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: WT and SNAP25 KO BEAS-2B cells were transfected with TETMR1-GFP and incubated with doxycycline overnight. 6-FP or NaOH (solvent control) was added the next day for at least 18 hours. (A-C) Cells were analyzed by flow cytometry. Representative of three independent experiments with pooled geometric mean fluorescence intensity (GeoMFI) of GFP+ cells on total MR1 (A), surface MR1 (B) and surface HLA-Ia expression (C). For BFA decay assays, cells were incubated with brefeldin A (BFA) for indicated time periods after induction of cells with doxycycline and 6-FP. (D) GeoMFI of GFP+ cells on total MR1 (left) and surface MR1 expression (right) were measured by flow cytometry. Representative of four independent experiments with pooled GeoMFI for each time point. Non-linear regression analysis on one-phase exponential decay curves and comparing one curve fit to both data sets by extra sum-of squares F test. 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: Transfection, Incubation, Solvent, Control, Flow Cytometry, Fluorescence, Expressing

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: WT and SNAP25 KO BEAS-2B cells were transfected with TETMR1-GFP and incubated with doxycycline overnight. 6-FP or NaOH (solvent control) was added the next day for at least 18 hours. (A-C) Cells were analyzed by flow cytometry. Representative of three independent experiments with pooled geometric mean fluorescence intensity (GeoMFI) of GFP+ cells on total MR1 (A), surface MR1 (B) and surface HLA-Ia expression (C). For BFA decay assays, cells were incubated with brefeldin A (BFA) for indicated time periods after induction of cells with doxycycline and 6-FP. (D) GeoMFI of GFP+ cells on total MR1 (left) and surface MR1 expression (right) were measured by flow cytometry. Representative of four independent experiments with pooled GeoMFI for each time point. Non-linear regression analysis on one-phase exponential decay curves and comparing one curve fit to both data sets by extra sum-of squares F test. 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: Transfection, Incubation, Solvent, Control, Flow Cytometry, Fluorescence, Expressing