Journal: bioRxiv

Article Title: SLC33A1 exports oxidized glutathione to maintain endoplasmic reticulum redox homeostasis

doi: 10.64898/2026.02.01.703113

Figure Lengend Snippet: a. Top, schematic of engineered GshF construct targeted to ER ( ER-GshF ), with signal peptide (SP) from ERP44 at N-terminus, V5 tag and ER retention signal KDEL at C-terminus. Bottom left, schematic of GSH synthesis in ER catalyzed by ER-GshF. Bottom right, immunoblot analysis of ER-GshF expression from HEK293T cells expressing inducible ER-GshF (iER-GshF HEK293T) treated with 1 µg/ml doxycycline for 24 hours. b. Immunofluorescence analysis of ER-GshF (V5, red) and calnexin (CANX, green) in iER-GshF HEK293T cells treated with 1 µg/ml doxycycline for 48 hours. Micrographs are representative of two independent experiments. c. Percent labeled glutathione from HEK293T cells expressing a vector control or inducible ER-GshF pre-treated with or without BSO and doxycycline. Cells pretreated with or without 1 mM BSO and 1 µg/ml doxycycline for 24 hours, were switched to cystine free media with 200 µM isotope labeled cystine (³CL, ¹LNL) for 8 hours before harvesting the cells, BSO and doxycycline were kept the same as pretreatment during labeling. d. Schematic of the ER-focused CRISPR genetic screens in iER-GshF HEK293T cells cultured in the presence or absence of 1 µg/ml doxycycline for 14 doublings. e. Left, CRISPR gene scores in iER-GshF HEK293T cells cultured in the presence or absence of 1 µg/ml doxycycline for 14 doublings. Top scoring hits color-coded. Pearson correlation coefficient, two-sided. Right, differential gene score from iER-GshF HEK293T cells cultured with doxycycline compared to untreated. Top genes sensitizing iER-GshF HEK293T cells under doxycycline treatment are shown. f. Left, percent cell number from SLC33A1 knockout HEK293T cells expressing inducible ER-GshF complemented with a vector control or SLC33A1 cDNA under different concentrations of doxycycline for 4 days. Numbers under doxycycline treated are normalized to untreated. Right, representative images of the indicated cells. g. Left, volcano plot showing relative fold change (log 2 ) in ER metabolite abundance versus −log (P values) from SLC33A1 knockout ER-tag HEK293T cells expressing a vector control or SLC33A1 cDNA. Statistical significance was determined by multiple two-tailed unpaired t-tests. The dotted line represents P =0.01. Right, volcano plot showing relative fold change (log 2 ) in whole-cell metabolite abundance versus −log (P values) from SLC33A1 knockout ER-tag HEK293T cells expressing a vector control or SLC33A1 cDNA. Statistical significance was determined by multiple two-tailed unpaired t-tests. The dotted line represents P =0.01. h. Left, volcano plot showing relative fold change (log 2 ) in ER metabolite abundance versus −log (P values) from Slc33a1 knockout ER-tag KPK cells expressing a vector control or SLC33A1 cDNA. Statistical significance was determined by multiple two-tailed unpaired t-tests. The dotted line represents P =0.01. Right, volcano plot showing relative fold change (log 2 ) in whole-cell metabolite abundance versus −log (P values) from Slc33a1 knockout ER-tag KPK cells expressing a vector control or SLC33A1 cDNA. Statistical significance was determined by multiple two-tailed unpaired t-tests. The dotted line represents P =0.01. i. Relative metabolite abundance of indicated whole cell or ER metabolites from SLC33A1 knockout ER-tag HEK293T, HeLa and KPK cells expressing a vector control compared to those expressing SLC33A1 cDNA. ER UDP-GlcNAc abundance is shown to indicate ER amount.

Article Snippet: Human cells lines HeLa, HEK293T cells were purchased from the ATCC.

Techniques: Construct, Western Blot, Expressing, Immunofluorescence, Labeling, Plasmid Preparation, Control, CRISPR, Cell Culture, Knock-Out, Two Tailed Test

Journal: bioRxiv

Article Title: Chemical control of 2’-hydroxyl-dependent Cas9 target engagement enables CRISPR RNA ribose replacement

doi: 10.64898/2026.01.26.701763

Figure Lengend Snippet: ( A ) Schematic of the crRNA sequence used for targeting an EGFP reporter gene and chemical modifications used to probe 2’-OH contacts. In vitro cleavage activity (orange) and cell-based EGFP editing (blue) is shown on the right. 2’-OH contacts with SpCas9 are indicated with red asterisks below. n = 3 or more experimental replicates. Error bars are S.E.M. ( B ) Time-course of in vitro cleavage activity using select crRNAs from panel A. Curves were fitted to an exponential two-phase decay equation. Error is reported as S.E.M. ( C ) Thermal denaturation of Cas9 RNP complexes assayed by absorbance at 280 nm to measure melting temperature ( T m ). n = 2 experimental replicates. Error bars are S.E.M. ( D ) Target DNA binding by dCas9 RNP measured by dot blot filter binding of radiolabeled target DNA. Curves were fit to a one-site binding curve. n = 2 experimental replicates. Error bars are S.E.M. ( E ) CRISPRa-based assay to measure dCas9-VPR binding guided by crTREa crRNA to a Tet-On 3G promoter driving EGFP in HeLa cells. Unmodified crTREa is shown as a control. EGFP expression was quantified by flow cytometry. N = 3 experimental replicates, Error is S.E.M.

Article Snippet: HeLa Tet-On 3G cells (Takara, Cat. #631183) were transduced according to the manufacturer’s recommended protocol.

Techniques: Sequencing, In Vitro, Activity Assay, Binding Assay, Dot Blot, Control, Expressing, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Interaction between HIV-1 Tat and EBV Zta favours immune escape of B cells by downregulating HLA-ABC expression

doi: 10.1007/s00018-025-06029-5

Figure Lengend Snippet: HIV-1 Tat and EBV Zta physically interact in B cells and human serum. (A) Immunoprecipitation of Tat in B cells. The RPMI8866 lymphoblastoid cell line inducibly expressing unconjugated HIV-1 Tat (RPMI8866 Tat i cell line) upon treatment with Doxycycline (Tat) was treated or not with 1 µg/ml recombinant Zta protein. Cell lysates were subjected to immunoprecipitation using anti-Tat antibodies. Input and immunoprecipitated fractions were analysed by western blotting staining for Tat, Zta, Galectin 9 (negative control) and β-actin (control of protein load). (B) Immunoprecipitation of Zta in B cells. The RPMI8866 lymphoblastoid cells were transfected with YFP (lane 1), YFP-Tat (Tat, lane 2), mCherry-Zta (Zta, lane 3), YFP-Tat and mCherry-Zta (lane 4), YFP-Zta and mCherry-Tat (lane 5), followed by immunoprecipitation of Zta using anti-Zta antibodies after 24 h. Input and immunoprecipitated fractions were analysed by western blotting staining for Tat, Zta, Galectin 9 (negative control) and GAPDH (control of protein load). ( C) A healthy donor (HIV-negative) serum sample supplemented with Tat and Zta (at 250 ng/ml and 1000 ng/ml, respectively, HIV-neg. + Tat + Zta) and a serum sample from an HIV-positive individual (HIV-pos.) were immunoprecipitated using Protein G magnetic beads cross-linked with anti-Zta or anti-Tat antibodies. Immunoprecipitated (IP) and non-immunoprecipitated flowthrough (FT) fractions were analysed by Western blotting staining for Zta, Tat, transferrin (negative control) and transthyretin (negative control). (D , E) Analysis of Tat and Zta interaction by an in vitro binding assay. Equal amounts of recombinant HIV-1 Tat protein linked to AminoLink agarose beads or BSA control agarose beads were incubated with increasing amounts of recombinant Zta protein. (D) Bound protein was separated on SDS-PAGE gel and bound Zta was then analysed by Western blotting staining. A representative image is shown. (E) The non-linear fit of Zta binding to Tat (densitometry analysis of western blotting band intensity of bound Zta) against Zta concentration in solution with one site-specific binding model. Zta binding at a concentration of 0.2 µM was set as 1 (fraction bound). (F , G) Tat and Zta interaction analysed by YFP reconstitution. (F) The modular composition of the fusion proteins used in this study (above). A representative image of HeLa cells after transfection with YFP1-Tat and YFP2-Zta plasmids, nuclei were counterstained with DAPI, scale bar 10 μm (below). (G) The percentage of YFP-positive cells in HeLa, RPMI8866 or Jurkat cells transfected with YFP1-Tat + YFP2-Zta or YFP1-ERGIC-53 and YFP2-MCFD2 (positive control) as analysed by flow cytometry. (H-J) The analysis of Tat and Zta interaction by FRET with the acceptor photobleaching method. (H) Representative images of RPMI8866 cells, transfected with different combinations of CFP, YFP, Tat-CFP and YFP-Zta. Scale bar 10 μm. (I-J) FRET efficiencies for each combination in RPMI8866 (I) and HeLa (J) cells. Data are presented as mean ± SEM

Article Snippet: Human cervical carcinoma cell line HeLa (American Type Culture Collection), Human Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line RPMI8866 (ECACC General Cell Collection), freshly EBV-transformed B lymphoblastoid cell line from healthy donor AS (BLAS, established by EBV (B95-8) immortalization of mature B cells and characterized by Genethon (Evry, France)), human immortalized T cell line Jurkat (American Type Culture Collection) and their derivatives were used in the study.

Techniques: Immunoprecipitation, Expressing, Recombinant, Western Blot, Staining, Negative Control, Control, Transfection, Magnetic Beads, In Vitro, Binding Assay, Incubation, SDS Page, Concentration Assay, Positive Control, Flow Cytometry

Journal: bioRxiv

Article Title: Regulation of Type VI secretion systems of Burkholderia thailandensis during transition to intracellular lifestyle

doi: 10.64898/2026.01.30.702787

Figure Lengend Snippet: (A) Scheme depicting the phagosomal membrane markers when B. thailandensis enters a host cell. (B) LAMP-1 immunofluorescence staining of HeLa cells stably expressing Rab5 and Rab7-mApple infected with B. thailandensis at MOI 50. Cells were fixed and permeabilized with ice-cold methanol at 3 hpi and stained with anti-LAMP1 antibody at 1 ug/mL for 1 hour at room temperature. Then the slide was washed with PBS, and stained with anti-mouse-AlexaFluor647 overnight at 4 degrees. Yellow arrows indicate TssB-5 expressing bacteria. Blue arrows indicate the presence of phagosomal markers. Scale bar represents 2 µm. (C) Quantification of percentage of T6SS-5 expressing bacteria co-localizing with Rab5, Rab7, or LAMP-1 positive vacuoles at 3 hpi. Each data point represents one biological replicate. More than 100 bacteria were counted in each biological replicate. (D) Time-lapse imaging of A549 cells treated with bafilomycin A1 (100nM) or DMSO infected with with B. thailandensis expressing cytosolic mCherry2 under ribosomal promoter pSC12 (cyan) and TssB-5-msfGFP (grey) at MOI of 200. A549 cells were pre-treated with 0.25 mg/mL of fluorescent dextran for 18 hours to identify intra-phagosomal bacteria. Host cells were pre-treated 1 hour before infection, infected with B. thailandensis for 1 hour without treatment, and bafilomycin A1 or DMSO was added and kept in the media with 300 µg/mL kanamycin from 1 hpi onwards. Host cell membrane was stained with CellMask DeepRed (magenta). Blue arrows indicate bacteria that do not express T6SS-1 and yellow arrows indicate T6SS-5 expressing bacteria. Scale bar represents 5 µm. (E) Percentage of intracellular B. thailandensis expressing T6SS-5 when A549 cells were treated with bafilomycin A1 or DMSO. A549 cells were pre-treated with 0.25 mg/mL of fluorescent dextran for 18 hours to identify intra-phagosomal bacteria. A total of 109 bacteria were identified and tracked for T6SS-5 expression across 3 biological replicates. An unpaired t-test were carried out (** P<0.001). (F) Fluorescence intensity of A549 cells treated with either 100nM bafilomycin A1 or DMSO stained with Lysotracker DeepRed. Fluorescent images were adjusted with the same threshold values and integrated density values were measured with ImageJ. An unpaired t-test were carried out (*** P<0.0001).

Article Snippet: The cell lines HeLa cells (CCL-2, ATCC), A549 lung cells (CCL-185, ATCC) were used in this study.

Techniques: Membrane, Immunofluorescence, Staining, Stable Transfection, Expressing, Infection, Bacteria, Imaging, Fluorescence