Con A group.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: Protein-Protein interactions, Immunohistochemistry, Western Blot

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Effects of ROF on Th1/Th17 cells differentiation in vitro (A) The purity of isolated CD4 + T cells was measured by flow cytometry. (B) The viability of CD4 + T cells following 5–20 μM ROF treatments for 24 h was assessed utilizing the CCK-8 assay. (C, D) CD4 + T cells were pre-exposed for 1 h to ROF (20 μM), TOF (1 μM), or ROF (20 μM) + TOF (1 μM), followed by stimulation with Con A for 24 h. The levels of p-JAK2, p-JAK3, p-STAT1, and p-STAT3 were monitored by Western blotting and normalized to each protein’s total concentration in the graphs. (E) The generation of IFN-γ or IL-17 in the cellular supernatants of Th1 or Th17 subtypes were measured using ELISA. (F) The gene expression levels of T-bet or RORγt in differentiated T cells were evaluated by PCR. The values are presented as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001 vs the Con A group. # P < 0.05, ## P < 0.01, ### P < 0.001 vs the ROF only group or TOF only group.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: In Vitro, Isolation, Flow Cytometry, CCK-8 Assay, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Effects of ROF on primary hepatocytes apoptosis in vitro (A) Primary hepatocytes were pretreated with ROF (5, 10, 20 μM) for 6 h and treated with or without Con A (10 μg/mL) for 24 h, the cell viability of each group was analyzed by CCK-8 assay. (B) Flow cytometry analysis of apoptosis in primary hepatocytes. (C) Primary hepatocytes were pretreated with ROF (20 μM), TOF (1 μM), or ROF (20 μM) + TOF (1 μM) for 6 h, and subsequently triggered with Con A (10 μg/mL) for 24 h. The IL-6, p-JAK2, p-STAT1, p-STAT3, BNIP3, and Bax expression levels were evaluated by using Western blotting. (D) The quantification and standardization of the protein expression were performed using ImageJ. The results are presented as mean ± SEM of three independent experiments. ** P < 0.01, *** P < 0.001 vs the Con A group. # P < 0.05, ## P < 0.01 vs the ROF only group or TOF only group.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: In Vitro, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot