Journal: bioRxiv

Article Title: Galectin-3 recruitment at the Mycobacterium tuberculosis -containing phagosome is critical in macrophage but dispensable in epithelial cells

doi: 10.64898/2026.06.04.730029

Figure Lengend Snippet: Galectin-3 recruitment to intracellular bacteria and associated cellular phenotypes during infection. THP-1 cells were grown in 384-well plates, infected with Mtb H37Rv pMRF1 (MOI = 1) or heat-killed (HK) H37Rv pMRF1 for 5 days and imaged by automated confocal microscopy. (A) Representative images of THP-1 (nucleus; blue) infected with H37Rv pMRF1 (yellow) over 5 days. Scale bar = 50 μm (20X). (B) Representative images of THP-1 infected with live and HK H37Rv pMRF1 (MOI = 1; yellow) and labelled by immunofluorescence for Galectin-3 (Gal3; red) at day 3 post-infection. (Left) The focused image highlights a live bacterium colocalized with a Gal3 spot, indicating phagosomal membrane damage, while (right) no colocalization of HK bacterium with Gal3 spot is shown. The magnified inset of bystander cells under conditions of infection with both live and HK bacteria show Gal3 spots, suggesting cellular damage. Scale bar = 50 μm (20X). (C, D, E, F, G) Quantitative image analysis of infected and bystander HPAEpiC cells infected with live and HK H37Rv pMRF1 and in non-infected conditions over 5 days. (C) Percentage of bacteria colocalizing with Gal3 spots. Live bacteria display significantly higher rate of colocalization with Gal3 than HK bacteria from day 1 post-infection (Mann-Whitney, P≤0.05). (D) Intracellular bacterial area (pixel²) per infected cells containing at least one bacterium that is positive for Gal3 (Gal3+, rupture) compared to cells infected with bacteria not colocalized with Gal3 spot (Gal3-, non-rupture). The cells exhibiting colocalization with Gal3 show significantly greater bacterial area from 1 day after infection (Wilcoxon test, P≤0.05). (E) Total cell number. No significant difference is detected on day 0, while cell numbers obtained for live-infection conditions and HK controls are significantly different from day 1 post-infection (One-way Anova, P≤0.05). (F) Percentage of infected and bystander Gal3-positive THP-1 cells for infection with live bacteria. Infected cells show significant higher rate of Gal3-positive cells than bystander cells (Paired t-test, P≤0.05). (G) Percentage of infected and bystander Gal3-positive THP-1 cells for infection with HK bacteria. Infected cells exhibit a significantly higher proportion of Gal3-positive cells up to the first day following infection, whereas no significant difference is observed from the day 2 (Paired t-test, P≤0.05). (H) Mean cellular Gal3 fluorescence intensity. Infected cells exhibit significantly reduced Gal3 intensity relative to bystanders from day 1 (Paired t-test, P≤0.05), whereas bystander and uninfected cells do not differ (Unpaired t-test). (I) Mean cellular Gal3 fluorescence intensity over time in infected and bystander cells obtained for infection with HK H37Rv pMRF1, and in uninfected controls. No significant differences (paired and unpaired t-test, P≤0.05).

Article Snippet: We analyzed Gal3 dynamics over a five-day infection course in THP-1 differentiated human macrophages with a virulent red fluorescent strain of Mtb (H37Rv) followed by immunostaining with an anti-Galectin 3 (Gal3) antibody and nuclear labeling with DAPI ( ).

Techniques: Bacteria, Infection, Confocal Microscopy, Immunofluorescence, Membrane, MANN-WHITNEY, Fluorescence

Journal: bioRxiv

Article Title: Galectin-3 recruitment at the Mycobacterium tuberculosis -containing phagosome is critical in macrophage but dispensable in epithelial cells

doi: 10.64898/2026.06.04.730029

Figure Lengend Snippet: ESX-1–dependent phagosomal damage using BCG and BCG::ESX-1 Mmar strains. THP-1 cells were grown in 384-well plates, infected with BCG pMRF1 and BCG::ESX-1 Mmar pMRF1 (MOI = 2) for 6 days and imaged with confocal microscope. (A) Representative images of THP-1 (nuclei; blue) infected with BCG and BCG::ESX-1Mmar (yellow), showing bacterial colocalization of BCG::ESX-1 Mmar with a Gal3 spot (red). Scale bar = 10 μm (60X). (B) Percentage of bacteria colocalizing with Gal3 spots over 6 days. BCG::ESX-1 Mmar display a significantly higher rate of colocalization with Gal3 than BCG from day 2 post-infection (Mann-Whitney test, P≤0.05). (C) Quantification of total cell number under conditions of BCG and BCG::ESX-1 Mmar infection. No significant difference is observed between the two bacterial strains (Unpaired t-test). (D) Flow cytometry gating strategy for BCG and BCG::ESX-1 Mmar -infected samples. Singlet cells were first selected based on size (Forward Scatter, FSC) vs. granularity (Side Scatter, SSC) and on FSC-H vs. FSC-A, followed by separation of infected populations (DsRed+) displaying two distinct phenotypes based on blue and green fluorescence signals of the CCF4-AM staining. (E): Percentage of CCF4-positive (blue) infected cells at 3 days post-infection, which is significantly increased under BCG::ESX-1 Mmar infection conditions than under BCG infection conditions (Unpaired t-test, P≤0.05).

Article Snippet: We analyzed Gal3 dynamics over a five-day infection course in THP-1 differentiated human macrophages with a virulent red fluorescent strain of Mtb (H37Rv) followed by immunostaining with an anti-Galectin 3 (Gal3) antibody and nuclear labeling with DAPI ( ).

Techniques: Infection, Microscopy, Bacteria, MANN-WHITNEY, Flow Cytometry, Fluorescence, Staining

Journal: bioRxiv

Article Title: Galectin-3 recruitment at the Mycobacterium tuberculosis -containing phagosome is critical in macrophage but dispensable in epithelial cells

doi: 10.64898/2026.06.04.730029

Figure Lengend Snippet: Functional impact of LGALS3 silencing on Galectin-3 levels and infection phenotypes. THP-1 cells were transfected with SiRNA in 384-well plates, infected with H37Rv pMRF1 (MOI = 1) for 3 days and imaged by automated confocal microscopy. (A) Representative images of THP-1 cells transfected with scramble control (left), siLGALS3 (middle) and siVPS18 (right) and infected. Scale bar = 50 μm (20X). (B, C, D, E) Quantitative image analysis of non-transfected (NT), scramble, siLGALS3 and siVPS18 conditions on day 3 post-infection. (B) Mean Gal3 fluorescence intensity. Gal3 signal is 4-fold reduced for siLGALS3-treated cells relative to scramble and siVPS18-treated ones (Brown–Forsythe ANOVA, P≤0.01). (C) Intracellular bacterial area (pixel²) per infected cells containing at least one bacterium that is positive for Gal3 (Gal3+, rupture) compared to cells infected with bacteria not colocalized with Gal3 spot (Gal3-, non-rupture). Bacterial area is significantly increased under both siLGALS3 and siVPS18 conditions by comparison with scramble condition (Unpaired t-test, P≤0.05). (D) Percentage of bacteria colocalizing with Gal3 spots. No significant difference is observed between NT and scramble controls. Bacteria and Gal3 colocalization is significantly decreased for siLGALS3-transfected cells and significantly increased for siVPS18-transfected ones relative to scramble condition (Mann-Whitney test, P≤0.05). (E) Total cell number for transfected and non transfected conditions. No significant difference is observed (Unpaired t-test).

Article Snippet: We analyzed Gal3 dynamics over a five-day infection course in THP-1 differentiated human macrophages with a virulent red fluorescent strain of Mtb (H37Rv) followed by immunostaining with an anti-Galectin 3 (Gal3) antibody and nuclear labeling with DAPI ( ).

Techniques: Functional Assay, Infection, Transfection, Confocal Microscopy, Control, Fluorescence, Bacteria, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Galectin-3 recruitment at the Mycobacterium tuberculosis -containing phagosome is critical in macrophage but dispensable in epithelial cells

doi: 10.64898/2026.06.04.730029

Figure Lengend Snippet: Infection phenotype and Galectin-3 distribution in human alveolar epithelial cells (HPAEpiC). HPAEpiC cells were grown in 384-well plates, infected with Mtb H37Rv pMRF1 (MOI = 5) for 3 days and imaged by automated confocal microscopy. (A) Representative image of HPAEpiC cells infected with live H37Rv pMRF1 bacteria (yellow) and labelled by immunofluorescence for Galectin-3 (Gal3; red). The focused image shows a bacterium which colocalized with a Gal3 spot. Scale bar = 20 μm (40X). (B, C, D, E) Quantitative image analysis of infected and bystander HPAEpiC cells infected with live H37Rv pMRF1 or uninfected over 3 days. (B) Percentage of infected, bystander and uninfected Gal3-positive HPAEpiC cells. No significant difference is observed between infected and bystander cells (Paired t-test) and between uninfected and bystander cells or uninfected and infected cells (Mann-Whitney test). (C) Mean Gal3 fluorescence intensity in infected, bystander and uninfected HPAEpiC cells. No significant difference is observed between cells infected with H37Rv pMRF1 and bystander cells (Paired t-tests) or uninfected conditions (Unpaired t-test). (D) Intracellular bacterial area (pixel²) per infected cells containing at least one bacterium that is positive for Gal3 (rupture) versus infected cells without colocalization of bacterium with Gal3 spot (non-rupture). The cells exhibiting colocalization with Gal3 or not show non significant difference of bacterial area from 2 days after infection. (Mann-Whitney test). (E) Total cell number for infected and non infected conditions. No significant difference observed between the conditions (Unpaired t-test).

Article Snippet: We analyzed Gal3 dynamics over a five-day infection course in THP-1 differentiated human macrophages with a virulent red fluorescent strain of Mtb (H37Rv) followed by immunostaining with an anti-Galectin 3 (Gal3) antibody and nuclear labeling with DAPI ( ).

Techniques: Infection, Confocal Microscopy, Bacteria, Immunofluorescence, MANN-WHITNEY, Fluorescence

Journal: bioRxiv

Article Title: Galectin-3 recruitment at the Mycobacterium tuberculosis -containing phagosome is critical in macrophage but dispensable in epithelial cells

doi: 10.64898/2026.06.04.730029

Figure Lengend Snippet: Human alveolus-on-chip (AoC) model and Galectin-3 responses to infection. HPAEpiC cells and human lung endothelial cells were seeded in in-house AoC devices, respectively in top and bottom channels, and grown over 7 days to reconstitute the alveolar interface. Human macrophages were introduced into the top channel on the day 7 (M-AoC) and then infected with Mtb H37Rv pMRF1 (MOI = 5) on day 8. M-AoC was imaged at 1 and 5 days post-infection by confocal microscopy and analyzed using a 3D image analysis software (IMARIS v10, Oxford Instruments). (A) Photograph of the in-house alveolus-on-chip (AoC) device. (B) Representative 3D-imaging of infected M-AoC at 1 day post-infection, used for the determination of H37Rv pMRF1 infection (red) inside the alveolar barrier model (nuclei, blue; actin, gray; macrophages, green). Scale bar = 50 μm (20X). (C) Representative immunofluorescence image of the alveolar barrier co-cultured with macrophages (CFSE labeled, green) and infected with H37Rv pMRF1 (yellow). The focused image highlights a bacterium within a macrophage colocalized with a strong Gal3 signal (red), consistent with phagosomal rupture. (D) Percentage of infected Gal3-positive HPAEpiC cells and macrophages in AoC and M-AoC models at days 1 and 5 post-infection. Infected macrophages exhibit significant higher rate than epithelial cells in M-AoC (Paired t-test) and epithelial cells in AoC models (Unpaired t-test, P≤0.05). Quantifications were obtained from pooled datasets comprising ∼10 infected macrophages and ∼40 infected epithelial cells per experiment.

Article Snippet: We analyzed Gal3 dynamics over a five-day infection course in THP-1 differentiated human macrophages with a virulent red fluorescent strain of Mtb (H37Rv) followed by immunostaining with an anti-Galectin 3 (Gal3) antibody and nuclear labeling with DAPI ( ).

Techniques: Infection, Confocal Microscopy, Software, 3D Imaging, Immunofluorescence, Cell Culture, Labeling

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Time-dependent nuclear localization of Galectin-9 and electrotransfected plasmid DNA in super-resolution images. Stably Gal9-GFP–expressing C2C12 cells were electrotransfected with Cy5-labeled plasmid DNA (red) and imaged by confocal fluorescence microscopy at 3, 6, and 9 h post-ET. Hoechst (blue) was used to stain nuclei, and Gal9-GFP (green) was used to monitor Gal9 localization. Scale bars: 10 μm.

Article Snippet: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

Techniques: Plasmid Preparation, Stable Transfection, Expressing, Labeling, Fluorescence, Microscopy, Staining

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Galectin-9 nuclear localization in untreated and mimosine-treated cells. (A) Quantification of cell proliferation with EdU. C2C12 cells were pretreated with or without mimosine for 24 h, followed by electrotransfection of pLuc and incubation with EdU for 16 h. The cells were analyzed with flow cytometry. The histograms of EdU signal were used to quantify the percentages of cells in G0/G1 phase, early S phase, and late S phase. (B) Typical super-resolution images of Cy5-pLuc and Gal9-GFP post-ET in untreated and mimosine-treated cells. Hoechst-stained nuclei (blue), Cy5-pDNA signals (white), colocalized Gal9-GFP with pDNA in the inner nucleus (green), and pDNA localized on the nuclear edge (red). Scale bars: 10 μm. (C) Quantification of pDNA, Gal9, and pDNA-Gal9 colocalization in untreated and mimosine-treated cells. n = 20. mean ± SEM; * P < 0.05, ** P < 0.01, ns = not significant (Mann–Whitney U test).

Article Snippet: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

Techniques: Incubation, Flow Cytometry, Staining, MANN-WHITNEY

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Galectin-9 colocalizes with transcriptional nuclear hubs and accumulates in speckle-like regions following pDNA electrotransfection. (A) Representative confocal fluorescence images of C2C12 cells electrotransfected with Cy5-labeled pDNA, acquired 1 h post-electroporation. Hoechst (blue) stains the nucleus, Gal9-GFP (green) shows Gal9 localization, SC35 (red) is detected using a specific antibody, and Cy5-pDNA is shown in white. The merged image highlights areas of colocalization between Gal9 and SC35 (yellow arrow). (B) Representative confocal image showing the association of Gal9, pDNA, and SC35 at 3 h post-electroporation (left panel). The yellow arrow indicates a triple colocalization among Gal9, SC35, and pDNA. The right panel shows a super-resolution enlargement of the boxed region from the left panel. Scale bars: 10 μm.

Article Snippet: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

Techniques: Fluorescence, Labeling, Electroporation

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Time-dependent colocalization of Galectin-9 with plasmid DNA following electrotransfection and Lipofectamine-mediated transfection. (A) Quantification of Gal9-GFP and pDNA colocalization in C2C12 cells following electrotransfection. Cells transfected with pDNA were fixed at different time points (3, 6, 9, 12, 16, and 24 h) and analyzed by in-situ hybridization. (B) Quantification of Gal9-GFP and pDNA colocalization in cells transfected with Lipofectamine 3000. Cells were fixed at 3, 6, 24, 36, and 48 h post-transfection and assessed by in-situ hybridization. Data are shown as mean ± SEM ( n = 15–25, each time point); Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD test. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. (C) Representative confocal images of Gal9-GFP expressing C2C12 cells electrotransfected with Cy5-labeled nucleic acid cargos. C2C12 cells were pulsed with pDNA, minicircle DNA, mRNA, or without cargo (ET only) and imaged by confocal fluorescence microscopy at 1 h post-ET. Confocal images showing Gal9 (green), nucleic acid cargos (red), and nuclei (blue). Scale bars: 10 μm.

Article Snippet: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

Techniques: Plasmid Preparation, Transfection, In Situ Hybridization, Expressing, Labeling, Fluorescence, Microscopy

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Knockdown of Galectin-9 reduces electrotransfection efficiency and pDNA transcription. (A) Western blot analysis confirming Gal9 knockdown in C2C12 cells treated with Gal9-specific siRNA compared to WT and negative siRNA (Neg) controls. β-actin was used as an internal control. (B) Representative confocal images showing nuclear distribution of Gal9, SC35, and pDNA in WT and Gal9 knockdown (KD) cells. Gal9-GFP (green), pDNA (white), SC35 (red), and nuclei stained with Hoechst (blue). The yellow arrow indicates colocalization of Gal9, pDNA, and SC35 in WT cells. Scale bars: 10 μm. (C) Quantification of electrotransfection efficiency (eTE, left) and EGFP reporter expression level (right) by flow cytometry in WT, negative control siRNA, and Gal9 siRNA knockdown groups. Data are shown as mean ± SEM ( n = 6); one-way ANOVA followed by Tukey’s HSD test was performed: **** P < 0.0001, ns, not significant.

Article Snippet: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

Techniques: Knockdown, Western Blot, Control, Staining, Expressing, Flow Cytometry, Negative Control

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Overexpression of Galectin-9 enhances electrotransfection efficiency and transgene expression. (A) Western blot confirming Gal9 overexpression (OE) in C2C12 cells compared to wide type (WT) controls. β-actin served as an internal control. (B) Quantification of electrotransfection efficiency (eTE, left) and EGFP reporter expression level (right) by flow cytometry in WT and Gal9-overexpressing cells. Data are shown as mean ± SEM ( n = 4); Mann–Whitney U test: * P < 0.05.

Article Snippet: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

Techniques: Over Expression, Expressing, Western Blot, Control, Flow Cytometry, MANN-WHITNEY

Journal: NAR Molecular Medicine

Article Title: Functional role of Galectin-9 in nucleic acid trafficking and transcription post-electrotransfection

doi: 10.1093/narmme/ugag020

Figure Lengend Snippet: Correlation between Galectin-9 expression and pDNA electrotransfection efficacy across different cell lines. (A) Western blot analysis of Gal9 protein levels in B16F10, C2C12, and 4T1 cells, with β-actin used as a control. (B) Comparison of eTE, EGFP expression level, cell viability, and apparent expression level in cells electrotransfected with an EGFP-encoding plasmid. Data are shown as mean ± SEM ( n = 3); one-way ANOVA followed by Tukey’s HSD test was performed: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, ns, not significant.

Article Snippet: Gal9 proteins were detected using a primary anti-Galectin-9 antibody and an HRP-conjugated secondary antibody (Invitrogen).

Techniques: Expressing, Western Blot, Control, Comparison, Plasmid Preparation