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

Journal: Cell Communication and Signaling : CCS

Article Title: ER-phagy mediates the anti-tumoral synergism between HDAC inhibition and chemotherapy

doi: 10.1186/s12964-025-02198-9

Figure Lengend Snippet: ISX and Gemcitabine synergistically induce ER-stress ( A ) Scheme of the IRE1-selective ER-stress reporter. ( B ) Immunofluorescence images of Panc1 cells transiently transfected with IRE1 reporter (green), treated with DMSO, 20 µM ISX, 1.0 µM Gem and the combination ISX/Gem (I/G) for 48h. Nuclei appear blue (DAPI). Scale bar: 100µm. Shown is one representative of n=3. ( C ) Quantification of Panc1 cells transiently expressing IRE1 reporter. Cells were treated as described in ( B ) or with 1.0 μM Thapsigargin (Th) for 48h, analyzed using the number of positive cells per viewing field normalized to the values of the DMSO samples. Each dot represents one image (from n=2 independent experiments; mean ± SD). ( D ) Rel. mRNA expression of ER-stress genes selective for the IRE1 pathway ( XBP1s , DNAJB9 ). Panc1 cells were treated as described in ( C ) (mean of n=3 ± SD). ( E ) Scheme of the PERK-selective ER-stress reporter. ( F ) Immunofluorescence pictures of Panc1 cells transiently expressing PERK reporter (red) and treated as in ( B ). Nuclei appear blue (DAPI). Scale bar: 100µm. Shown is one representative of n=3. ( G ) Quantification of PERK pathway activity in Panc1 cells stably expressing a PERK reporter construct. Treatment as in ( C ). Shown are the numbers of positive cells per viewing field normalized to the DMSO condition. Each dot represents one analyzed viewing field (mean ± SD of n=2). ( H ) Rel. mRNA expression of ER-stress genes selective for the PERK pathway ( CHOP , PERK ) in Panc1 cells treated as described in ( C ) Quantification of ATF6 activity in Panc1 cells transiently expressing an ATF6 luciferase reporter, treated as described in ( C ) (mean of n=3 ± SD). ( K ) Rel. mRNA expression of HERPUD1 , an ER-stress gene selective for the ATF6 pathway (Panc1 cells treated as in ( B ); mean of n=3 ± SD)

Article Snippet: The following plasmids were used: IRE1 reporter/pLHCX-XBP1 mNeonGreen NLS (Addgene 115971; David Andrews), PERK reporter/pLHCX-ATF4 mScarlet NLS (Addgene, 115,970; David Andrews), ER-Keima reporter/GST-Keima-cb5 (Addgene, 137,755; Carol Mercer), mCherry-LC3B (Addgene 40827, David Rubinsztein), ATF6 reporter/p5xATF6-GL3 (Addgene, 11,976, Ron Prywes).

Techniques: Immunofluorescence, Transfection, Expressing, Activity Assay, Stable Transfection, Construct, Luciferase

Journal: Cell Communication and Signaling : CCS

Article Title: ER-phagy mediates the anti-tumoral synergism between HDAC inhibition and chemotherapy

doi: 10.1186/s12964-025-02198-9

Figure Lengend Snippet: The impact of HDACi/Gem on apoptosis and its link to clinical data. ( A ) Rel. mRNA expression of FAM134B and CHOP (mean of n=3 ± SD). Panc1 cells were treated with DMSO or the combination of 20 µM ISX and 1.0 µM Gem for different time periods. ( B ) Rel. mRNA expression of FAM134B and PERK (mean of n=3 ± SD). Panc1 cells were treated with DMSO, 20 µM ISX, 1.0 µM Gem, dual treatment (I/G), or 1.0 µM Thapsigargin (Th) in calcium-free or calcium-containing DMEM medium for 48h. ( C ) Flow cytometric quantification of IRE1 activity in Panc1 cells stably expressing an IRE1 reporter construct. Treatment as in ( B ) (n=3 ± SD). ( D ) Caspase 3/7 activity in Panc1 cells exposed to ISX (I, 20 µM) and Gem (G, 1.0 µM) for various time periods. Staurosporine (1.0 µM) was included as a positive control. The mean ± SD of two representative experiments measured in triplicates is shown. ( E ) Caspase 3/7 activity in Panc1 cells treated with the indicated drugs for 60h. ISX (I, 20 µM); Gem (G, 1.0 µM); IRE1-inh. 4µ8c (30µM); PERK-inh. ISRIB (1.0µM); ATF6-inh. Ceapin-A7 (6.0µM). Shown is the mean (± SD) of two representative experiments measured in quadruplicate each. Each dot represents one biological sample. ( F ) Western blot analysis of FAM134B protein levels in Panc1 cells treated for 48h with DMSO, 20 µM Loperamide, or 5/10 µM Nelfinavir. Shown is one representative experiment of n=5. β-Actin was used as a loading control. ( G , H , I ) FAM134B mRNA expression in bulk tissue from PDAC patients and corresponding healthy pancreatic tissue. Each dot represents one patient. ( J , K ) Kaplan-Meier plots representing the overall survival of patients with PDAC as a function of FAM134B expression (Scan-split; log-rank p). ( L ) Scheme illustrating the ER-phagy-induced mechanism by which HDACi and Gemcitabine synergize in their anti-tumor effectiveness

Article Snippet: The following plasmids were used: IRE1 reporter/pLHCX-XBP1 mNeonGreen NLS (Addgene 115971; David Andrews), PERK reporter/pLHCX-ATF4 mScarlet NLS (Addgene, 115,970; David Andrews), ER-Keima reporter/GST-Keima-cb5 (Addgene, 137,755; Carol Mercer), mCherry-LC3B (Addgene 40827, David Rubinsztein), ATF6 reporter/p5xATF6-GL3 (Addgene, 11,976, Ron Prywes).

Techniques: Expressing, Activity Assay, Stable Transfection, Construct, Positive Control, Western Blot, Control