Journal: PLOS Pathogens

Article Title: N-linked glycosylation of NS1 protein modulates progeny virion assembly in orthoflaviviruses

doi: 10.1371/journal.ppat.1014408

Figure Lengend Snippet: (A) Detection of cell viability. CCK-8 assay showing the viability of BHK-21 cells after treatment with different concentrations of tunicamycin (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 μg/ml) for 6 h in BHK-21 cells. Means and SDs from three independent experiments are presented. (B) Effects of NS1 deglycosylation on ER stress. BHK-21 cells in 12-well plates were transfected with the indicated NS1-expression plasmids (1000 ng/well). Cell lysate was harvested at 48 h post transfection for western blot analysis using anti-GRP78 pAb (1:3000) or anti-GAPDH mAb (1:5000). Cells treated with 10 mg/ml tunicamycin for 6 h were served as a positive control. The intensities of the indicated protein bands were quantified by using ImageJ software. Data were shown as the fold-change of GRP78/GAPDH. (C) Effects of NS1 deglycosylation on the three UPR signaling branches. Hela cells in 6-well plates were infected with the rTMUV or rTMUV-NS1 N130A/N175A/N207A at 10 4 TCID 50 . Cell lysates were harvested at 48 h post infection and subjected to western blot analysis using anti-GRP78 pAb (1:3000), anti-PERK mAb (1:1000), anti-p-PERK mAb (1:1000), anti-XBP1s mAb (1:1000), anti-ATF6 mAb (1:1000), anti-NS1 mAb (clone 10F12) or anti-GAPDH mAb (1:5000). The intensities of the indicated protein bands were quantified by using ImageJ software. Data were shown as the fold-change of GRP78/GAPDH, p-PERK/PERK, XBP1s/GAPDH and ATF6/GAPDH, respectively. (D) Detection of viral titers after overexpression with NS1 or NS1 N130A/N175A/N207A . BHK-21 cells in 12-well plates were transfected with the indicated NS1-expression plasmids (1000 ng/well). At 12 h post transfection, cells were infected with rTMUV at 300 TCID 50 . Cell culture supernatants were collected at 48 h post infection and viral titers were then determined by TCID 50 assays. Means and SDs from three independent experiments are presented. *, P < 0.05; ns, not significant. (E) Detection of intracellular and extracellular NS1 after treatment with tunicamycin. BHK-21 cells in 6-well plates were transfected with the indicated NS1-expression plasmids (2000 ng/well). At 42 h post transfection, cells were treated with 5 mg/ml or 10 mg/ml tunicamycin in 0.1% DMSO for 6 h. Cell culture supernatant and cell lysate was harvested and subjected to western blot analysis using anti-Flag mAb (1:5000) or anti-β-tubulin mAb (1:5000). Empty triangles represent glycosylated NS1, while filled triangles correspond to unglycosylated NS1. (F and G) Detection of levels of NS1 and viral titers in supernatants from TMUV-infected cells after treatment with tunicamycin. BHK-21 cells in 12-well plates were infected with the rTMUV at 300 TCID 50 . At 42 h post infection, cells were treated with either 0.1% DMSO or 10 mg/ml tunicamycin in 0.1% DMSO for 6 h. Cell culture supernatant was harvested for indirect ELISA (F) or TCID 50 assays (G) . Means and SDs from three independent experiments are presented. **, P < 0.01; ***, P < 0.001.

Article Snippet: Anti-GAPDH, anti-His tag, anti-β-tubulin, anti-GM130, anti-GFP tag, anti-PERK, anti-p-PERK mAbs and anti-GRP78 pAbs, were purchased from ABclonal (Wuhan, China).

Techniques: CCK-8 Assay, Transfection, Expressing, Western Blot, Positive Control, Software, Infection, Over Expression, Cell Culture, Indirect ELISA

Journal: PLOS Pathogens

Article Title: N-linked glycosylation of NS1 protein modulates progeny virion assembly in orthoflaviviruses

doi: 10.1371/journal.ppat.1014408

Figure Lengend Snippet: (A) Growth kinetics of rWNV Kunjin , rYFV, and their mutants in BHK-21 cells. BHK-21 cells in 12-well plates were infected with rWNV Kunjin , rYFV or their various mutants at 300 TCID 50 , and the culture supernatants were collected at the indicated time points. The viral titers were then determined by TCID 50 assays. Means and SDs from three independent experiments are presented. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. (B) Plaque morphology of rWNV Kunjin , rYFV and their mutants on BHK-21 cells. BHK-21 cells in 12-well plates were infected with 50 TCID 50 of the indicated viruses and incubated in overlay medium supplemented with 2% FBS and 1% methyl cellulose. After 6 days, cells were fixed with 4% formaldehyde for 20 min and stained with 1% crystal violet for 1 min. Plaque sizes were measured via ImageJ software. (C) Effects of deglycosylated WNV Kunjin or YFV NS1 on ER stress. BHK-21 cells in 12-well plates were transfected with the indicated NS1-expression plasmids (1000 ng/well). Cell lysate was harvested at 48 h post transfection and subjected to western blot analysis using anti-GRP78 pAb (1:3000) or anti-GAPDH mAb (1:5000). Cells treated with 10 mg/ml tunicamycin for 6 h were served as a positive control. Data were shown as the fold-change of GRP78/GAPDH. (D) Co-localization of WNV Kunjin or YFV NS1 with the ER and the Golgi apparatus in transfected cells. BHK-21 cells in 12-well plates were co-transfected with pDsRed2-ER/pDsRed2-Golgi and the indicated WNV Kunjin -NS1- or YFV-NS1-expression plasmids for 36 h. Then cells were incubated with mouse anti-Flag mAb, followed by staining with Alexa Fluor 488-conjugated anti-mouse IgG as the secondary antibody. The nuclei were stained with DAPI. The fluorescence signals were observed and imaged using confocal microscopy. The fluorescence intensity profile of NS1 (green) and ER/Golgi apparatus (red) was measured along the line drawn by ImageJ software. Scale bar, 10 μm or 2 μm. (E) Detection the interaction between exogenous E proteins and NS1 of WNV Kunjin or YFV. HEK-293T cells in 60-mm culture dishes were co-transfected with WNV Kunjin -E-GFP or YFV-E-GFP (2000 ng/dish) and the indicated NS1-expression plasmids (2000 ng/dish) for 24 h. The cell lysate then was subjected to coimmunoprecipitation with anti-GFP mAb and western blot analysis. (F) Stability of orthoflavivirus E protein. CHX chase analysis was performed to evaluate the stability of WNV Kunjin or YFV E protein in transfected cells. HEK-293T cells in 6-well plates were co-transfected with the E-2*Strep (1000 ng/well) and indicated NS1-expression plasmids (1000 ng/well). After 24 h post-transfection, 50 μg/ml of CHX with DMSO or 10 μM MG132 were added to the cell culture. Samples were collected at the indicated time points and then were subjected to western blot analysis using anti-Flag mAb (1:5000), anti-Strep mAb (1:3000) or anti-GAPDH mAb (1:5000). The intensities of the indicated protein bands were quantified by using ImageJ software. Data were shown as the fold-change of E/GAPDH.

Article Snippet: Anti-GAPDH, anti-His tag, anti-β-tubulin, anti-GM130, anti-GFP tag, anti-PERK, anti-p-PERK mAbs and anti-GRP78 pAbs, were purchased from ABclonal (Wuhan, China).

Techniques: Infection, Incubation, Staining, Software, Transfection, Expressing, Western Blot, Positive Control, Fluorescence, Confocal Microscopy, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Overexpression of Circular RNA circTTN Promotes Ferroptosis in Sepsis‐Induced Cardiomyopathy

doi: 10.1111/jcmm.71271

Figure Lengend Snippet: circTTN interacts with HSPA5. (A) Docking assay of the interaction of circTTN with HSPA5; (B) The binding site between HSPA5 and circTTN; (C) Immunofluorescence and FISH colocalisation analysis for HSPA5 and circTTN in HL‐1; (D) RIP‐qPCR detection of HSPA5 and circTTN; (E) Western blot detection of HSPA5 in RNA pull‐down of wild type (WT) and mutated (Mut) circTTN overexpressed HL‐1 cell samples. Three biological repeats were performed for all qPCR detections. Student's t ‐test was used for two‐group comparison, * p < 0.05, ** p < 0.05, *** p < 0.001.

Article Snippet: Primary antibodies for GAPDH (1:6000, M20006S, Abmaret), H4 (1:800, PTM‐1009, PTM Bio), GPX4 (1:600, 67,763–1‐IG, Proteintech), SLC7A11 (1:600, 26,864–1‐AP, Proteintech), β‐Actin (1:40000, 66,009–1‐Ig, Proteintech), and HSPA5 (1:8000, A23453, ABClonal) were incubated overnight at 4°C.

Techniques: Docking Assay, Binding Assay, Immunofluorescence, Western Blot, Comparison

Journal: Journal of Advanced Research

Article Title: Tankyrase activity is essential for asymmetric division and chromosome segregation in oocyte meiosis

doi: 10.1016/j.jare.2025.07.008

Figure Lengend Snippet: TNKS affects distributions of Formin2 and ER for chromosome migration in mouse oocytes. (A) Representative images of Formin2 in MI stage oocyte from the control and JW55 treatment groups. Green, Formin2. Cyan, DNA. Bar = 20 μm. (B) Relative intensity of Formin2 at spindle poles in oocytes from the control (n = 47) and JW55 treatment (n = 51) groups. ***, P < 0.001. (C) Band intensity analysis of Formin2 expression in the MI stage oocytes from control and JW55 treatment groups. *, P < 0.05. (D) Representative images of ER-tracker in MI stage oocyte from the control and JW55 treatment groups. Black, ER-tracker. Bar = 20 μm. (E) The percentage of abnormal ER distribution in oocytes from the control (n = 50) and JW55-treated (n = 56) groups. *, P < 0.05. (F) Relative intensity of ER-tracker in oocytes from the control (n = 50) and JW55-treated (n = 56) groups. ***, P < 0.001. (G) Western blot of GRP78 in MI stage oocytes from control and JW55 treatment groups. (H) Band intensity analysis of GRP78 in the control and JW55 treatment groups. *, P < 0.05. (I) Representative images of a relative position change between chromosomes and Formin2 in ATI stage oocytes from the control and JW55 treatment groups. Green, Formin2. Cyan, DNA. Bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Rabbit anti-α-Tubulin antibody (11224-1-AP), rabbit polyclonal anti-GAPDH antibody (10494-1-AP), rabbit anti-GRP78 antibody (11587-1-AP), rabbit anti-TNKS1 antibody (18030-1-AP), rabbit anti-Ran antibody (10469-1-AP), and rabbit anti-Rab11a antibody (67902-1-Ig), rabbit anti-Securin antibody (18040-1-AP), rabbit anti-CDC25C antibody (66912-1-Ig), rabbit anti-Formin2 antibody (11259-1-AP), rabbit anti-BubR1 (11504-2-AP) were from Proteintech.

Techniques: Migration, Control, Expressing, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Glutathione S-transferase inhibitor NBDHEX induces ROS-dependent immunogenic cell death and enhances PD-1 blockade in DLBCL

doi: 10.1186/s12964-026-02810-6

Figure Lengend Snippet: ROS-dependent ER stress mediates NBDHEX-induced cell death in DLBCL cells. A – B Apoptosis of SU-DHL-2 and SU-DHL-4 cells pretreated with or without NAC (5 mM) for 1 h, followed by NBDHEX treatment for 24 h. Vehicle (DMSO) and NAC-only groups were included. Representative flow cytometry plots are shown in ( A ), with quantification in ( B ) ( n = 3). C Representative immunofluorescence images of γ-H2A.X foci in SU-DHL-2 and SU-DHL-4 cells pretreated with or without NAC (5 mM) for 1 h, followed by NBDHEX treatment for 12 h. Scale bar: 10 μm. D Quantification of γ-H2A.X foci in panel C. Cells with ≥ 10 foci were considered positive ( n = 3). E Western blot analysis of apoptosis-related protein levels in SU-DHL-2 and SU-DHL-4 cells pretreated with or without NAC, followed by NBDHEX treatment for 24 h. GAPDH served as a loading control. F Time-dependent changes in ER stress-related protein levels (p-PERK, p-eIF2α, GRP78, ATF4, and CHOP) in SU-DHL-2 and SU-DHL-4 cells after NBDHEX treatment, assessed by western blot. G Western blot analysis of ER stress-related proteins in cells treated with increasing concentrations of NBDHEX for 24 h. H Western blot detection of ER stress-related proteins in cells pretreated with NAC followed by NBDHEX exposure. I Apoptosis analysis by flow cytometry in SU-DHL-2 and SU-DHL-4 cells pretreated with or without 4-PBA (50 µM) for 1 h, followed by NBDHEX treatment for 24 h. n.s., not significant; *** p < 0.001

Article Snippet: Primary antibodies used in our study were: Phospho-Histone H2A.X (Ser139) (#9718, Cell Signaling Technology, 1:1000 dilution), GAPDH (#60004-1-Ig, Proteintech), PARP (#A19596, Abclonal), Cleaved Caspase-3 (#9664, Cell Signaling Technology), Bcl2 (#12789-1AP, Proteintech), BAX (#50599-2-Ig, Proteintech), Cytochrome C (#A4912, Abclonal), GRP78 (#A0241, Abclonal), Phospho-PERK (Thr980) (#3179, Cell Signaling Technology), PERK (#3192, Cell Signaling Technology), eIF2a (#5324, Cell Signaling Technology), Phospho-eIF2a (Ser51) (#3398, Cell Signaling Technology), CHOP (#5554, Cell Signaling Technology), ATF4 (#A0201, Abclonal).

Techniques: Flow Cytometry, Immunofluorescence, Western Blot, Control

Journal: Veterinary Research

Article Title: The CP312R protein of African swine fever virus inhibits host protein translation via the BiP/PERK/eIF2 α pathway

doi: 10.1186/s13567-025-01688-5

Figure Lengend Snippet: The ectopically expressed pCP312R inhibits the expression of BiP . A pCP312R is localized in the endoplasmic reticulum (ER) of the cell. HEK293T cells cotransfected with pCA-CP312R-Flag (0.2 μg) and pDsRed-calnexin (expressing calnexin, an ER marker) (0.5 μg). At 18 h post-transfection (hpt), the cells were analyzed by laser confocal microscopy. Scale bars: 2 μm. B pCP312R is localized in the ER in the ASFV-infected PAMs. PAMs were infected with ASFV-WT (MOI = 1). At 12 h post-infection (hpi), the cells were immunostained with anti-pCP312R and anti-KDEL antibodies. Cell nuclei were stained with DAPI. Scale bars: 2 μm. C ASFV infection induces in the differential expression of BiP. PAMs were infected with ASFV-WT (MOI = 1). At 12, 18, and 24 hpi, the cells were collected for western blotting analysis using anti-BiP, anti-pCP312R, or anti- β -actin antibodies. D The ectopic expression of pCP312R leads to a reduction in BiP expression. HEK293T cells were transfected with an increasing amount of pCA-CP312R-Flag (0, 0.5, 1, 1.5, or 2 μg). At 18 hpt, the cells were collected for western blotting analysis using anti-BiP, anti-Flag, or anti- β -actin antibodies. E Knockdown of CP312R partially restores the expression of BiP during ASFV infection. PAMs were transfected with the siCP312R or a siNC. At 6 hpt, the cells were infected with ASFV-WT (MOI = 0.1). At 12 hpi, the cells were collected for western blotting analysis using anti-CP312R, anti-BiP, or anti- β -actin antibodies. F pCP312R reduces BiP expression through the proteasomal pathway. HEK293T cells were transfected with pCAGGS or pCA-CP312R-Flag (1 μg each). At 18 hpt, the cells were incubated with 10 µM bafilomycin A1 (BafA1, an autolysosome inhibitor), 10 µM Z-VAD-FMK (ZVF, a caspase inhibitor), or 10 µM MG-132 (a proteasome inhibitor) for 6 h. The cells were subjected to western blotting analysis using anti-BiP, anti-Flag, or anti- β -actin antibodies.

Article Snippet: Mouse anti-p72 monoclonal antibody (MAb) (catalog no. Anti PPA 1BC11, INGENASA), mouse anti-puromycin MAb (catalog no. MABE343, Sigma-Aldrich), rabbit anti- β -actin PAbs (catalog no. AC026, ABclonal), mouse anti-Flag MAb (catalog no. M8823, Sigma-Aldrich), mouse anti-GST MAb (catalog no. K200006M, Solarbio), rabbit anti-Myc PAbs (catalog no. YN5506, ImmunoWay), rabbit anti-KDEL PAbs (catalog no. ab214714, Abcam), rabbit anti-BiP PAbs (catalog no. A0241, ABclonal), mouse anti-PERK MAb (catalog no. 3192S, CST), rabbit anti-p-PERK PAbs (catalog no. BS66100, Bioworld), rabbit anti-p-eIF2 α PAbs (catalog no. 3398S, CST), and rabbit anti-eIF2 α PAbs (catalog no. 9722S, CST) are commercially available.

Techniques: Expressing, Marker, Transfection, Confocal Microscopy, Infection, Staining, Quantitative Proteomics, Western Blot, Knockdown, Incubation