Journal: bioRxiv

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1101/2025.09.11.675615

Figure Lengend Snippet: (A) A549 cells were treated with DMSO, KIRA6 (10 μM), or Ceapin-A7 (6 μM) for 24 hours. Alamar blue assay was used to assess cell viability based on cellular metabolic activity. (B-C) A549 cells were primed with DMSO or Tg (0.05 μM) in the absence or presence of KIRA6 (10 μM) or Ceapin-A7 (6 μM). Cell lysates were collected at 24 hours post-treatment. Xbp1s (B) or HERPUD1 (C) protein expression was assessed by western blot. (D-F) A549 cells were primed with DMSO or Tg (0.05 μM) in the presence or absence of KIRA6 (10 μM), prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or KIRA6 (10 μM). (D-F) Cell lysates were collected at 24 hpi to assess XBP1s and N gene expression by RT-qPCR (D, F) , or Xbp1s and HCoV-229E N protein expression by western blot (E) . (G-I) A549 cells were primed with DMSO or Tg (0.05 μM) in the presence or absence of Ceapin-A7 (6 μM) prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or Ceapin-A7 (6 μM). (G-I) Cell lysates were collected at 24 hpi to assess HERPUD1 and N by RT-qPCR (G, I) , or HERPUD1 and HCoV-229E N protein expression by western blot (H) . RT-qPCR data are normalized to actin and set relative to DMSO. (J) A549 cells were primed with DMSO or Tg (0.05 μM) in the presence or absence of KIRA6 (10 μM) or Ceapin-A7 (6 μM), alone or in combination, prior to infection with HCoV-229E (MOI 0.05). Inoculum was removed 2 hours later and replaced with media containing the respective inhibitor(s). Viral supernatants were collected 24 hpi and titrated by plaque assay on Huh7 cells. Graphs show mean +/− SEM from three independent experiments performed in triplicate (*p<0.05, ****p<0.0001). Representative western blots are shown.

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1:1000), ATF6 (CST 8089, dilution 1:1000), PERK (CST 5683, dilution 1:1000), XBP1s (CST 12782T, dilution 1:1000), HERPUD1 (Abcam ab150424, dilution 1:1000), eIF2α-P (Ser-51) (CST 3398S, dilution 1:1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1:10,000), beta-actin (Abcam ab8226, dilution 1:5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1:5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1:1000) were used for western blotting.

Techniques: Alamar Blue Assay, Activity Assay, Expressing, Western Blot, Infection, Gene Expression, Quantitative RT-PCR, Plaque Assay

Journal: Nature Communications

Article Title: Endoplasmic reticulum stress drives proteinuria-induced kidney lesions via Lipocalin 2

doi: 10.1038/ncomms10330

Figure Lengend Snippet: LCN2 expression evaluated by ( a ) immunohistochemistry (scale bar, 100 μm), ( b ) quantitative reverse transcriptase–PCR and ( c ) western blotting in kidneys in WT1 +/mut mice at 3 ( n =5), 4 ( n =6) and 6 ( n =7) weeks after birth and their control littermates ( n =5). LCN2 overexpression occurred before apparition of tubular lesions. ( d , e ) LCN2 protein (upper panel) and mRNA (lower panel) abundance in ( d ) mIMCD-3 cells and ( e ) in mouse renal primary cultured cells exposed to 1% albumin (Alb) for 24 h ( n =3). ( f ) Representative western blotting (upper panel) and quantification (lower panel) of LCN2 protein abundance in mIMCD-3 cells exposed to 1% albumin for 24 h, treated with NAC, Cd 2+ or Gd 3+ ( n =5). ( g ) LCN2 protein expression in mouse renal primary cultured cells exposed to 1% albumin or heat-denatured (denatured) 1% albumin for 24 h ( n =3). ( h ) CHOP and LCN2 protein, and ( i ) Lcn2 mRNA expression in mice injected with thapsigargin (Thap), tunicamycin (Tuni) or the vehicle (Ctrl). ( n =5 per group; scale bar, 100 μm; insert scale bar 10 μm). ( j ) LCN2 expression in mIMCD-3 cells exposed to Thap (left panel) or Tuni (right panel) for 24 h ( n =3). The different apparent size of LCN2 protein due to the inhibition of LCN2 glycosylation by Tuni is worth noting. ( k ) LCN2 protein (upper panel) and mRNA abundance (lower panel) in mIMCD-3 cells transiently transfected with different constructs (control, ATF4, ATF6, CHOP, unspliced XBP1 (XBP1u) and spliced XBP1 (XBP1p)). ( n =3). ( l ) Luciferase activity in mIMCD-3 cells co-transfected with different transcription factors and a Lcn2 promoter luciferase reporter ( n =3). ( m ) LCN2 protein and mRNA expression in wild-type ATF4 +/+ and ATF4 −/− MEFs exposed to 1% albumin or 0.5 μM thapsigargin (Th) for 24 h ( n =3). ( n ) LCN2 mRNA expression in ATF4 −/− MEFs transfected with empty vector (EV) or ATF4 construct and exposed to 1% albumin or 0.5 μM thapsigargin (Th) for 24 h ( n =3). Statistical analysis: one way analysis of variance followed by Tukey–Kramer test; Mann–Whitney test when only two groups are compared; * P <0.05, ** P <0.01, *** P <0.001 versus controls.

Article Snippet: The following plasmids were used: ATF4 (Addgene Inc., ID 21845), ATF6 (Addgene Inc., ID 11975), CHOP (Addgene Inc., ID 21898), XBP1u (Addgene Inc., ID 21832) and XBP1p (Addgene Inc., ID 21833). pcDNA3.1 vector was used as a control.

Techniques: Expressing, Immunohistochemistry, Reverse Transcription, Western Blot, Control, Over Expression, Cell Culture, Quantitative Proteomics, Injection, Inhibition, Glycoproteomics, Transfection, Construct, Luciferase, Activity Assay, Plasmid Preparation, MANN-WHITNEY

Journal: European Journal of Histochemistry : EJH

Article Title: SRSF3 promotes the generation of XBP1s to stabilize autophagy and enhance hypoxia adaptation in glioma

doi: 10.4081/ejh.2026.4530

Figure Lengend Snippet: Hypoxic glioma cells exhibit increased cell death accompanied by elevated endoplasmic reticulum stress levels and blocked autophagic flux. A ) Western blot analysis of HIF-1α expression in U87 cells. B ) LDH release assay to detect cell death. C ) Western blot analysis of cleaved-caspase3 expression in U87 cells. D ) Western blot analysis of GRP78, CHOP, and ATF4 expression in U87 cells. E ) RT-PCR detection of XBP1u and XBP1s expression. F ) Western blot analysis of XBP1u and XBP1s expression in U87 cells. G ) Western blot analysis of LC3-II and p62 expression in U87 cells. H ) mRFP-GFP-LC3 staining to assess autophagic flux. n=3 independent experiments; * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: The primary antibodies used were against the following: SRSF3 (1:1000; ab198291; Abcam, Cambridge, UK), HIF-1α (1:1000; ab179483; Abcam), GRP78 (1:2000; 11587-1-AP; Proteintech, Rosemont, IL, USA), CHOP (1:1000; 15204-1-AP; Proteintech), ATF4 (1:1000; 10835-1-AP; Proteintech), LC3 (1:2000; ab192890; Abcam), p62 (1:1000; ab109012; Abcam), cleaved-caspase3 (1:1000; #9661; Cell Signaling Technology, Inc., Danvers, MA, USA), XBP1u (1:1000; 25997-1-AP; Proteintech), XBP1s (1:1000; 24868-1-AP; Proteintech), and β-actin (1:1000; ab8227; Abcam).

Techniques: Western Blot, Expressing, Lactate Dehydrogenase Assay, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: SRSF3 promotes the generation of XBP1s to stabilize autophagy and enhance hypoxia adaptation in glioma

doi: 10.4081/ejh.2026.4530

Figure Lengend Snippet: SRSF3 promotes XBP1s formation. A ) RT‒PCR detection of XBP1u and XBP1s expression in each group of cells. B ) Western blot detection of XBP1u and XBP1s expression in each group of cells. n=3 independent experiments; * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: The primary antibodies used were against the following: SRSF3 (1:1000; ab198291; Abcam, Cambridge, UK), HIF-1α (1:1000; ab179483; Abcam), GRP78 (1:2000; 11587-1-AP; Proteintech, Rosemont, IL, USA), CHOP (1:1000; 15204-1-AP; Proteintech), ATF4 (1:1000; 10835-1-AP; Proteintech), LC3 (1:2000; ab192890; Abcam), p62 (1:1000; ab109012; Abcam), cleaved-caspase3 (1:1000; #9661; Cell Signaling Technology, Inc., Danvers, MA, USA), XBP1u (1:1000; 25997-1-AP; Proteintech), XBP1s (1:1000; 24868-1-AP; Proteintech), and β-actin (1:1000; ab8227; Abcam).

Techniques: Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: A Conserved Structural Determinant Located at the Interdomain Region of Mammalian Inositol-requiring Enzyme 1α

doi: 10.1074/jbc.m111.273714

Figure Lengend Snippet: FIGURE 2. The P830L mutation abolishes IRE1 phosphorylation and activation. A–C, Western blot analysis of IRE1 in cell lysates of IRE1/ MEFs stably expressing WT or mutant IRE1. Cells were treated with 60 nM Tg for 3 h. In C, lysates were treated with PPase. Phos-tag gels are indicated with a straight line on the left side. HSP90, a loading control. Note the difference of IRE1 pattern in Phos-tag gels versus regular SDS-polyacrylamide gels. D and E, quantitation of p-IRE1 in Phos-tag gels shown in A–C. Results are expressed as mean S.E. (error bars). All p values 0.05 used unpaired Student’s t test comparing the P830L with other mutants or WT (except S769F and K599A). For simplicity, the p values are not indicated in the figures. F, Western blot analysis of XBP1 protein in IRE1/ MEFs stably expressing WT or mutant IRE1. Cells were treated with 60 nM Tg for 3 h. *, a nonspecific band. Data are representative of at least two repeats with two independent stable cell lines for each mutant.

Article Snippet: Antibodies used in this study included XBP1 and HSP90 (Santa Cruz Biotechnology), IRE1 (Cell Signaling) and PARP (a gift from Lee Kraus, University of Texas SouthwesternMedical Center).

Techniques: Mutagenesis, Phospho-proteomics, Activation Assay, Western Blot, Stable Transfection, Expressing, Control, Quantitation Assay