Journal: bioRxiv

Article Title: Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

doi: 10.64898/2026.02.17.706344

Figure Lengend Snippet: A. Schematic of the XBP1-Venus IRE1 splicing reporter. B . Expression, measured by qPCR, of the IRE1/XBP1s target genes XBP1s and ERDJ4/DNAJB9 in HEK293 cells expressing non-targeting or SLC33A1 shRNA and treated for 4 h with Tg (0.5 µM) or IXA4 (10 µM). C . Expression, measured by qPCR, of the IRE1 target genes Xbp1s and Erdj4/Dnajb9 , the ATF6 target gene BiP , and the PERK target gene Chop in MEF cells CRISPR-deleted of Slc33a1 using two distinct sgRNA and treated for 4 h with IXA4 (10 µM). MEF cells expressing two distinct non-targeting sgRNAs are shown as a control. D . Expression, measured by qPCR, of the IRE1 target gene XBP1s in non-targeting HEK293 cells or HEK293 cells lacking SLC33A1 transfected with mock or wild-type SLC33A1, as indicated, and then treated for 4 h with Tg (0.5 µM) or IXA4 (10 µM). E . Immunoblots of the indicated proteins in HEK293T cells expressing non-targeting or SLC33A1 sgRNAs treated for 4 h with IXA4 (10 µM). Black asterisks in ( C ) represent comparisons between vehicle-treated control cells expressing NT sgRNA #1 and mutant cells treated with or without IXA4. Red asterisks in ( C ) indicate comparisons between vehicle and IXA4-treated samples. *p<0.05, **p<0.01, ***p<0.005, one-way ANOVA.

Article Snippet: To clone individual sgRNAs, top and bottom oligonucleotides (IDT) were annealed and ligated to lentiviral sgRNA expression vectors LentiGuide-puromycin (Addgene #52963), LentiCRISPRv2-puromycin (Addgene #98290), or LentiCRISPRv2-blasticidin (Addgene #98293)).

Techniques: Expressing, shRNA, CRISPR, Control, Transfection, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

doi: 10.64898/2026.02.17.706344

Figure Lengend Snippet: A . XBP1-Venus reporter signal, measured by flow cytometry, in HEK293 cells stably expressing Cas9 and transduced with lentivirus encoding non-targeting (NT) or IRE1 sgRNA treated for 14 h with vehicle or IXA4 (10 µM). B . CRISPR screen used to identify genes whose deletion blocks IXA4-induced IRE1 activation. C . Plot showing gene score (DMSO) vs. gene score (IXA4), quantified as previously defined , from genome-wide CRISPR screen. D . XBP1-Venus signal, measured by flow cytometry, in HEK293 cells stably expressing Cas9 and transduced with lentivirus encoding non-targeting (NT) or SLC33A1 sgRNA treated for 14 h with vehicle or IXA4 (10 µM). E . Expression, measured by qPCR, of the IRE1 target genes XBP1s and DNAJB9 , the ATF6 target gene BiP, and the PERK target gene CHOP in HEK293 cells stably expressing Cas9 transduced with lentivirus encoding NT or SLC33A1 sgRNA treated for 4 h with vehicle, IXA4 (10 µM) or Tg (0.5 µM). *p<0.05, ***p<0.005 one-way ANOVA.

Article Snippet: To clone individual sgRNAs, top and bottom oligonucleotides (IDT) were annealed and ligated to lentiviral sgRNA expression vectors LentiGuide-puromycin (Addgene #52963), LentiCRISPRv2-puromycin (Addgene #98290), or LentiCRISPRv2-blasticidin (Addgene #98293)).

Techniques: Flow Cytometry, Stable Transfection, Expressing, Transduction, CRISPR, Activation Assay, Genome Wide

Journal: bioRxiv

Article Title: Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

doi: 10.64898/2026.02.17.706344

Figure Lengend Snippet: A. XBP1-Rluc signal in HEK293 cells stably expressing XBP1-RLuc treated with the indicated concentration of IXA4 or PTG2018 in the presence or absence of the IRE1 RNAse inhibitor 4µ8c (32 µM) for 14 h. Data are normalized to the XBP1-RLuc signal seen in cells treated with thapsigargin (Tg; 0.5 µM). Error bars show SEM for n=3 replicates. B . Dose-dependent proteome labeling with PTG2018 (left, rhodamine fluorescence; right, Coomassie stain). PTG2018-labeled proteins were identified by appending TAMRA-azide to PTG2018 via click chemistry. C . Streptavidin affinity purification and proteomes (input) from HEK293T cells overexpressing FLAG-tagged EPHX1 and treated with the indicated concentration of PTG2018 and IXA4. PTG2018 labeling of FLAG-tagged EPHX1 was visualized by appending a biotin to the alkyne handle of PTG2018 using click chemistry and monitoring the recovery of FLAG-tagged protein in streptavidin isolates. Mock transfected cells are shown as a control. D . Streptavidin affinity purification and proteomes (input) from HEK293T cells treated with the indicated concentration of PTG2018 and IXA4. PTG2018 labeling of SOAT1 was visualized by appending biotin to the alkyne handle of PTG2018 via click chemistry and monitoring the recovery the recovery of endogenous SOAT1 in streptavidin isolates. E , F . XBP1-Venus signal, measured by flow cytometry, in HEK293 cell stably expressing Cas9 and transduced with lentivirus encoding non-targeting (NT), SOAT1 or EPHX1 sgRNA treated for 14 h with vehicle or IXA4 (10 µM).

Article Snippet: To clone individual sgRNAs, top and bottom oligonucleotides (IDT) were annealed and ligated to lentiviral sgRNA expression vectors LentiGuide-puromycin (Addgene #52963), LentiCRISPRv2-puromycin (Addgene #98290), or LentiCRISPRv2-blasticidin (Addgene #98293)).

Techniques: Stable Transfection, Expressing, Concentration Assay, Labeling, Fluorescence, Staining, Affinity Purification, Transfection, Control, Flow Cytometry, Transduction

Journal: bioRxiv

Article Title: Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

doi: 10.64898/2026.02.17.706344

Figure Lengend Snippet: A. [ 3 H]-acetyl-CoA uptake by ER microsomes (50 µg protein) isolated from HEK293T cells treated with/without IXA4 (10 µM) during the uptake assay. B . [ 3 H]-acetyl-CoA uptake by ER microsomes (50 µg protein) isolated from HEK293T cells expressing Cas9 and non-targeting (NT) or SLC33A1 sgRNA. C , D . Relative acetyl-CoA abundance ( C ) and oxidized glutathione abundance (GSSG; D ) in whole cell or ER isolated from HEK293T cells with non-targeting (NT) sgRNA or sgRNA targeting SLC33A1. Cells were treated with IXA4 (10 µM) for 4 h before ER isolation and LC/MS analysis. E . Immunoblot of DNAJC10 and PDIA4 in proteomes prepared from HeLa cells treated for 1 h with IXA4 (10 µM), DTT (5 mM), or diamide (500 µM). Following lysis in the presence of alkylating agents, disulfides within these proteins were reduced and then labeled with PEG(5K)-maleimide (2 mM), allowing visualization of oxidized and reduced protein populations. F . Immunoblot of proteomes from Ire1 -/- MEFs reconstituted with wild-type or a C148S IRE1 mutant, treated for 2 h with IXA4 (10 µM) or thapsigargin (Tg; 0.5 µM), and separated on a non-reducing SDS-PAGE gel. Oxidized and reduced IRE1 populations are shown. G . XBP1s levels, measured by qPCR, in HEK293T cells pre-treated for 18 h with BSO (50 µM) and then treated for 3 h with IXA4 (10 µM). H . Viability, assessed with CellTiter-Glo, of KP and KPK cells treated for 3 days with IXA4 (10 µM) or thapsigargin (Tg; 0.5 µM). I . Viability, assessed with CellTiter-Glo, of KP and KPK cells treated for 3 days with IXA4 (10 µM), BSO (50 µM), and 4µ8c (32 µM). *p<0.05, ***p<0.005, one-way ANOVA.

Article Snippet: To clone individual sgRNAs, top and bottom oligonucleotides (IDT) were annealed and ligated to lentiviral sgRNA expression vectors LentiGuide-puromycin (Addgene #52963), LentiCRISPRv2-puromycin (Addgene #98290), or LentiCRISPRv2-blasticidin (Addgene #98293)).

Techniques: Isolation, Expressing, Liquid Chromatography with Mass Spectroscopy, Western Blot, Lysis, Labeling, Mutagenesis, SDS Page

Journal: bioRxiv

Article Title: Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

doi: 10.64898/2026.02.17.706344

Figure Lengend Snippet: A . In-gel fluorescence of acetylated proteins in ER and mitochondrial fractions isolated from HEK293T cells treated for 3 h with IXA4 (10 µM) and then incubated for 1 h with 3-butynoic acid. Acetylated proteins were identified by appending a rhodamine tag onto the 3-butynoic acid probe via click chemistry. B . In-gel fluorescence of acetylated proteins in ER fractions isolated from HEK293T Cas9 cells expressing non-targeting (NT) or SLC33A1 sgRNA incubated for 1 h with the 3-butynoic acid probe. Acetylated proteins were then identified by appending a rhodamine tag onto the 3-butynoic acid probe via click chemistry. C . Expression (published DNA microarrays) of IRE1/XBP1s, ATF6, and PERK target genesets in HEK293T cells overexpressing the ER oxidase ERO1. Source data available in GEO (GSE40601). D . XBP1 mRNA splicing (RT-PCR) in IRE1 -deficient HeLa cells transfected with wild-type IRE1 or a C109S/C148S/C332S IRE1 triple mutant and treated for 2 h with IXA4 (10 µM) or thapsigargin (Tg; 0.5 µM). Unspliced (u), spliced (s), and hybrid (*), XBP1 are shown. E . Crystal violet stained KPK cells treated for 2 days with thapsigargin (Tg; 0.5 µM) or IXA4 (10 µM). F . Crystal violet stained KPK cells incubated for 2 days with IXA4 (10 µM) and/or BSO (50 µM). p<0.05, **p<0.01, ***p<0.005, one-way ANOVA.

Article Snippet: To clone individual sgRNAs, top and bottom oligonucleotides (IDT) were annealed and ligated to lentiviral sgRNA expression vectors LentiGuide-puromycin (Addgene #52963), LentiCRISPRv2-puromycin (Addgene #98290), or LentiCRISPRv2-blasticidin (Addgene #98293)).

Techniques: Fluorescence, Isolation, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Mutagenesis, Staining