Journal: iScience

Article Title: DNAJB9 promotes epithelial-mesenchymal transition in the nasal epithelium of chronic rhinosinusitis by enhancing TRIM22-mediated IκBα degradation

doi: 10.1016/j.isci.2026.116681

Figure Lengend Snippet: DNAJB9 promotes epithelial-mesenchymal transition in nasal epithelial cells (A) Comparison of primary nasal epithelial cell morphology between control and 0.5 μg/mL tunicamycin treated for 48 h (magnification: 400×; scale bars, 1000 μm). Tunicamycin treatment induced the transition of cells from epithelial-like to mesenchymal-like morphology. (B) hNECs were treated with tunicamycin at concentrations ranging from 0 to 0.5 μg/mL for 48 h. Western blot analysis was used to detect the protein expression levels of DNAJB9 and EMT-related markers. GAPDH was used as a control. (C) Quantitative analysis of DNAJB9 and EMT-related markers protein levels. (D) Quantitative analysis of the relative mRNA expression of DNAJB9 and EMT-related markers. (E) BEAS-2B cells were treated for 48 h under the conditions indicated in the figure, and western blot analysis was used to detect the protein expression levels of DNAJB9 and EMT-related markers. GAPDH was used as a control. (F) Quantitative analysis of DNAJB9 and EMT-related markers protein levels. Data in (C), (D), and (F) are presented as mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Tunicamycin , MCE , Cat# HY-A0098.

Techniques: Comparison, Control, Western Blot, Expressing, Standard Deviation

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: The Cell Counting Kit-8 (CCK-8) (MedChemExpress, Shanghai, China) was used to evaluate the viability of BHK-21 cells when exposed to tunicamycin (Tun) (MedChemExpress, Shanghai, China), following the instructions provided by the manufacturer.

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: The Cell Counting Kit-8 (CCK-8) (MedChemExpress, Shanghai, China) was used to evaluate the viability of BHK-21 cells when exposed to tunicamycin (Tun) (MedChemExpress, Shanghai, China), following the instructions provided by the manufacturer.

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

Journal: Cell Death & Disease

Article Title: The OAS1-FASN axis promotes pancreatic cancer by coordinating lipogenic stress and the unfolded protein response

doi: 10.1038/s41419-026-08922-8

Figure Lengend Snippet: Transmission electron microscopy images of ER ultrastructure ( A ) and statistical analysis of ER surface density ( B ) in indicated groups of Patu-8988 based on OAS1 knockdown and Tunicamycin treatment (scale bar: 500 nm). BODIPY lipid staining and BiP-ATF4 IF staining images ( C ), statistical analysis of BiP intensity ( D ), and ATF4 nuclear translocation ratio ( E ) of indicated groups of Patu-8988 based on OAS1 knockdown and Tunicamycin treatment with DAPI (scale bar: 20 μm). F Western blotting of BiP and OAS1 expression across indicated groups of Patu-8988 and PDC0034. Western blotting analysis ( G ) and corresponding gray density heatmaps ( H ) showing the protein levels of OAS1 and ATF4 in the indicated groups. Representative images ( I ) and statistical analysis ( J ) of colony formation assay of Patu-8988 based on ATF4 knockdown upon OAS1 deficiency. Representative images ( K ) and statistical analysis ( L ) of the Transwell assay of Patu-8988 and PDC0034 based on ATF4 knockdown upon OAS1 deficiency. M Immunofluorescence staining of ATF4, ER-Tracker, and BODIPY in Patu-8988 cells across the four experimental groups (scale bar: 20 μm). N BODIPY lipid staining, ER Tracker staining, and BiP-ATF4 IF staining images of indicated groups of Patu-8988 based on OAS1 knockdown and TVB-2640 treatment with DAPI (scale bar: 20 μm). For IF staining in ( C , M , N ), primary antibodies were diluted as specified in Supplementary Table (OAS1 1:200, BiP 1:100, ATF4 1:200) and incubated overnight at 4 °C, followed by either FITC-conjugated Goat anti-rabbit IgG (1:100) or Cy3-conjugated Goat anti-rabbit IgG (1:100) incubation for 1 h at room temperature. BODIPY and ER tracker ( M , N ) were incubated at room temperature for 20 min. For WB assay in ( F , G ), primary antibodies (OAS1 1:1000, BiP 1:1000, ATF4 1:1000, β-actin 1:1000) were diluted and incubated overnight at 4 °C, followed by Goat Anti-Rabbit IgG H&L (HRP) (1:5000) incubation for 1 h at room temperature.

Article Snippet: ER stress inducer Tunicamycin (2.5 μM for 24 h; HY-A0098, MedChemExpress), ER pathways inhibitors ISRIB (0.5 μM for 24 h; HY-12495A, MedChemExpress), 4μ8C (50 μM for 24 h; HY-19707, MedChemExpress), and Ceapin-A7 (2 μM for 24 h; HY-108434, MedChemExpress) were used as pharmacological interventions of ER stress.

Techniques: Transmission Assay, Electron Microscopy, Knockdown, Staining, Translocation Assay, Western Blot, Expressing, Colony Assay, Transwell Assay, Immunofluorescence, Incubation