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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 <t>microscope.</t> (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).
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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 <t>microscope.</t> (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).
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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).

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: AoCs were imaged using a spinning disk confocal microscope (CSU-W1 LiveSR, Gataca Systems coupled to Nikon) equipped with a 20× dry Nikon objective.

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