human lgals8  (R&D Systems)

Journal: Science Advances

Article Title: Mycobacterium tuberculosis phagosome Ca 2+ leakage triggers multimembrane ATG8/LC3 lipidation to restrict damage in human macrophages

doi: 10.1126/sciadv.adt3311

Figure Lengend Snippet: ( A ) GAL8 staining in THP-1 macrophages stably expressing GFP-LC3B after Mtb WT infection (2.5 hpi, MOI: 2) under treatment with BAPTA-AM and EGTA-AM. ( B ) GAL8 and GAL3 staining in THP-1 macrophages after Mtb WT and Mtb ΔRD1 infection (2.5 hpi, MOI: 2) under indicated treatments. ( C ) Percentage of Mtb-LC3-TVS positive for GAL8 related to (A) and fig. S3D. n (number of infected cells) = 51 to 72; data points correspond to individual technical replicates from three independent experiments. ( D ) Percentage of infected cells with Mtb-GAL3 positive, Mtb-GAL8 positive, and Mtb-GAL3/GAL8 double positive compartments. n (number of infected cells) = 96 to 151; data points correspond to individual technical replicates from three independent experiments. ( E ) Percentage of infected cells showing the Mtb-GAL3 positive, Mtb-GAL8 positive, and Mtb-GAL3/GAL8 double positive structures at 2.5, 8, and 24 hpi. n (number of infected cells) = 101 to 151; data points correspond to individual technical replicates from three independent experiments. The datasets for 2.5 hpi matches data from (D). Scale bars, in (A) and (B): 10 μm (main images) and 1 μm (inserted area).

Article Snippet: The antibodies used are as follows: GAL3 (BioLegend, 125410), GAL8 (R&D Systems, AF1305), LC3B (Sigma-Aldrich, L7543), p62 (CST, 88588), ATP6V1D (Abcam, ab157458), and CD63 (Abcam, ab217345).

Techniques: Staining, Stable Transfection, Expressing, Infection

Journal: Science Advances

Article Title: Mycobacterium tuberculosis phagosome Ca 2+ leakage triggers multimembrane ATG8/LC3 lipidation to restrict damage in human macrophages

doi: 10.1126/sciadv.adt3311

Figure Lengend Snippet: ( A ) Mtb infection (MOI: 2) in THP-1 macrophages stably expressing RFP-GFP-LC3B at 2.5 hpi under BafA1 and CQ treatment. ( B ) Percentage of infected cells showing Mtb-LC3-TVS under BafA1 and CQ treatment, related to (A). The dataset for control cells matches data from . Diagram shows endolysosome pH after BafA1and CQ treatment. n (number of infected cells) = 122 to 195; data points correspond to individual technical replicates from three independent experiments. ( C ) GAL8 staining in THP-1 macrophages stably expressing GFP-ATG16L1 at 2.5 hpi during Mtb infection (MOI: 2) under BafA1 treatment. ( D ) Quantification shows the percentage of Mtb-GAL8 positive structures positive for ATG16L1 under BafA1 treatment, related to (C). Diagram shows the function of BafA1 in blocking VATPase-ATG16L1 complex assembly. n (number of infected cells) = 178 and 202; data points correspond to individual technical replicates from 3 independent experiments. ( E ) ATP6V1D staining in THP-1 macrophages stably expressing GFP-ATG16L1 at 2.5 hpi during Mtb WT and Mtb ΔRD1 infection (MOI: 2) under indicated treatments. ( F and G ) Percentage of infected cells showing Mtb-ATP6V1D positive (F) and Mtb-ATG16L1 positive (G) structures under the indicated treatments, related to (E). n (number of infected cells) = 93 to 130; data points correspond to individual technical replicates from three independent experiments. ( H ) GAL8 and ATP6V1D staining in THP-1 macrophages infected with Mtb WT and Mtb ΔRD1 at 2.5 hpi. ( I ) Percentage of Mtb WT– and Mtb ΔRD1–infected cells showing the GAL8– and ATP6V1D–double positive structures, related to (H). n (number of infected cells) = 215 and 175; data points correspond to individual technical replicates from three independent experiments. Scale bars, 10 μm (main images) and 1 μm (inserted area).

Article Snippet: The antibodies used are as follows: GAL3 (BioLegend, 125410), GAL8 (R&D Systems, AF1305), LC3B (Sigma-Aldrich, L7543), p62 (CST, 88588), ATP6V1D (Abcam, ab157458), and CD63 (Abcam, ab217345).

Techniques: Infection, Stable Transfection, Expressing, Control, Staining, Blocking Assay

Journal: Science Advances

Article Title: Mycobacterium tuberculosis phagosome Ca 2+ leakage triggers multimembrane ATG8/LC3 lipidation to restrict damage in human macrophages

doi: 10.1126/sciadv.adt3311

Figure Lengend Snippet: ( A ) GAL8 and GAL3 staining in WT and ATG16L1 KO and FIP200 KO THP-1 macrophages after Mtb WT and Mtb ΔRD1 infection (2.5 hpi, MOI: 2). ( B and C ) Percentage of infected cells showing the Mtb-GAL8 positive and Mtb-GAL3 positive structures in WT and ATG7 KO, ATG16L1 KO, ATG13 KO, and FIP200 KO THP-1 macrophages related to (A) and fig. S5A. The datasets for WT cells are same as . n (number of infected cells) = 96 and 174; data points correspond to individual technical replicates from three independent experiments. ( D ) Representative micrographs at indicated time points of ATG16L1 KO and FIP200 KO THP-1 macrophages infected with Mtb WT (red) in the presence of PI+ necrotic cells (yellow). Bright-field images show the localization of macrophages. Data are representative from one of three independent experiments. ( E ) Quantification shows the fold change of Mtb area at 70 hpi with WT Mtb in WT, ATG7 KO, ATG16L1 KO, and FIP200 KO THP-1 macrophages. Data are from three independent biological replicates, each of which represents the mean of three technical replicates. ( F ) Quantification shows the fold change in the number of PI-positive cells 70 hpi with WT Mtb, compared to noninfected cells, in WT, ATG7 KO, ATG16L1 KO, and FIP200 KO THP-1 macrophages. Data are from three independent biological replicates, each of which represents the mean of three technical replicates. Scale bars, in (A): 10 μm; in (D): 50 μm.

Article Snippet: The antibodies used are as follows: GAL3 (BioLegend, 125410), GAL8 (R&D Systems, AF1305), LC3B (Sigma-Aldrich, L7543), p62 (CST, 88588), ATP6V1D (Abcam, ab157458), and CD63 (Abcam, ab217345).

Techniques: Staining, Infection