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: iScience

Article Title: RNF126 mediates fetal growth restriction via ubiquitination-dependent degradation of the MYH9/MYH10 complex

doi: 10.1016/j.isci.2026.116285

Figure Lengend Snippet: ER Stress mediates the effect of RNF126 on trophoblast function (A) Western blot confirms stable overexpression of Flag-RNF126 (F-RNF126) in BeWo cells. Representative blot is shown from three independent experiments. (B and C) Stable expression of RNF126 inhibited proliferation (B) and promoted apoptosis (C) in BeWo cells. Data are shown as representative images and quantitative analyses ( n = 3 independent experiments). Scale bars, 50 μm. (D and E) The GO and KEGG enrichment analysis plot of the differentially expressed genes (DEGs; |Log2 FoldChange|>0.58, p < 0.05) between two groups. (F) Gene set enrichment analysis of RNA-seq data shows the enrichment of unfolded-protein-response-signaling related genes in OE-RNF126 cells compared to OE-NC cells. (G and H) Representative western blots and quantification show increased GRP78/BiP, pPERK, and CHOP with RNF126 overexpression, and reduced levels upon RNF126 knockdown under TM- or TG-induced ER stress for 24h ( n = 3 independent experiments). (I and J) Representative images and quantification show that 24h treatment with 4-PBA (1 mM) reverses RNF126 overexpression-induced changes in cell proliferation (I) and apoptosis (J) ( n = 3 independent experiments). Scale bars, 50 μm. Data in B, C, H, I, and J are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, n.s. not significant.

Article Snippet: GRP78/Bip , HUABIO , Cat#HA722202, RRID: AB_3718306.

Techniques: Western Blot, Over Expression, Expressing, RNA Sequencing, Knockdown