Journal: bioRxiv

Article Title: Opposing roles for SNAP23 and SNAP25 in mediating MR1 trafficking and antigen presentation

doi: 10.64898/2026.02.18.706493

Figure Lengend Snippet: (A) Relative gene expression levels of SNAP23, SNAP25, SNAP29 , and SNAP47 compared to GAPDH in BEAS-2B cells. (B) Knockdown efficiency after 48 hours of knockdown with missense (Mis) or gene-specific (KD) siRNA for SNAP23, SNAP25, SNAP29, and SNAP47. (C-F) IFN-γ response by T cell clones (MAIT and HLA-B45-restricted) co-cultured with BEAS-2B cells following siRNA knockdown of (C) SNAP23, (D) SNAP25, (E) SNAP29, or (F) SNAP47. Cells were either infected overnight with H37Rv Mtb (MOI=8) or incubated with exogenously added antigens ( M. smeg supernatant and CFP10 2-9 peptide). IFN-γ response is measured by ELISpot and represented as spot forming units (SFU). All data are plotted as mean±SEM and pooled from three independent experiments. Experiments were performed in parallel for (C-D) and (E-F). For (C-F), the means of technical duplicates were pooled and normalized to Control (Missense) at the highest antigen concentration. Non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test.

Article Snippet: Taqman FAM-MGB probes for SNAP23 (Hs01047496_m1, Hs01047498_m1), SNAP25 (Hs00938957_m1, Hs00938966_m1), SNAP29 (Hs00191150_m1), and SNAP47 (Hs00369605_m1) were obtained from ThermoFisher Scientific.

Techniques: Gene Expression, Knockdown, Clone Assay, Cell Culture, Infection, Incubation, Enzyme-linked Immunospot, Control, Concentration Assay

Journal: bioRxiv

Article Title: Opposing roles for SNAP23 and SNAP25 in mediating MR1 trafficking and antigen presentation

doi: 10.64898/2026.02.18.706493

Figure Lengend Snippet: Clonal SNAP23 KO and SNAP25 KO cells lines were generated from BEAS-2B cells (Figure S1). (A) Cells were either infected overnight with H37Rv Mtb (MOI=8) or incubated with M. smeg supernatant. IFN-γ response is measured by ELISpot and represented as spot forming units (SFU). The means of technical duplicates were pooled from three independent experiments and normalized to WT at the highest antigen concentration. Non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test. (B) Percent of cells with Mtb (left) or beads (right) that are GFP+ after infection with mEmeraldRFP-AuxMtb (MOI=8) or incubation with yellow-green fluorescent beads (ratio=8) overnight. (C) Colony forming units (CFU) of H37Rv Mtb (MOI=8) in cells after overnight infection. (D) GeoMFI of GFP of cells treated with pHrodo dextran green. (E-F) Cells were either infected with microbes or incubated with supernatants of C. albicans and M. avium . Cells were infected with C. albicans (MOI=0.2) for 90 minutes or with M. avium (MOI=22) for overnight. 50 µL of supernatants were added for both C. albicans and M. avium . IFN-γ response is measured by ELISpot and represented as spot forming units (SFU). The means of technical duplicates were pooled from three independent experiments, and non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test. For (B-F), ordinary one-way ANOVA with Dunnett’s multiple comparisons test were used to analyze significant differences. All data are plotted as mean±SEM.

Article Snippet: Taqman FAM-MGB probes for SNAP23 (Hs01047496_m1, Hs01047498_m1), SNAP25 (Hs00938957_m1, Hs00938966_m1), SNAP29 (Hs00191150_m1), and SNAP47 (Hs00369605_m1) were obtained from ThermoFisher Scientific.

Techniques: Generated, Infection, Incubation, Enzyme-linked Immunospot, Concentration Assay

Journal: bioRxiv

Article Title: Opposing roles for SNAP23 and SNAP25 in mediating MR1 trafficking and antigen presentation

doi: 10.64898/2026.02.18.706493

Figure Lengend Snippet: A clonal SNAP23 KO cell line was generated in the background of BEAS-2B MR1KO:tetMR1-GFP clone D4 (WT) cells (Figure S2). (A) Cells were infected overnight with H37Rv Mtb (MOI=8). IFN-γ response by T cell clones (MR1- and HLA-B45-restricted) is measured by ELISpot and represented as spot forming units (SFU). The means of technical duplicates were pooled and normalized to WT at the highest antigen concentration. Non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test. For MR1 surface expression, cells were incubated with doxycycline overnight. 6-FP or NaOH (solvent control) was added the next day for at least 18 hours. (B) Cells were then analyzed for geometric mean fluorescence intensity (GeoMFI) of GFP+ cells on surface MR1 and surface HLA-Ia expression. (C-E) Representative images of WT and SNAP23 KO BEAS-2B MR1KO:tetMR1-GFP cells at (C) steady state, (D) infected overnight with CellLight Rab5a (top) or LAMP1 (bottom), (E) incubated overnight with beads (top) or infected with AuxMtb (bottom). (C, E) Total number of MR1 vesicles in each cell. (D) Percent co-localization of Rab5a and LAMP1 with MR1 vesicles. Each dot represents one cell. p-values were analyzed by two-tailed unpaired Student’s t-test. All data points are pooled and representative of three independent experiments, and plotted as mean±SEM. All scale bars represent 10 µm.

Article Snippet: Taqman FAM-MGB probes for SNAP23 (Hs01047496_m1, Hs01047498_m1), SNAP25 (Hs00938957_m1, Hs00938966_m1), SNAP29 (Hs00191150_m1), and SNAP47 (Hs00369605_m1) were obtained from ThermoFisher Scientific.

Techniques: Generated, Infection, Clone Assay, Enzyme-linked Immunospot, Concentration Assay, Expressing, Incubation, Solvent, Control, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Opposing roles for SNAP23 and SNAP25 in mediating MR1 trafficking and antigen presentation

doi: 10.64898/2026.02.18.706493

Figure Lengend Snippet: (A) Relative gene expression levels of SNAP23, SNAP25, SNAP29 , and SNAP47 compared to GAPDH in BEAS-2B cells. (B) Knockdown efficiency after 48 hours of knockdown with missense (Mis) or gene-specific (KD) siRNA for SNAP23, SNAP25, SNAP29, and SNAP47. (C-F) IFN-γ response by T cell clones (MAIT and HLA-B45-restricted) co-cultured with BEAS-2B cells following siRNA knockdown of (C) SNAP23, (D) SNAP25, (E) SNAP29, or (F) SNAP47. Cells were either infected overnight with H37Rv Mtb (MOI=8) or incubated with exogenously added antigens ( M. smeg supernatant and CFP10 2-9 peptide). IFN-γ response is measured by ELISpot and represented as spot forming units (SFU). All data are plotted as mean±SEM and pooled from three independent experiments. Experiments were performed in parallel for (C-D) and (E-F). For (C-F), the means of technical duplicates were pooled and normalized to Control (Missense) at the highest antigen concentration. Non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test.

Article Snippet: Taqman FAM-MGB probes for SNAP23 (Hs01047496_m1, Hs01047498_m1), SNAP25 (Hs00938957_m1, Hs00938966_m1), SNAP29 (Hs00191150_m1), and SNAP47 (Hs00369605_m1) were obtained from ThermoFisher Scientific.

Techniques: Gene Expression, Knockdown, Clone Assay, Cell Culture, Infection, Incubation, Enzyme-linked Immunospot, Control, Concentration Assay

Journal: bioRxiv

Article Title: Opposing roles for SNAP23 and SNAP25 in mediating MR1 trafficking and antigen presentation

doi: 10.64898/2026.02.18.706493

Figure Lengend Snippet: Clonal SNAP23 KO and SNAP25 KO cells lines were generated from BEAS-2B cells (Figure S1). (A) Cells were either infected overnight with H37Rv Mtb (MOI=8) or incubated with M. smeg supernatant. IFN-γ response is measured by ELISpot and represented as spot forming units (SFU). The means of technical duplicates were pooled from three independent experiments and normalized to WT at the highest antigen concentration. Non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test. (B) Percent of cells with Mtb (left) or beads (right) that are GFP+ after infection with mEmeraldRFP-AuxMtb (MOI=8) or incubation with yellow-green fluorescent beads (ratio=8) overnight. (C) Colony forming units (CFU) of H37Rv Mtb (MOI=8) in cells after overnight infection. (D) GeoMFI of GFP of cells treated with pHrodo dextran green. (E-F) Cells were either infected with microbes or incubated with supernatants of C. albicans and M. avium . Cells were infected with C. albicans (MOI=0.2) for 90 minutes or with M. avium (MOI=22) for overnight. 50 µL of supernatants were added for both C. albicans and M. avium . IFN-γ response is measured by ELISpot and represented as spot forming units (SFU). The means of technical duplicates were pooled from three independent experiments, and non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test. For (B-F), ordinary one-way ANOVA with Dunnett’s multiple comparisons test were used to analyze significant differences. All data are plotted as mean±SEM.

Article Snippet: Taqman FAM-MGB probes for SNAP23 (Hs01047496_m1, Hs01047498_m1), SNAP25 (Hs00938957_m1, Hs00938966_m1), SNAP29 (Hs00191150_m1), and SNAP47 (Hs00369605_m1) were obtained from ThermoFisher Scientific.

Techniques: Generated, Infection, Incubation, Enzyme-linked Immunospot, Concentration Assay

Journal: bioRxiv

Article Title: Opposing roles for SNAP23 and SNAP25 in mediating MR1 trafficking and antigen presentation

doi: 10.64898/2026.02.18.706493

Figure Lengend Snippet: A clonal SNAP23 KO cell line was generated in the background of BEAS-2B MR1KO:tetMR1-GFP clone D4 (WT) cells (Figure S2). (A) Cells were infected overnight with H37Rv Mtb (MOI=8). IFN-γ response by T cell clones (MR1- and HLA-B45-restricted) is measured by ELISpot and represented as spot forming units (SFU). The means of technical duplicates were pooled and normalized to WT at the highest antigen concentration. Non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test. For MR1 surface expression, cells were incubated with doxycycline overnight. 6-FP or NaOH (solvent control) was added the next day for at least 18 hours. (B) Cells were then analyzed for geometric mean fluorescence intensity (GeoMFI) of GFP+ cells on surface MR1 and surface HLA-Ia expression. (C-E) Representative images of WT and SNAP23 KO BEAS-2B MR1KO:tetMR1-GFP cells at (C) steady state, (D) infected overnight with CellLight Rab5a (top) or LAMP1 (bottom), (E) incubated overnight with beads (top) or infected with AuxMtb (bottom). (C, E) Total number of MR1 vesicles in each cell. (D) Percent co-localization of Rab5a and LAMP1 with MR1 vesicles. Each dot represents one cell. p-values were analyzed by two-tailed unpaired Student’s t-test. All data points are pooled and representative of three independent experiments, and plotted as mean±SEM. All scale bars represent 10 µm.

Article Snippet: Taqman FAM-MGB probes for SNAP23 (Hs01047496_m1, Hs01047498_m1), SNAP25 (Hs00938957_m1, Hs00938966_m1), SNAP29 (Hs00191150_m1), and SNAP47 (Hs00369605_m1) were obtained from ThermoFisher Scientific.

Techniques: Generated, Infection, Clone Assay, Enzyme-linked Immunospot, Concentration Assay, Expressing, Incubation, Solvent, Control, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A ) Illustration of the VAMP-Stx-SNAP protein interaction for the formation of the SNARE complex initiating exocytotic processes. Created in BioRender. Dersch, P. (2026). ( B - J ) Caco-2 monolayer was infected with 2,5 x 10 6 bacteria of Y. pseudotuberculosis wildtype YPIII pFU92 ( gfp + ). 3.5 h post-infection, the Caco-2 cell monolayers were analyzed by CLSM to detect Yersinia (GFP-green), the cell plasma membranes (CellMask Plasma Membrane Red-CMPR), the nuclei (DAPI-blue), and SNARE proteins (yellow) colocalized with the bacteria using antibodies against VAMP3 ( B ), VAMP7 ( C,D ), Stx3 ( E ), Stx4 ( F,G ), SNAP25 ( H ), and SNAP23 ( I,J ). Orthogonal XZ and YZ planes are shown.

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Infection, Bacteria, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A-D ) Caco-2, and the isogenic cell lines Caco-2ΔStx3, Caco-2ΔStx4, Caco-2ΔSNAP23, and were seeded into transwells and cultivated until the TEER had reached > 400 Ω•cm². Whole-cell extracts were prepared and analyzed by Western blotting using antibodies specific to Stx3 ( A ), Stx4 ( B ), and SNAP23 ( C ), and actin as a loading control ( A-C ). M: protein marker. ( D ) Monolayers of wildtype Caco-2 cells (wt), and ΔStx3, ΔStx4, and ΔSNAP23 mutant derivatives were infected with Y. pseudotuberculosis wildtype strain YPIII ( cnfY + ) or YP147 (Δ cnfY ). The CFUs of egressed bacteria were determined 3.5 h after infection. The mean +/- SEM of three independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: **** < 0.0001). ( E ) Illustration of the CNF Y -triggered activation of Yersinia egress through the induction of the Cdc42-dependent Ca 2+ -regulated exocytosis machinery. Created in BioRender. Dersch, P. (2026). Upon uptake of Y. pseudotuberculosis, the bacteria reside in a membrane-bound vacuole, to which the v-SNARE protein VAMP-3 is initially recruited. Later, during transcytosis, VAMP3 is replaced by VAMP7. The CNF Y toxin is secreted by the intracellular bacteria (indicated by red arrows), leading to the activation of Cdc42 at the cell membrane by the deamidation of Gln61 of the Rho GTPase. This activates PLC-γ1, leading to the cleavage of phosphatidylinositol-4,5-bisphosphate (PIP 2 ), producing inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 interacts with the IP 3 receptor (IP 3 -R) in the endoplasmic reticulum (ER), which triggers the activation of adjacent Ca 2+ channels and the subsequent formation of a functional SNARE complex of VAMP7, Stx4, and SNAP25, and allows egress of Yersinia by exocytosis.

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Western Blot, Control, Marker, Mutagenesis, Infection, Bacteria, Activation Assay, Membrane, Functional Assay

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A ) Illustration of the VAMP-Stx-SNAP protein interaction for the formation of the SNARE complex initiating exocytotic processes. Created in BioRender. Dersch, P. (2026). ( B - J ) Caco-2 monolayer was infected with 2,5 x 10 6 bacteria of Y. pseudotuberculosis wildtype YPIII pFU92 ( gfp + ). 3.5 h post-infection, the Caco-2 cell monolayers were analyzed by CLSM to detect Yersinia (GFP-green), the cell plasma membranes (CellMask Plasma Membrane Red-CMPR), the nuclei (DAPI-blue), and SNARE proteins (yellow) colocalized with the bacteria using antibodies against VAMP3 ( B ), VAMP7 ( C,D ), Stx3 ( E ), Stx4 ( F,G ), SNAP25 ( H ), and SNAP23 ( I,J ). Orthogonal XZ and YZ planes are shown.

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Infection, Bacteria, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A-D ) Caco-2, and the isogenic cell lines Caco-2ΔStx3, Caco-2ΔStx4, Caco-2ΔSNAP23, and were seeded into transwells and cultivated until the TEER had reached > 400 Ω•cm². Whole-cell extracts were prepared and analyzed by Western blotting using antibodies specific to Stx3 ( A ), Stx4 ( B ), and SNAP23 ( C ), and actin as a loading control ( A-C ). M: protein marker. ( D ) Monolayers of wildtype Caco-2 cells (wt), and ΔStx3, ΔStx4, and ΔSNAP23 mutant derivatives were infected with Y. pseudotuberculosis wildtype strain YPIII ( cnfY + ) or YP147 (Δ cnfY ). The CFUs of egressed bacteria were determined 3.5 h after infection. The mean +/- SEM of three independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: **** < 0.0001). ( E ) Illustration of the CNF Y -triggered activation of Yersinia egress through the induction of the Cdc42-dependent Ca 2+ -regulated exocytosis machinery. Created in BioRender. Dersch, P. (2026). Upon uptake of Y. pseudotuberculosis, the bacteria reside in a membrane-bound vacuole, to which the v-SNARE protein VAMP-3 is initially recruited. Later, during transcytosis, VAMP3 is replaced by VAMP7. The CNF Y toxin is secreted by the intracellular bacteria (indicated by red arrows), leading to the activation of Cdc42 at the cell membrane by the deamidation of Gln61 of the Rho GTPase. This activates PLC-γ1, leading to the cleavage of phosphatidylinositol-4,5-bisphosphate (PIP 2 ), producing inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 interacts with the IP 3 receptor (IP 3 -R) in the endoplasmic reticulum (ER), which triggers the activation of adjacent Ca 2+ channels and the subsequent formation of a functional SNARE complex of VAMP7, Stx4, and SNAP25, and allows egress of Yersinia by exocytosis.

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Western Blot, Control, Marker, Mutagenesis, Infection, Bacteria, Activation Assay, Membrane, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: Selective peptide–guided transcytosis enhances extracellular vesicle–mediated siRNA delivery across the blood–brain barrier

doi: 10.1016/j.jbc.2025.110942

Figure Lengend Snippet: RVG-sEV transcytosis efficiency in BBB endothelial cells. A, fluorescence images of sEV-EGFP ( green ) and Cy5-siRNA ( red ) in hCMEC/D3 cells treated with chlorpromazine (CME inhibitor, 20 μM, 30 min) or genistein (CAV inhibitor, 200 μM, 30 min). Nuclei: DAPI ( blue ). The scale bar represents 25 μm. B, quantification of sEV-EGFP ( upper panel ) and Cy5-siRNA (l ower panel ) fluorescence intensity normalized to DAPI ( n = 5 per group). C, fluorescence images showing colocalization of sEV-EGFP ( green ) with early endosome marker EEA1 (red , left ) or late endosome marker Rab7 (red , right ). Nuclei: DAPI ( blue ). The scale bar represents 10 μm. Individual channels are shown in A and S3B. D, quantification of colocalization of sEV-EGFP with EEA1 or Rab7 normalized to DAPI ( n = 5 per group). E, fluorescence images showing colocalization of Cy5-siRNA ( red ) with EEA1 ( green , left ) or Rab7 ( green , right ). Nuclei: DAPI ( blue ). The scale bar represents 10 μm. Individual channels are shown in , A and B . F, quantification of colocalization of Cy5-siRNA with EEA1 or Rab7 normalized to DAPI ( n = 5 per group). G, fluorescence images showing colocalization of sEV-EGFP ( green ) with SNAP23 ( red ). Nuclei: DAPI ( blue ). The scale bar represents 10 μm. Individual channels are shown in C . H, quantification of colocalization of sEV-EGFP with SNAP23 normalized to DAPI ( n = 5 per group). I, fluorescence images showing colocalization of Cy5-siRNA ( red ) with SNAP23 ( green ). Nuclei: DAPI ( blue ). The scale bar represents 10 μm. Individual channels are shown in C . J, quantification of colocalization of siRNA with SNAP23 normalized to DAPI ( n = 5 per group). Data are presented as the mean ± SEM. p Values were determined using two-way ANOVA in B and one-way ANOVA followed by Tukey’s multiple comparison test in D , F , H , and J , ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also . BBB, blood–brain barrier; CAV, caveolin; DAPI, 4′,6-diamidino-2-phenylindole; EEA1, early endosome antigen 1; hCMEC, human cerebral microvascular endothelial cell; RVG, rabies virus glycoprotein; sEV, small extracellular vesicle.

Article Snippet: The following antibodies were used in this study: anti-nAChR, anti-p75NTR, anti-EGFP, anti-GFAP, anti-NeuN, and anti-CD31 antibodies were purchased from Abcam, and the anti-ZO1, anti-EEA1, anti-Rab7, anti-CD63, anti-TSG101, anti-Alix antibodies, horseradish peroxidase–conjugated Goat Anti-Mouse IgG (H + L), and horseradish peroxidase–conjugated Goat Anti-Rabbit IgG (H + L) were obtained from Proteintech; anti-SNAP23 antibody was purchased from Santa Cruz Biotechnology.

Techniques: Fluorescence, Marker, Comparison, Virus