Journal: bioRxiv

Article Title: The active secretion of a subunit of IL-12 by tissue cells is regulated by Valosin-Containing Protein and intracellular calcium redistribution

doi: 10.64898/2026.01.28.702376

Figure Lengend Snippet: (A) Percentage of UPR+ 7-AAD-L cells (GFP+ for XBP-1 binding to Unfolded Protein Response Element) after treatment with vehicle (DMSO), 3.12μM ionomycin, or 1.56μM CB-5083 then analyzed with flow cytometry. (B) Luciferase assay of supernatants from p35NL cells pre-treated with PERK inhibitor AMG PERK 44 for 2 hours then stimulated with 3.12 μM ionomycin for 24 hours. (C) Luciferase assay of supernatants from p35NL cells pre-treated with PERK inhibitor AMG PERK 44 for 2 hours then stimulated with 1.56μM CB-5083 for 24 hours. (D) Luciferase assay of supernatants from p35NL cells pre-treated with IRE1 inhibitor MKC8866 for 2 hours then stimulated with 3.12 μM ionomycin for 24 hours. (E) Luciferase assay of supernatants from p35NL cells pre-treated with IRE1 inhibitor MKC8866 for 2 hours then stimulated with 1.56μM CB-5083 for 24 hours. (F) Luciferase assay of supernatants from p35NL cells pre-treated with ATF6 inhibitor Ceapin-A7 for 2 hours then stimulated with 3.12 μM ionomycin for 24 hours. (G) Luciferase assay of supernatants from p35NL cells pre-treated with ATF6 inhibitor Ceapin-A7 for 2 hours then stimulated with 1.56μM CB-5083 for 24 hours. (n=3). Data is represented as raw Relative Luminescence Units (RLUs). Significant changes in luminescence were determined through ordinary one-way ANOVAs of cells stimulated with ionomycin only or CB-5083 only (control). *, p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant

Article Snippet: The following inhibitors were used to target specific arms of the UPR response: AMG PERK 44 for PERK, MKC8866 for IRE1α, and Ceapin-A7 for ATF6 (Med Chem Express).

Techniques: Binding Assay, Flow Cytometry, Luciferase, Control

Journal: mBio

Article Title: Quantitative proteomic analysis identifies the unfolded protein response as a host pathway co-opted by ASFV to promote replication

doi: 10.1128/mbio.03242-25

Figure Lengend Snippet: D117L activates three UPR pathways through targeting key UPR components. ( A ) Heatmap of ASFV proteins identified by proteomics analysis across infection. ( B ) PPI network analysis of the virus-host interaction. ( C ) HEK293T cells were transfected with vector or Flag-D117L for 24 h, and mRNA expression of Ddit3, Dnajc3, and Dnajb9 was detected by RT-qPCR. ( D ) HEK293T cells were transfected with vector, Flag-D117L, Flag-D117LΔ38-60, Flag-D117LΔ67-76, or Flag-D117LΔ96-117; each group was also co-transfected with ATF6-LUC and Renilla-TK. After 24 h post-transfection, luciferase activities were examined. ( E ) Mass spectrometry was performed to identify the interacting proteins of D117L and D117LΔ38-60, and the top 10 proteins were listed. ( F ) Kyoto Encyclopedia of Gene and Genomes pathway enrichment analysis of D117L interacting proteins. ( G and H ) Immunofluorescence analysis of the colocalization of Flag-tagged D117L (green) and Myc-tagged or endogenous HSPA5 (red) in iPAM cells. ( I ) Immunoprecipitation (IP) assay examining the interaction between HSPA5 and D117L. ( J and K ) IP assays assessing the interaction of D117L (WT) or D117LΔ38-60, D117LΔ67-76, and D117LΔ96-117 mutants with CANX or HSPA5 in HEK293T cells.Data are presented as mean + SD. PP values were calculated by a two-tailed unpaired t-test. ** P < 0.01, *** P < 0.001.

Article Snippet: The commercial antibodies used in this study included GRP78/BIP Rabbit Polyclonal Antibody (11587-1-AP, 1:1,000; Proteintech), Rabbit Anti-Phospho-PERK (Thr980) antibody (bs-3330R, 1:1,000; Bioss), PERK Rabbit pAb (A18196, 1:1,000; ABclonal), XBP1S-specific Polyclonal antibody (24868-1-AP, 1:1,000; Proteintech), Rabbit Monoclonal (EPR5253) to IRE1 (phosphoS724) ( AB124945 , 1:1,000; abcam), IRE1; ERN1 Polyclonal antibody (27528-1-AP, 1:1,000; Proteintech), ATF6 Polyclonal antibody (24169-1-AP, 1:1,000; Proteintech), Monoclonal Anti-Flag M2 antibody produced in mouse (F1804, 1:1,000; Sigma-Aldrich), Myc-Tag Rabbit mAb (C-terminal) (AE070, 1:1,000; ABclonal), MYC tag mouse monoclonal antibody (60003-2-1g, 1:1,000; Proteintech), Goat Anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, AlexaFluor 488 (A-11008, 1:1,000; Thermo Fisher Scientific), Goat Anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A11037, 1:1,000; Thermo Fisher Scientific), Goat Anti-mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A11005,1:1000, Thermo Fisher Scientific), Goat Anti-Mouse IgG (H + L) Superclonal Secondary Antibody, Alexa Fluor 488 (A28175, 1:1,000; Thermo Fisher Scientific), GAPDH mouse monoclonal antibody (TA802519M, 1:1,000; Origene), β-tubulin mouse monoclonal antibody (66240-1-1g, 1:1,000; Proteintech), and anti-β-actin monoclonal antibody (sc-8432, 1:1,000; Santa Cruz Biotechnology).

Techniques: Infection, Virus, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Luciferase, Mass Spectrometry, Immunofluorescence, Immunoprecipitation, Two Tailed Test

Journal: mBio

Article Title: Quantitative proteomic analysis identifies the unfolded protein response as a host pathway co-opted by ASFV to promote replication

doi: 10.1128/mbio.03242-25

Figure Lengend Snippet: Activation of UPR facilitates ASFV replication. ( A ) PAM cells were treated with or without inhibitor 4-PBA (50 µM), followed by ASFV challenge at 0.1 MOI for 24 h; P72 and P54 proteins were detected by Western blotting; and tubulin was used as a loading control. ( B ) PAM cells were treated with or without inhibitors, 4μ8C (5 µM), GSK2606414 (0.5 µM), or Ceapin-A7 (5 µM), and then infected with ASFV at 0.1 MOI for 24 h. P72 and P54 proteins were detected by Western blotting. ( C ) PAM cells were transfected with siRNA-NC (negative control), siRNA-Eif2AK3, siRNA-ERN1, or siRNA-ATF6 for 24 h and then challenged with ASFV at 0.1 MOI. P72 and P30 proteins were detected by Western blotting at 24 h; GAPDH was used as a loading control. ( D ) Fluorescence analysis of ASFV replication in PAM cells pretreated with or without inhibitors, 4μ8C (5 µM), GSK2606414 (0.5 µM), or Ceapin-A7 (5 µM). ( E–L ) The antiviral activities of the inhibitors against ASFV mRNA transcription. Relative mRNA expression levels of ASFV genes B646L and CP204L in PAM cells were quantified. PAM cells were treated with or without inhibitors or transfected with siRNAs and then infected with GFP-ASFV at 0.1 MOI for 24 h. Flow cytometry was used to analyze the percentage of infected positive cells ( M and O ). Viral titers were measured by TCID 50 assay at 72 h ( N and P ). Data presented as means + SD. P values were calculated by a two-tailed unpaired t -test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The commercial antibodies used in this study included GRP78/BIP Rabbit Polyclonal Antibody (11587-1-AP, 1:1,000; Proteintech), Rabbit Anti-Phospho-PERK (Thr980) antibody (bs-3330R, 1:1,000; Bioss), PERK Rabbit pAb (A18196, 1:1,000; ABclonal), XBP1S-specific Polyclonal antibody (24868-1-AP, 1:1,000; Proteintech), Rabbit Monoclonal (EPR5253) to IRE1 (phosphoS724) ( AB124945 , 1:1,000; abcam), IRE1; ERN1 Polyclonal antibody (27528-1-AP, 1:1,000; Proteintech), ATF6 Polyclonal antibody (24169-1-AP, 1:1,000; Proteintech), Monoclonal Anti-Flag M2 antibody produced in mouse (F1804, 1:1,000; Sigma-Aldrich), Myc-Tag Rabbit mAb (C-terminal) (AE070, 1:1,000; ABclonal), MYC tag mouse monoclonal antibody (60003-2-1g, 1:1,000; Proteintech), Goat Anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, AlexaFluor 488 (A-11008, 1:1,000; Thermo Fisher Scientific), Goat Anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A11037, 1:1,000; Thermo Fisher Scientific), Goat Anti-mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A11005,1:1000, Thermo Fisher Scientific), Goat Anti-Mouse IgG (H + L) Superclonal Secondary Antibody, Alexa Fluor 488 (A28175, 1:1,000; Thermo Fisher Scientific), GAPDH mouse monoclonal antibody (TA802519M, 1:1,000; Origene), β-tubulin mouse monoclonal antibody (66240-1-1g, 1:1,000; Proteintech), and anti-β-actin monoclonal antibody (sc-8432, 1:1,000; Santa Cruz Biotechnology).

Techniques: Activation Assay, Western Blot, Control, Infection, Transfection, Negative Control, Fluorescence, Expressing, Flow Cytometry, Two Tailed Test

Journal: Antioxidants

Article Title: Cross-Species Insights into In Vitro Maturation Defects of the Oocyte and Identification of Crucial Regulators for Sheep Oocyte Maturation

doi: 10.3390/antiox14121499

Figure Lengend Snippet: EFNA1 alleviates excessive misfolded protein accumulation in oocytes during IVM. ( A ) GSEA analysis of differential expression genes related to oxidative phosphorylation, glutathione metabolism, and reactive oxygen species in in vitro matured oocytes compared to in vivo . ( B ) Effect of 10 ng/mL EFNA1 on ROS and GSH levels in matured sheep oocytes. Intracellular ROS and GSH levels were measured using CM-H2DCFDA and CellTracker™ Blue CMF2HC fluorescent probes, respectively (Scale bar = 200 µm). ( C ) Effect of 10 ng/mL EFNA1 on protein aggregates content in matured sheep oocytes. The protein aggregates content was determined by proteoStat aggresome detection reagent labeling (Scale bar = 50 µm). ( D ) Effect of 10 ng/mL EFNA1 on proteasome activity in matured sheep oocytes. The proteasome activity was evaluated by Me4BodipyFL (Scale bar = 50 µm). ( E , F ) Immunofluorescence detection of ER stress-associated protein (GRP78, ATF6) in matured sheep oocytes cultured with or without EFNA1 (Scale bar = 50 µm). ( G ) Relative expression levels of ER stress-associated genes (GRP78, PERK, EIF2A, ATF4, ATF6, IRE1, XBP1) in matured sheep oocytes cultured with or without EFNA1 as analyzed by RT-PCR. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After IVM, oocytes were washed in PBS-PVA, fixed in 4% PFA for 30 min at room temperature, and permeabilized with 0.5% Triton X-100/0.5% BSA in PBS for 15 min. After blocking with 1% BSA-PBS for 1 h, oocytes were incubated overnight at 4 °C with primary antibodies against GRP78 (11587-1-AP; Proteintech, Wuhan, China), and ATF6 (24169-1-AP; Proteintech, Wuhan, China).

Techniques: Quantitative Proteomics, Phospho-proteomics, In Vitro, In Vivo, Labeling, Activity Assay, Immunofluorescence, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Antioxidants

Article Title: Cross-Species Insights into In Vitro Maturation Defects of the Oocyte and Identification of Crucial Regulators for Sheep Oocyte Maturation

doi: 10.3390/antiox14121499

Figure Lengend Snippet: NRXN1 mitigates aberrant lipid deposition and ER stress in oocytes during IVM. ( A ) Heatmaps showing scaled expression levels of lipid metabolism genes in oocytes from in vivo and in vitro matured oocytes. (Created in BioRender. Cui, J. (2025) https://BioRender.com/onf1fa2 accessed on 7 December 2025). ( B ) Effect of 100 ng/mL NRXN1 on lipid droplets content in matured sheep oocytes. Intracellular lipid droplets content was measured using Nile-red (Scale bar = 50 µm). ( C ) Effect of 100 ng/mL NRXN1 on fatty acid contents in matured sheep oocytes. The lipid content was determined by BODIPY 500/510 labeling (Scale bar = 50 µm). ( D , E ) Immunofluorescence detection of ER stress-associated protein (GRP78, ATF6) in oocytes cultured with or without NRXN1 (Scale bar = 50 µm). ( F ) Relative expression levels of ER stress-associated genes ( GRP78 , PERK , EIF2A , ATF4 , ATF6 , IRE1 , XBP1 ) in oocytes cultured with or without NRXN1 as analyzed by RT-PCR. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After IVM, oocytes were washed in PBS-PVA, fixed in 4% PFA for 30 min at room temperature, and permeabilized with 0.5% Triton X-100/0.5% BSA in PBS for 15 min. After blocking with 1% BSA-PBS for 1 h, oocytes were incubated overnight at 4 °C with primary antibodies against GRP78 (11587-1-AP; Proteintech, Wuhan, China), and ATF6 (24169-1-AP; Proteintech, Wuhan, China).

Techniques: Expressing, In Vivo, In Vitro, Labeling, Immunofluorescence, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Antioxidants

Article Title: Cross-Species Insights into In Vitro Maturation Defects of the Oocyte and Identification of Crucial Regulators for Sheep Oocyte Maturation

doi: 10.3390/antiox14121499

Figure Lengend Snippet: Combined EFNA1 and NRXN1 supplementation enhances oocyte developmental competence and rewires transcriptional landscape. ( A ) Effects of EFNA1 combined with NRXN1 (EN) supplementation during IVM on cleavage rate in abattoir-derived oocytes. ( B ) Representative images of day 6 and day 7 blastocysts in control and EN-treated groups (Scale bar = 400 µm). ( C , D ) Effects of EN supplementation during IVM blastocyst formation rate (per oocyte and per cleaved embryo) in abattoir-derived oocytes. ( E , F ) Representative images of blastocysts showing total cell number and quantification of total cell numbers (Scale bar = 100 µm). ( G – I ) Effects of EN supplementation during IVM on cleavage rate, blastocyst formation rate (per oocyte and per cleaved embryo) in LOPU-derived oocytes. Different letters denote significant differences, ns p > 0.05, * p < 0.05. ( J ) PCA showing the reproducibility between repetitive samples and the difference between groups. ( K ) DEGs between control and EN oocytes (co-treated with EFNA1 and NRXN1) were shown in the volcano map. Genes that expressed higher (up-regulated) in EN oocytes are shown in red, and genes that were lower expressed (down-regulated) are shown in blue. ( L ) GO enrichment of up-regulated DEGs and down-regulated DEGs. Red represents upregulation and blue represents downregulation. ( M ) Heatmaps showing scaled expression levels of ER stress-associated genes ( GRP78 , PERK , EIF2A , ATF4 , ATF6 , IRE1 , XBP1 ) in oocytes from control and EN-treated groups. Statistical significance: * p < 0.05.

Article Snippet: After IVM, oocytes were washed in PBS-PVA, fixed in 4% PFA for 30 min at room temperature, and permeabilized with 0.5% Triton X-100/0.5% BSA in PBS for 15 min. After blocking with 1% BSA-PBS for 1 h, oocytes were incubated overnight at 4 °C with primary antibodies against GRP78 (11587-1-AP; Proteintech, Wuhan, China), and ATF6 (24169-1-AP; Proteintech, Wuhan, China).

Techniques: Derivative Assay, Control, Expressing