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pex3 cdna r turbo infusion  (Sino Biological)


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    Sino Biological pex3 cdna r turbo infusion
    Generation and characterization of <t>PEX3</t> −/− iPSC and iPSDM clones. (A and B) Selection of PEX3 −/− clones. (A) PCR genotyping of the expanded clones, D2 and E3. (B) Top: Schematic representation of the PEX3 KO CRISPR strategy. PCR primers (blue), sgRNA (orange). Bottom: Sanger sequencing of the upper band for the D2 clones (*). (C) Immunofluorescence of iPSC PEX3 −/− for pluripotent markers (OCT3/4, TRA-1-60, and TRA-1-81). Scale bars: 100 µm. (D) Flow cytometry characterization of PEX3 +/+ and PEX3 −/− monocytes and macrophages. Names of the markers are indicated on the graph graphs. Black, negative sample; red, isotype control; blue, marker.
    Pex3 Cdna R Turbo Infusion, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pex3+cdna+orf/Human+PEX3+Gene+ORF+cDNA+clone+expression+plasmid/pmc10515436-151-14-25
    Average 91 stars, based on 1 article reviews
    pex3 cdna r turbo infusion - by Bioz Stars, 2026-10
    91/100 stars

    Images

    1) Product Images from "Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages"

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202303066

    Generation and characterization of PEX3 −/− iPSC and iPSDM clones. (A and B) Selection of PEX3 −/− clones. (A) PCR genotyping of the expanded clones, D2 and E3. (B) Top: Schematic representation of the PEX3 KO CRISPR strategy. PCR primers (blue), sgRNA (orange). Bottom: Sanger sequencing of the upper band for the D2 clones (*). (C) Immunofluorescence of iPSC PEX3 −/− for pluripotent markers (OCT3/4, TRA-1-60, and TRA-1-81). Scale bars: 100 µm. (D) Flow cytometry characterization of PEX3 +/+ and PEX3 −/− monocytes and macrophages. Names of the markers are indicated on the graph graphs. Black, negative sample; red, isotype control; blue, marker.
    Figure Legend Snippet: Generation and characterization of PEX3 −/− iPSC and iPSDM clones. (A and B) Selection of PEX3 −/− clones. (A) PCR genotyping of the expanded clones, D2 and E3. (B) Top: Schematic representation of the PEX3 KO CRISPR strategy. PCR primers (blue), sgRNA (orange). Bottom: Sanger sequencing of the upper band for the D2 clones (*). (C) Immunofluorescence of iPSC PEX3 −/− for pluripotent markers (OCT3/4, TRA-1-60, and TRA-1-81). Scale bars: 100 µm. (D) Flow cytometry characterization of PEX3 +/+ and PEX3 −/− monocytes and macrophages. Names of the markers are indicated on the graph graphs. Black, negative sample; red, isotype control; blue, marker.

    Techniques Used: Clone Assay, Selection, CRISPR, Sequencing, Immunofluorescence, Flow Cytometry, Control, Marker

    Human macrophages lacking peroxisomes are unable to restrict Mtb WT replication. (A) Confocal images of iPSDM PEX3 +/+ and PEX3 −/− for GFP-PTS1 (green), PEX14 (green), TOM20 (red), CAT (red), and PMP70 (green). Nuclear staining (blue). Scale bars: 10 µm. (B) Analysis of Mtb WT growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb WT. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom: 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data representative of one out of three independent experiments ( n = 4 independent wells). (C) Analysis of Mtb ΔRD1 growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb ΔRD1. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data are representative of one out of three independent experiments ( n = 4 technical replicate per each condition). Significance was determined by two-way ANOVA with Tukey’s multiple comparison post-test (B and C). P value 0.033 (*), 0.002 (**), <0.0001 (***).
    Figure Legend Snippet: Human macrophages lacking peroxisomes are unable to restrict Mtb WT replication. (A) Confocal images of iPSDM PEX3 +/+ and PEX3 −/− for GFP-PTS1 (green), PEX14 (green), TOM20 (red), CAT (red), and PMP70 (green). Nuclear staining (blue). Scale bars: 10 µm. (B) Analysis of Mtb WT growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb WT. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom: 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data representative of one out of three independent experiments ( n = 4 independent wells). (C) Analysis of Mtb ΔRD1 growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb ΔRD1. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data are representative of one out of three independent experiments ( n = 4 technical replicate per each condition). Significance was determined by two-way ANOVA with Tukey’s multiple comparison post-test (B and C). P value 0.033 (*), 0.002 (**), <0.0001 (***).

    Techniques Used: Staining, Infection, Control, Clone Assay, Comparison

    Characterization of PEX3 −/− iPSDM clones and uptake and growth of Mtb in PEX3 +/+ and PEX3 −/− iPSDM. (A) Rescue experiment with PEX3_turbo for 24 h. Snapshot of iPSDM PEX3 +/+ and PEX3 −/− . Nuclear staining (blue), PEX3_turbo (orange), and EGFP-PTS1 (green). Scale bars: 10 µm. (B) Western blot of peroxisomal related protein expressed in iPSDM PEX3 +/+ and PEX3 −/− at the steady state. (C) Quantification of CAT expression from B normalized with actin. (D) Analysis of Mtb growth in iPSDM PEX3 +/+ and PEX3 −/− . Violin plot representation of Mtb area (px2) per cells over time (2, 24, 48, and 72 hpi) in PEX3 +/+ and PEX3 −/− (clone 1 and 2) during infection with Mtb WT and Mtb ∆RD1. Significance was determined for the 2 hpi time point by two-way ANOVA with Tukey’s multiple comparison post-test. Source data are available for this figure: .
    Figure Legend Snippet: Characterization of PEX3 −/− iPSDM clones and uptake and growth of Mtb in PEX3 +/+ and PEX3 −/− iPSDM. (A) Rescue experiment with PEX3_turbo for 24 h. Snapshot of iPSDM PEX3 +/+ and PEX3 −/− . Nuclear staining (blue), PEX3_turbo (orange), and EGFP-PTS1 (green). Scale bars: 10 µm. (B) Western blot of peroxisomal related protein expressed in iPSDM PEX3 +/+ and PEX3 −/− at the steady state. (C) Quantification of CAT expression from B normalized with actin. (D) Analysis of Mtb growth in iPSDM PEX3 +/+ and PEX3 −/− . Violin plot representation of Mtb area (px2) per cells over time (2, 24, 48, and 72 hpi) in PEX3 +/+ and PEX3 −/− (clone 1 and 2) during infection with Mtb WT and Mtb ∆RD1. Significance was determined for the 2 hpi time point by two-way ANOVA with Tukey’s multiple comparison post-test. Source data are available for this figure: .

    Techniques Used: Clone Assay, Staining, Western Blot, Expressing, Infection, Comparison

    Peroxisome-dependent restriction of Mtb is associated with higher levels of ROS in the cytosol. (A–C) Cyto_Hyper reporter during Mtb infection. (A) Confocal images of iPSDM at 24 hpi of infection (Uninfected, Mtb WT, and ΔRD1). The left graph shows Cyto_Hyper in iPSDM PEX3 +/+ and right graph shows Cyto_Hyper in PEX3 −/− iPSDM. Top: Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Bottom: Ratiometric imaging of the Cyto_Hyper reporter. Scale bars: 10 µm. (B) Quantification of Cyto_Hyper ratio in iPSDM PEX3 +/+ and PEX3 −/− during infection at 20 and 40 hpi. Data representative from one out of two independent experiments ( n = 4 independent wells per replicate). Significance was determined using unpaired by two-way ANOVA with Šídák’s multiple comparisons post-test. P value (APA) 0.033 (*), 0.002 (**). (C) 3D surface (left) and line plot (right) of Cyto_Hyper reporter in iPSDM PEX3 +/+ (1 box) and PEX3 −/− (2 box) infected with Mtb WT. (D and E) Endo_Hyper reporter during Mtb infection. (D) Snapshot of Endo_Hyper reporter in iPSDM PEX3 +/+ and PEX3 −/− during infection over 24 hpi. Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Scale bars: 10 µm. (E) Analysis of Endo_Hyper ratio around Mtb (area 0.5 µm) in iPSDM PEX3 +/+ and PEX3 −/− infected with Mtb WT and ΔRD1 at 24 hpi. The red line represents the median of Mtb-Endo_Hyper ratio in PEX3 +/+ and the light blue line represents the median of Mtb-Endo_Hyper ratio in PEX3 −/− iPSDM. N > 500 Mtb regions of interest were quantified per each condition.
    Figure Legend Snippet: Peroxisome-dependent restriction of Mtb is associated with higher levels of ROS in the cytosol. (A–C) Cyto_Hyper reporter during Mtb infection. (A) Confocal images of iPSDM at 24 hpi of infection (Uninfected, Mtb WT, and ΔRD1). The left graph shows Cyto_Hyper in iPSDM PEX3 +/+ and right graph shows Cyto_Hyper in PEX3 −/− iPSDM. Top: Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Bottom: Ratiometric imaging of the Cyto_Hyper reporter. Scale bars: 10 µm. (B) Quantification of Cyto_Hyper ratio in iPSDM PEX3 +/+ and PEX3 −/− during infection at 20 and 40 hpi. Data representative from one out of two independent experiments ( n = 4 independent wells per replicate). Significance was determined using unpaired by two-way ANOVA with Šídák’s multiple comparisons post-test. P value (APA) 0.033 (*), 0.002 (**). (C) 3D surface (left) and line plot (right) of Cyto_Hyper reporter in iPSDM PEX3 +/+ (1 box) and PEX3 −/− (2 box) infected with Mtb WT. (D and E) Endo_Hyper reporter during Mtb infection. (D) Snapshot of Endo_Hyper reporter in iPSDM PEX3 +/+ and PEX3 −/− during infection over 24 hpi. Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Scale bars: 10 µm. (E) Analysis of Endo_Hyper ratio around Mtb (area 0.5 µm) in iPSDM PEX3 +/+ and PEX3 −/− infected with Mtb WT and ΔRD1 at 24 hpi. The red line represents the median of Mtb-Endo_Hyper ratio in PEX3 +/+ and the light blue line represents the median of Mtb-Endo_Hyper ratio in PEX3 −/− iPSDM. N > 500 Mtb regions of interest were quantified per each condition.

    Techniques Used: Infection, Imaging

    Peroxisome-dependent restriction of Mtb is spatial-temporally separated from the NADPH oxidase activity in human macrophages. (A) Workflow for nucleofection (nf) approach to KO CYBB (CYBB nf ) and PEX3 (PEX3 nf ) genes in HMDM. (B) Western blot of HMDM CTRL, PEX3 nf , and CYBB nf for gp91-phox protein (CYBB). (C) Left: Immunofluorescence of HMDM CTRL and PEX3 nf . Nuclear staining (blue), PMP70 (orange), and CAT (green). Scale bars: 20 µm. Right: Quantification of CAT and PMP70 puncta area per area of cells. Significance was determined by two-way ANOVA with Šidák’s test. P value <0.0001 (***). (D) Mtb growth in HMDM CTRL, PEX3 nf , and CYBB nf . Left: Confocal images of HMDM CTRL, PEX3 nf , and CYBB nf at 60 hpi infected with Mtb WT (top) and ΔRD1 (bottom). Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm. Right: Fold change of Mtb growth in HMDM CTRL (green), PEX3 nf (orange), and CYBB nf (light blue) over 98 h of infection. The top graph shows the fold change of Mtb WT and the bottom graph the fold change of Mtb ΔRD1. Data representative from one out of two independent experiments ( n = 3 independent wells per replicate). Significance was determined only for the last time point (98 hpi) by two-way ANOVA with Dunnett’s post-doc test. P value 0.033 (*).
    Figure Legend Snippet: Peroxisome-dependent restriction of Mtb is spatial-temporally separated from the NADPH oxidase activity in human macrophages. (A) Workflow for nucleofection (nf) approach to KO CYBB (CYBB nf ) and PEX3 (PEX3 nf ) genes in HMDM. (B) Western blot of HMDM CTRL, PEX3 nf , and CYBB nf for gp91-phox protein (CYBB). (C) Left: Immunofluorescence of HMDM CTRL and PEX3 nf . Nuclear staining (blue), PMP70 (orange), and CAT (green). Scale bars: 20 µm. Right: Quantification of CAT and PMP70 puncta area per area of cells. Significance was determined by two-way ANOVA with Šidák’s test. P value <0.0001 (***). (D) Mtb growth in HMDM CTRL, PEX3 nf , and CYBB nf . Left: Confocal images of HMDM CTRL, PEX3 nf , and CYBB nf at 60 hpi infected with Mtb WT (top) and ΔRD1 (bottom). Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm. Right: Fold change of Mtb growth in HMDM CTRL (green), PEX3 nf (orange), and CYBB nf (light blue) over 98 h of infection. The top graph shows the fold change of Mtb WT and the bottom graph the fold change of Mtb ΔRD1. Data representative from one out of two independent experiments ( n = 3 independent wells per replicate). Significance was determined only for the last time point (98 hpi) by two-way ANOVA with Dunnett’s post-doc test. P value 0.033 (*).

    Techniques Used: Activity Assay, Western Blot, Immunofluorescence, Staining, Infection

    Related Articles

    Sequencing:

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages
    Article Snippet: .. For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template. .. The In-Fusion cloning reaction was performed using the purified PCR product and the tdTurboRFP-Lysosomes-20 (plasmid #58061; Addgene) as backbone, following the vendor’s instructions (In-Fusion HD Cloning Plus, #638910).

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages.
    Article Snippet: .. For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers (Table S1) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template. .. The In-Fusion cloning reaction was performed using the purified PCR product and the tdTurboRFP-Lysosomes-20 (plasmid #58061; Addgene) as backbone, following the vendor’s instructions (In-Fusion HD Cloning Plus, #638910).

    Amplification:

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages
    Article Snippet: .. For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template. .. The In-Fusion cloning reaction was performed using the purified PCR product and the tdTurboRFP-Lysosomes-20 (plasmid #58061; Addgene) as backbone, following the vendor’s instructions (In-Fusion HD Cloning Plus, #638910).

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages.
    Article Snippet: .. For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers (Table S1) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template. .. The In-Fusion cloning reaction was performed using the purified PCR product and the tdTurboRFP-Lysosomes-20 (plasmid #58061; Addgene) as backbone, following the vendor’s instructions (In-Fusion HD Cloning Plus, #638910).

    Polymerase Chain Reaction:

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages
    Article Snippet: .. For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template. .. The In-Fusion cloning reaction was performed using the purified PCR product and the tdTurboRFP-Lysosomes-20 (plasmid #58061; Addgene) as backbone, following the vendor’s instructions (In-Fusion HD Cloning Plus, #638910).

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages.
    Article Snippet: .. For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers (Table S1) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template. .. The In-Fusion cloning reaction was performed using the purified PCR product and the tdTurboRFP-Lysosomes-20 (plasmid #58061; Addgene) as backbone, following the vendor’s instructions (In-Fusion HD Cloning Plus, #638910).



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    Image Search Results


    Generation and characterization of PEX3 −/− iPSC and iPSDM clones. (A and B) Selection of PEX3 −/− clones. (A) PCR genotyping of the expanded clones, D2 and E3. (B) Top: Schematic representation of the PEX3 KO CRISPR strategy. PCR primers (blue), sgRNA (orange). Bottom: Sanger sequencing of the upper band for the D2 clones (*). (C) Immunofluorescence of iPSC PEX3 −/− for pluripotent markers (OCT3/4, TRA-1-60, and TRA-1-81). Scale bars: 100 µm. (D) Flow cytometry characterization of PEX3 +/+ and PEX3 −/− monocytes and macrophages. Names of the markers are indicated on the graph graphs. Black, negative sample; red, isotype control; blue, marker.

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Generation and characterization of PEX3 −/− iPSC and iPSDM clones. (A and B) Selection of PEX3 −/− clones. (A) PCR genotyping of the expanded clones, D2 and E3. (B) Top: Schematic representation of the PEX3 KO CRISPR strategy. PCR primers (blue), sgRNA (orange). Bottom: Sanger sequencing of the upper band for the D2 clones (*). (C) Immunofluorescence of iPSC PEX3 −/− for pluripotent markers (OCT3/4, TRA-1-60, and TRA-1-81). Scale bars: 100 µm. (D) Flow cytometry characterization of PEX3 +/+ and PEX3 −/− monocytes and macrophages. Names of the markers are indicated on the graph graphs. Black, negative sample; red, isotype control; blue, marker.

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Clone Assay, Selection, CRISPR, Sequencing, Immunofluorescence, Flow Cytometry, Control, Marker

    Human macrophages lacking peroxisomes are unable to restrict Mtb WT replication. (A) Confocal images of iPSDM PEX3 +/+ and PEX3 −/− for GFP-PTS1 (green), PEX14 (green), TOM20 (red), CAT (red), and PMP70 (green). Nuclear staining (blue). Scale bars: 10 µm. (B) Analysis of Mtb WT growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb WT. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom: 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data representative of one out of three independent experiments ( n = 4 independent wells). (C) Analysis of Mtb ΔRD1 growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb ΔRD1. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data are representative of one out of three independent experiments ( n = 4 technical replicate per each condition). Significance was determined by two-way ANOVA with Tukey’s multiple comparison post-test (B and C). P value 0.033 (*), 0.002 (**), <0.0001 (***).

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Human macrophages lacking peroxisomes are unable to restrict Mtb WT replication. (A) Confocal images of iPSDM PEX3 +/+ and PEX3 −/− for GFP-PTS1 (green), PEX14 (green), TOM20 (red), CAT (red), and PMP70 (green). Nuclear staining (blue). Scale bars: 10 µm. (B) Analysis of Mtb WT growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb WT. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom: 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data representative of one out of three independent experiments ( n = 4 independent wells). (C) Analysis of Mtb ΔRD1 growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb ΔRD1. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data are representative of one out of three independent experiments ( n = 4 technical replicate per each condition). Significance was determined by two-way ANOVA with Tukey’s multiple comparison post-test (B and C). P value 0.033 (*), 0.002 (**), <0.0001 (***).

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Staining, Infection, Control, Clone Assay, Comparison

    Characterization of PEX3 −/− iPSDM clones and uptake and growth of Mtb in PEX3 +/+ and PEX3 −/− iPSDM. (A) Rescue experiment with PEX3_turbo for 24 h. Snapshot of iPSDM PEX3 +/+ and PEX3 −/− . Nuclear staining (blue), PEX3_turbo (orange), and EGFP-PTS1 (green). Scale bars: 10 µm. (B) Western blot of peroxisomal related protein expressed in iPSDM PEX3 +/+ and PEX3 −/− at the steady state. (C) Quantification of CAT expression from B normalized with actin. (D) Analysis of Mtb growth in iPSDM PEX3 +/+ and PEX3 −/− . Violin plot representation of Mtb area (px2) per cells over time (2, 24, 48, and 72 hpi) in PEX3 +/+ and PEX3 −/− (clone 1 and 2) during infection with Mtb WT and Mtb ∆RD1. Significance was determined for the 2 hpi time point by two-way ANOVA with Tukey’s multiple comparison post-test. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Characterization of PEX3 −/− iPSDM clones and uptake and growth of Mtb in PEX3 +/+ and PEX3 −/− iPSDM. (A) Rescue experiment with PEX3_turbo for 24 h. Snapshot of iPSDM PEX3 +/+ and PEX3 −/− . Nuclear staining (blue), PEX3_turbo (orange), and EGFP-PTS1 (green). Scale bars: 10 µm. (B) Western blot of peroxisomal related protein expressed in iPSDM PEX3 +/+ and PEX3 −/− at the steady state. (C) Quantification of CAT expression from B normalized with actin. (D) Analysis of Mtb growth in iPSDM PEX3 +/+ and PEX3 −/− . Violin plot representation of Mtb area (px2) per cells over time (2, 24, 48, and 72 hpi) in PEX3 +/+ and PEX3 −/− (clone 1 and 2) during infection with Mtb WT and Mtb ∆RD1. Significance was determined for the 2 hpi time point by two-way ANOVA with Tukey’s multiple comparison post-test. Source data are available for this figure: .

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Clone Assay, Staining, Western Blot, Expressing, Infection, Comparison

    Peroxisome-dependent restriction of Mtb is associated with higher levels of ROS in the cytosol. (A–C) Cyto_Hyper reporter during Mtb infection. (A) Confocal images of iPSDM at 24 hpi of infection (Uninfected, Mtb WT, and ΔRD1). The left graph shows Cyto_Hyper in iPSDM PEX3 +/+ and right graph shows Cyto_Hyper in PEX3 −/− iPSDM. Top: Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Bottom: Ratiometric imaging of the Cyto_Hyper reporter. Scale bars: 10 µm. (B) Quantification of Cyto_Hyper ratio in iPSDM PEX3 +/+ and PEX3 −/− during infection at 20 and 40 hpi. Data representative from one out of two independent experiments ( n = 4 independent wells per replicate). Significance was determined using unpaired by two-way ANOVA with Šídák’s multiple comparisons post-test. P value (APA) 0.033 (*), 0.002 (**). (C) 3D surface (left) and line plot (right) of Cyto_Hyper reporter in iPSDM PEX3 +/+ (1 box) and PEX3 −/− (2 box) infected with Mtb WT. (D and E) Endo_Hyper reporter during Mtb infection. (D) Snapshot of Endo_Hyper reporter in iPSDM PEX3 +/+ and PEX3 −/− during infection over 24 hpi. Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Scale bars: 10 µm. (E) Analysis of Endo_Hyper ratio around Mtb (area 0.5 µm) in iPSDM PEX3 +/+ and PEX3 −/− infected with Mtb WT and ΔRD1 at 24 hpi. The red line represents the median of Mtb-Endo_Hyper ratio in PEX3 +/+ and the light blue line represents the median of Mtb-Endo_Hyper ratio in PEX3 −/− iPSDM. N > 500 Mtb regions of interest were quantified per each condition.

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Peroxisome-dependent restriction of Mtb is associated with higher levels of ROS in the cytosol. (A–C) Cyto_Hyper reporter during Mtb infection. (A) Confocal images of iPSDM at 24 hpi of infection (Uninfected, Mtb WT, and ΔRD1). The left graph shows Cyto_Hyper in iPSDM PEX3 +/+ and right graph shows Cyto_Hyper in PEX3 −/− iPSDM. Top: Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Bottom: Ratiometric imaging of the Cyto_Hyper reporter. Scale bars: 10 µm. (B) Quantification of Cyto_Hyper ratio in iPSDM PEX3 +/+ and PEX3 −/− during infection at 20 and 40 hpi. Data representative from one out of two independent experiments ( n = 4 independent wells per replicate). Significance was determined using unpaired by two-way ANOVA with Šídák’s multiple comparisons post-test. P value (APA) 0.033 (*), 0.002 (**). (C) 3D surface (left) and line plot (right) of Cyto_Hyper reporter in iPSDM PEX3 +/+ (1 box) and PEX3 −/− (2 box) infected with Mtb WT. (D and E) Endo_Hyper reporter during Mtb infection. (D) Snapshot of Endo_Hyper reporter in iPSDM PEX3 +/+ and PEX3 −/− during infection over 24 hpi. Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Scale bars: 10 µm. (E) Analysis of Endo_Hyper ratio around Mtb (area 0.5 µm) in iPSDM PEX3 +/+ and PEX3 −/− infected with Mtb WT and ΔRD1 at 24 hpi. The red line represents the median of Mtb-Endo_Hyper ratio in PEX3 +/+ and the light blue line represents the median of Mtb-Endo_Hyper ratio in PEX3 −/− iPSDM. N > 500 Mtb regions of interest were quantified per each condition.

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Infection, Imaging

    Peroxisome-dependent restriction of Mtb is spatial-temporally separated from the NADPH oxidase activity in human macrophages. (A) Workflow for nucleofection (nf) approach to KO CYBB (CYBB nf ) and PEX3 (PEX3 nf ) genes in HMDM. (B) Western blot of HMDM CTRL, PEX3 nf , and CYBB nf for gp91-phox protein (CYBB). (C) Left: Immunofluorescence of HMDM CTRL and PEX3 nf . Nuclear staining (blue), PMP70 (orange), and CAT (green). Scale bars: 20 µm. Right: Quantification of CAT and PMP70 puncta area per area of cells. Significance was determined by two-way ANOVA with Šidák’s test. P value <0.0001 (***). (D) Mtb growth in HMDM CTRL, PEX3 nf , and CYBB nf . Left: Confocal images of HMDM CTRL, PEX3 nf , and CYBB nf at 60 hpi infected with Mtb WT (top) and ΔRD1 (bottom). Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm. Right: Fold change of Mtb growth in HMDM CTRL (green), PEX3 nf (orange), and CYBB nf (light blue) over 98 h of infection. The top graph shows the fold change of Mtb WT and the bottom graph the fold change of Mtb ΔRD1. Data representative from one out of two independent experiments ( n = 3 independent wells per replicate). Significance was determined only for the last time point (98 hpi) by two-way ANOVA with Dunnett’s post-doc test. P value 0.033 (*).

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Peroxisome-dependent restriction of Mtb is spatial-temporally separated from the NADPH oxidase activity in human macrophages. (A) Workflow for nucleofection (nf) approach to KO CYBB (CYBB nf ) and PEX3 (PEX3 nf ) genes in HMDM. (B) Western blot of HMDM CTRL, PEX3 nf , and CYBB nf for gp91-phox protein (CYBB). (C) Left: Immunofluorescence of HMDM CTRL and PEX3 nf . Nuclear staining (blue), PMP70 (orange), and CAT (green). Scale bars: 20 µm. Right: Quantification of CAT and PMP70 puncta area per area of cells. Significance was determined by two-way ANOVA with Šidák’s test. P value <0.0001 (***). (D) Mtb growth in HMDM CTRL, PEX3 nf , and CYBB nf . Left: Confocal images of HMDM CTRL, PEX3 nf , and CYBB nf at 60 hpi infected with Mtb WT (top) and ΔRD1 (bottom). Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm. Right: Fold change of Mtb growth in HMDM CTRL (green), PEX3 nf (orange), and CYBB nf (light blue) over 98 h of infection. The top graph shows the fold change of Mtb WT and the bottom graph the fold change of Mtb ΔRD1. Data representative from one out of two independent experiments ( n = 3 independent wells per replicate). Significance was determined only for the last time point (98 hpi) by two-way ANOVA with Dunnett’s post-doc test. P value 0.033 (*).

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Activity Assay, Western Blot, Immunofluorescence, Staining, Infection

    Generation and characterization of PEX3 −/− iPSC and iPSDM clones. (A and B) Selection of PEX3 −/− clones. (A) PCR genotyping of the expanded clones, D2 and E3. (B) Top: Schematic representation of the PEX3 KO CRISPR strategy. PCR primers (blue), sgRNA (orange). Bottom: Sanger sequencing of the upper band for the D2 clones (*). (C) Immunofluorescence of iPSC PEX3 −/− for pluripotent markers (OCT3/4, TRA-1-60, and TRA-1-81). Scale bars: 100 µm. (D) Flow cytometry characterization of PEX3 +/+ and PEX3 −/− monocytes and macrophages. Names of the markers are indicated on the graph graphs. Black, negative sample; red, isotype control; blue, marker.

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Generation and characterization of PEX3 −/− iPSC and iPSDM clones. (A and B) Selection of PEX3 −/− clones. (A) PCR genotyping of the expanded clones, D2 and E3. (B) Top: Schematic representation of the PEX3 KO CRISPR strategy. PCR primers (blue), sgRNA (orange). Bottom: Sanger sequencing of the upper band for the D2 clones (*). (C) Immunofluorescence of iPSC PEX3 −/− for pluripotent markers (OCT3/4, TRA-1-60, and TRA-1-81). Scale bars: 100 µm. (D) Flow cytometry characterization of PEX3 +/+ and PEX3 −/− monocytes and macrophages. Names of the markers are indicated on the graph graphs. Black, negative sample; red, isotype control; blue, marker.

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Clone Assay, Selection, CRISPR, Sequencing, Immunofluorescence, Flow Cytometry, Control, Marker

    Human macrophages lacking peroxisomes are unable to restrict Mtb WT replication. (A) Confocal images of iPSDM PEX3 +/+ and PEX3 −/− for GFP-PTS1 (green), PEX14 (green), TOM20 (red), CAT (red), and PMP70 (green). Nuclear staining (blue). Scale bars: 10 µm. (B) Analysis of Mtb WT growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb WT. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom: 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data representative of one out of three independent experiments ( n = 4 independent wells). (C) Analysis of Mtb ΔRD1 growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb ΔRD1. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data are representative of one out of three independent experiments ( n = 4 technical replicate per each condition). Significance was determined by two-way ANOVA with Tukey’s multiple comparison post-test (B and C). P value 0.033 (*), 0.002 (**), <0.0001 (***).

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Human macrophages lacking peroxisomes are unable to restrict Mtb WT replication. (A) Confocal images of iPSDM PEX3 +/+ and PEX3 −/− for GFP-PTS1 (green), PEX14 (green), TOM20 (red), CAT (red), and PMP70 (green). Nuclear staining (blue). Scale bars: 10 µm. (B) Analysis of Mtb WT growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb WT. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom: 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data representative of one out of three independent experiments ( n = 4 independent wells). (C) Analysis of Mtb ΔRD1 growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb ΔRD1. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data are representative of one out of three independent experiments ( n = 4 technical replicate per each condition). Significance was determined by two-way ANOVA with Tukey’s multiple comparison post-test (B and C). P value 0.033 (*), 0.002 (**), <0.0001 (***).

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Staining, Infection, Control, Clone Assay, Comparison

    Characterization of PEX3 −/− iPSDM clones and uptake and growth of Mtb in PEX3 +/+ and PEX3 −/− iPSDM. (A) Rescue experiment with PEX3_turbo for 24 h. Snapshot of iPSDM PEX3 +/+ and PEX3 −/− . Nuclear staining (blue), PEX3_turbo (orange), and EGFP-PTS1 (green). Scale bars: 10 µm. (B) Western blot of peroxisomal related protein expressed in iPSDM PEX3 +/+ and PEX3 −/− at the steady state. (C) Quantification of CAT expression from B normalized with actin. (D) Analysis of Mtb growth in iPSDM PEX3 +/+ and PEX3 −/− . Violin plot representation of Mtb area (px2) per cells over time (2, 24, 48, and 72 hpi) in PEX3 +/+ and PEX3 −/− (clone 1 and 2) during infection with Mtb WT and Mtb ∆RD1. Significance was determined for the 2 hpi time point by two-way ANOVA with Tukey’s multiple comparison post-test. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Characterization of PEX3 −/− iPSDM clones and uptake and growth of Mtb in PEX3 +/+ and PEX3 −/− iPSDM. (A) Rescue experiment with PEX3_turbo for 24 h. Snapshot of iPSDM PEX3 +/+ and PEX3 −/− . Nuclear staining (blue), PEX3_turbo (orange), and EGFP-PTS1 (green). Scale bars: 10 µm. (B) Western blot of peroxisomal related protein expressed in iPSDM PEX3 +/+ and PEX3 −/− at the steady state. (C) Quantification of CAT expression from B normalized with actin. (D) Analysis of Mtb growth in iPSDM PEX3 +/+ and PEX3 −/− . Violin plot representation of Mtb area (px2) per cells over time (2, 24, 48, and 72 hpi) in PEX3 +/+ and PEX3 −/− (clone 1 and 2) during infection with Mtb WT and Mtb ∆RD1. Significance was determined for the 2 hpi time point by two-way ANOVA with Tukey’s multiple comparison post-test. Source data are available for this figure: .

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Clone Assay, Staining, Western Blot, Expressing, Infection, Comparison

    Peroxisome-dependent restriction of Mtb is associated with higher levels of ROS in the cytosol. (A–C) Cyto_Hyper reporter during Mtb infection. (A) Confocal images of iPSDM at 24 hpi of infection (Uninfected, Mtb WT, and ΔRD1). The left graph shows Cyto_Hyper in iPSDM PEX3 +/+ and right graph shows Cyto_Hyper in PEX3 −/− iPSDM. Top: Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Bottom: Ratiometric imaging of the Cyto_Hyper reporter. Scale bars: 10 µm. (B) Quantification of Cyto_Hyper ratio in iPSDM PEX3 +/+ and PEX3 −/− during infection at 20 and 40 hpi. Data representative from one out of two independent experiments ( n = 4 independent wells per replicate). Significance was determined using unpaired by two-way ANOVA with Šídák’s multiple comparisons post-test. P value (APA) 0.033 (*), 0.002 (**). (C) 3D surface (left) and line plot (right) of Cyto_Hyper reporter in iPSDM PEX3 +/+ (1 box) and PEX3 −/− (2 box) infected with Mtb WT. (D and E) Endo_Hyper reporter during Mtb infection. (D) Snapshot of Endo_Hyper reporter in iPSDM PEX3 +/+ and PEX3 −/− during infection over 24 hpi. Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Scale bars: 10 µm. (E) Analysis of Endo_Hyper ratio around Mtb (area 0.5 µm) in iPSDM PEX3 +/+ and PEX3 −/− infected with Mtb WT and ΔRD1 at 24 hpi. The red line represents the median of Mtb-Endo_Hyper ratio in PEX3 +/+ and the light blue line represents the median of Mtb-Endo_Hyper ratio in PEX3 −/− iPSDM. N > 500 Mtb regions of interest were quantified per each condition.

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Peroxisome-dependent restriction of Mtb is associated with higher levels of ROS in the cytosol. (A–C) Cyto_Hyper reporter during Mtb infection. (A) Confocal images of iPSDM at 24 hpi of infection (Uninfected, Mtb WT, and ΔRD1). The left graph shows Cyto_Hyper in iPSDM PEX3 +/+ and right graph shows Cyto_Hyper in PEX3 −/− iPSDM. Top: Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Bottom: Ratiometric imaging of the Cyto_Hyper reporter. Scale bars: 10 µm. (B) Quantification of Cyto_Hyper ratio in iPSDM PEX3 +/+ and PEX3 −/− during infection at 20 and 40 hpi. Data representative from one out of two independent experiments ( n = 4 independent wells per replicate). Significance was determined using unpaired by two-way ANOVA with Šídák’s multiple comparisons post-test. P value (APA) 0.033 (*), 0.002 (**). (C) 3D surface (left) and line plot (right) of Cyto_Hyper reporter in iPSDM PEX3 +/+ (1 box) and PEX3 −/− (2 box) infected with Mtb WT. (D and E) Endo_Hyper reporter during Mtb infection. (D) Snapshot of Endo_Hyper reporter in iPSDM PEX3 +/+ and PEX3 −/− during infection over 24 hpi. Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Scale bars: 10 µm. (E) Analysis of Endo_Hyper ratio around Mtb (area 0.5 µm) in iPSDM PEX3 +/+ and PEX3 −/− infected with Mtb WT and ΔRD1 at 24 hpi. The red line represents the median of Mtb-Endo_Hyper ratio in PEX3 +/+ and the light blue line represents the median of Mtb-Endo_Hyper ratio in PEX3 −/− iPSDM. N > 500 Mtb regions of interest were quantified per each condition.

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Infection, Imaging

    Peroxisome-dependent restriction of Mtb is spatial-temporally separated from the NADPH oxidase activity in human macrophages. (A) Workflow for nucleofection (nf) approach to KO CYBB (CYBB nf ) and PEX3 (PEX3 nf ) genes in HMDM. (B) Western blot of HMDM CTRL, PEX3 nf , and CYBB nf for gp91-phox protein (CYBB). (C) Left: Immunofluorescence of HMDM CTRL and PEX3 nf . Nuclear staining (blue), PMP70 (orange), and CAT (green). Scale bars: 20 µm. Right: Quantification of CAT and PMP70 puncta area per area of cells. Significance was determined by two-way ANOVA with Šidák’s test. P value <0.0001 (***). (D) Mtb growth in HMDM CTRL, PEX3 nf , and CYBB nf . Left: Confocal images of HMDM CTRL, PEX3 nf , and CYBB nf at 60 hpi infected with Mtb WT (top) and ΔRD1 (bottom). Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm. Right: Fold change of Mtb growth in HMDM CTRL (green), PEX3 nf (orange), and CYBB nf (light blue) over 98 h of infection. The top graph shows the fold change of Mtb WT and the bottom graph the fold change of Mtb ΔRD1. Data representative from one out of two independent experiments ( n = 3 independent wells per replicate). Significance was determined only for the last time point (98 hpi) by two-way ANOVA with Dunnett’s post-doc test. P value 0.033 (*).

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Peroxisome-dependent restriction of Mtb is spatial-temporally separated from the NADPH oxidase activity in human macrophages. (A) Workflow for nucleofection (nf) approach to KO CYBB (CYBB nf ) and PEX3 (PEX3 nf ) genes in HMDM. (B) Western blot of HMDM CTRL, PEX3 nf , and CYBB nf for gp91-phox protein (CYBB). (C) Left: Immunofluorescence of HMDM CTRL and PEX3 nf . Nuclear staining (blue), PMP70 (orange), and CAT (green). Scale bars: 20 µm. Right: Quantification of CAT and PMP70 puncta area per area of cells. Significance was determined by two-way ANOVA with Šidák’s test. P value <0.0001 (***). (D) Mtb growth in HMDM CTRL, PEX3 nf , and CYBB nf . Left: Confocal images of HMDM CTRL, PEX3 nf , and CYBB nf at 60 hpi infected with Mtb WT (top) and ΔRD1 (bottom). Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm. Right: Fold change of Mtb growth in HMDM CTRL (green), PEX3 nf (orange), and CYBB nf (light blue) over 98 h of infection. The top graph shows the fold change of Mtb WT and the bottom graph the fold change of Mtb ΔRD1. Data representative from one out of two independent experiments ( n = 3 independent wells per replicate). Significance was determined only for the last time point (98 hpi) by two-way ANOVA with Dunnett’s post-doc test. P value 0.033 (*).

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Activity Assay, Western Blot, Immunofluorescence, Staining, Infection

    Generation and characterization of PEX3 −/− iPSC and iPSDM clones. (A and B) Selection of PEX3 −/− clones. (A) PCR genotyping of the expanded clones, D2 and E3. (B) Top: Schematic representation of the PEX3 KO CRISPR strategy. PCR primers (blue), sgRNA (orange). Bottom: Sanger sequencing of the upper band for the D2 clones (*). (C) Immunofluorescence of iPSC PEX3 −/− for pluripotent markers (OCT3/4, TRA-1-60, and TRA-1-81). Scale bars: 100 µm. (D) Flow cytometry characterization of PEX3 +/+ and PEX3 −/− monocytes and macrophages. Names of the markers are indicated on the graph graphs. Black, negative sample; red, isotype control; blue, marker.

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Generation and characterization of PEX3 −/− iPSC and iPSDM clones. (A and B) Selection of PEX3 −/− clones. (A) PCR genotyping of the expanded clones, D2 and E3. (B) Top: Schematic representation of the PEX3 KO CRISPR strategy. PCR primers (blue), sgRNA (orange). Bottom: Sanger sequencing of the upper band for the D2 clones (*). (C) Immunofluorescence of iPSC PEX3 −/− for pluripotent markers (OCT3/4, TRA-1-60, and TRA-1-81). Scale bars: 100 µm. (D) Flow cytometry characterization of PEX3 +/+ and PEX3 −/− monocytes and macrophages. Names of the markers are indicated on the graph graphs. Black, negative sample; red, isotype control; blue, marker.

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Clone Assay, Selection, CRISPR, Sequencing, Immunofluorescence, Flow Cytometry, Control, Marker

    Human macrophages lacking peroxisomes are unable to restrict Mtb WT replication. (A) Confocal images of iPSDM PEX3 +/+ and PEX3 −/− for GFP-PTS1 (green), PEX14 (green), TOM20 (red), CAT (red), and PMP70 (green). Nuclear staining (blue). Scale bars: 10 µm. (B) Analysis of Mtb WT growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb WT. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom: 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data representative of one out of three independent experiments ( n = 4 independent wells). (C) Analysis of Mtb ΔRD1 growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb ΔRD1. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data are representative of one out of three independent experiments ( n = 4 technical replicate per each condition). Significance was determined by two-way ANOVA with Tukey’s multiple comparison post-test (B and C). P value 0.033 (*), 0.002 (**), <0.0001 (***).

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Human macrophages lacking peroxisomes are unable to restrict Mtb WT replication. (A) Confocal images of iPSDM PEX3 +/+ and PEX3 −/− for GFP-PTS1 (green), PEX14 (green), TOM20 (red), CAT (red), and PMP70 (green). Nuclear staining (blue). Scale bars: 10 µm. (B) Analysis of Mtb WT growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb WT. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom: 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data representative of one out of three independent experiments ( n = 4 independent wells). (C) Analysis of Mtb ΔRD1 growth in iPSDM lacking peroxisomes. Left: Confocal images of iPSDM PEX3 +/+ and PEX3 −/− (clone 1 and 2) at 24 and 72 hpi infected with Mtb ΔRD1. Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm, zoom 10 µm. Right: Growth index of Mtb WT in iPSDM control (PEX3 +/+ ) or KO for PEX3 (PEX3 −/− clones 1 and 2). Data are representative of one out of three independent experiments ( n = 4 technical replicate per each condition). Significance was determined by two-way ANOVA with Tukey’s multiple comparison post-test (B and C). P value 0.033 (*), 0.002 (**), <0.0001 (***).

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Staining, Infection, Control, Clone Assay, Comparison

    Characterization of PEX3 −/− iPSDM clones and uptake and growth of Mtb in PEX3 +/+ and PEX3 −/− iPSDM. (A) Rescue experiment with PEX3_turbo for 24 h. Snapshot of iPSDM PEX3 +/+ and PEX3 −/− . Nuclear staining (blue), PEX3_turbo (orange), and EGFP-PTS1 (green). Scale bars: 10 µm. (B) Western blot of peroxisomal related protein expressed in iPSDM PEX3 +/+ and PEX3 −/− at the steady state. (C) Quantification of CAT expression from B normalized with actin. (D) Analysis of Mtb growth in iPSDM PEX3 +/+ and PEX3 −/− . Violin plot representation of Mtb area (px2) per cells over time (2, 24, 48, and 72 hpi) in PEX3 +/+ and PEX3 −/− (clone 1 and 2) during infection with Mtb WT and Mtb ∆RD1. Significance was determined for the 2 hpi time point by two-way ANOVA with Tukey’s multiple comparison post-test. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Characterization of PEX3 −/− iPSDM clones and uptake and growth of Mtb in PEX3 +/+ and PEX3 −/− iPSDM. (A) Rescue experiment with PEX3_turbo for 24 h. Snapshot of iPSDM PEX3 +/+ and PEX3 −/− . Nuclear staining (blue), PEX3_turbo (orange), and EGFP-PTS1 (green). Scale bars: 10 µm. (B) Western blot of peroxisomal related protein expressed in iPSDM PEX3 +/+ and PEX3 −/− at the steady state. (C) Quantification of CAT expression from B normalized with actin. (D) Analysis of Mtb growth in iPSDM PEX3 +/+ and PEX3 −/− . Violin plot representation of Mtb area (px2) per cells over time (2, 24, 48, and 72 hpi) in PEX3 +/+ and PEX3 −/− (clone 1 and 2) during infection with Mtb WT and Mtb ∆RD1. Significance was determined for the 2 hpi time point by two-way ANOVA with Tukey’s multiple comparison post-test. Source data are available for this figure: .

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Clone Assay, Staining, Western Blot, Expressing, Infection, Comparison

    Peroxisome-dependent restriction of Mtb is associated with higher levels of ROS in the cytosol. (A–C) Cyto_Hyper reporter during Mtb infection. (A) Confocal images of iPSDM at 24 hpi of infection (Uninfected, Mtb WT, and ΔRD1). The left graph shows Cyto_Hyper in iPSDM PEX3 +/+ and right graph shows Cyto_Hyper in PEX3 −/− iPSDM. Top: Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Bottom: Ratiometric imaging of the Cyto_Hyper reporter. Scale bars: 10 µm. (B) Quantification of Cyto_Hyper ratio in iPSDM PEX3 +/+ and PEX3 −/− during infection at 20 and 40 hpi. Data representative from one out of two independent experiments ( n = 4 independent wells per replicate). Significance was determined using unpaired by two-way ANOVA with Šídák’s multiple comparisons post-test. P value (APA) 0.033 (*), 0.002 (**). (C) 3D surface (left) and line plot (right) of Cyto_Hyper reporter in iPSDM PEX3 +/+ (1 box) and PEX3 −/− (2 box) infected with Mtb WT. (D and E) Endo_Hyper reporter during Mtb infection. (D) Snapshot of Endo_Hyper reporter in iPSDM PEX3 +/+ and PEX3 −/− during infection over 24 hpi. Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Scale bars: 10 µm. (E) Analysis of Endo_Hyper ratio around Mtb (area 0.5 µm) in iPSDM PEX3 +/+ and PEX3 −/− infected with Mtb WT and ΔRD1 at 24 hpi. The red line represents the median of Mtb-Endo_Hyper ratio in PEX3 +/+ and the light blue line represents the median of Mtb-Endo_Hyper ratio in PEX3 −/− iPSDM. N > 500 Mtb regions of interest were quantified per each condition.

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Peroxisome-dependent restriction of Mtb is associated with higher levels of ROS in the cytosol. (A–C) Cyto_Hyper reporter during Mtb infection. (A) Confocal images of iPSDM at 24 hpi of infection (Uninfected, Mtb WT, and ΔRD1). The left graph shows Cyto_Hyper in iPSDM PEX3 +/+ and right graph shows Cyto_Hyper in PEX3 −/− iPSDM. Top: Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Bottom: Ratiometric imaging of the Cyto_Hyper reporter. Scale bars: 10 µm. (B) Quantification of Cyto_Hyper ratio in iPSDM PEX3 +/+ and PEX3 −/− during infection at 20 and 40 hpi. Data representative from one out of two independent experiments ( n = 4 independent wells per replicate). Significance was determined using unpaired by two-way ANOVA with Šídák’s multiple comparisons post-test. P value (APA) 0.033 (*), 0.002 (**). (C) 3D surface (left) and line plot (right) of Cyto_Hyper reporter in iPSDM PEX3 +/+ (1 box) and PEX3 −/− (2 box) infected with Mtb WT. (D and E) Endo_Hyper reporter during Mtb infection. (D) Snapshot of Endo_Hyper reporter in iPSDM PEX3 +/+ and PEX3 −/− during infection over 24 hpi. Merge of GFP_UV (green), GFP (red), and Mtb-E2-Crimson (cyan). Scale bars: 10 µm. (E) Analysis of Endo_Hyper ratio around Mtb (area 0.5 µm) in iPSDM PEX3 +/+ and PEX3 −/− infected with Mtb WT and ΔRD1 at 24 hpi. The red line represents the median of Mtb-Endo_Hyper ratio in PEX3 +/+ and the light blue line represents the median of Mtb-Endo_Hyper ratio in PEX3 −/− iPSDM. N > 500 Mtb regions of interest were quantified per each condition.

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Infection, Imaging

    Peroxisome-dependent restriction of Mtb is spatial-temporally separated from the NADPH oxidase activity in human macrophages. (A) Workflow for nucleofection (nf) approach to KO CYBB (CYBB nf ) and PEX3 (PEX3 nf ) genes in HMDM. (B) Western blot of HMDM CTRL, PEX3 nf , and CYBB nf for gp91-phox protein (CYBB). (C) Left: Immunofluorescence of HMDM CTRL and PEX3 nf . Nuclear staining (blue), PMP70 (orange), and CAT (green). Scale bars: 20 µm. Right: Quantification of CAT and PMP70 puncta area per area of cells. Significance was determined by two-way ANOVA with Šidák’s test. P value <0.0001 (***). (D) Mtb growth in HMDM CTRL, PEX3 nf , and CYBB nf . Left: Confocal images of HMDM CTRL, PEX3 nf , and CYBB nf at 60 hpi infected with Mtb WT (top) and ΔRD1 (bottom). Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm. Right: Fold change of Mtb growth in HMDM CTRL (green), PEX3 nf (orange), and CYBB nf (light blue) over 98 h of infection. The top graph shows the fold change of Mtb WT and the bottom graph the fold change of Mtb ΔRD1. Data representative from one out of two independent experiments ( n = 3 independent wells per replicate). Significance was determined only for the last time point (98 hpi) by two-way ANOVA with Dunnett’s post-doc test. P value 0.033 (*).

    Journal: The Journal of Cell Biology

    Article Title: Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages

    doi: 10.1083/jcb.202303066

    Figure Lengend Snippet: Peroxisome-dependent restriction of Mtb is spatial-temporally separated from the NADPH oxidase activity in human macrophages. (A) Workflow for nucleofection (nf) approach to KO CYBB (CYBB nf ) and PEX3 (PEX3 nf ) genes in HMDM. (B) Western blot of HMDM CTRL, PEX3 nf , and CYBB nf for gp91-phox protein (CYBB). (C) Left: Immunofluorescence of HMDM CTRL and PEX3 nf . Nuclear staining (blue), PMP70 (orange), and CAT (green). Scale bars: 20 µm. Right: Quantification of CAT and PMP70 puncta area per area of cells. Significance was determined by two-way ANOVA with Šidák’s test. P value <0.0001 (***). (D) Mtb growth in HMDM CTRL, PEX3 nf , and CYBB nf . Left: Confocal images of HMDM CTRL, PEX3 nf , and CYBB nf at 60 hpi infected with Mtb WT (top) and ΔRD1 (bottom). Nuclear staining (blue) and Mtb-E2-Crimson (red). Scale bars: 100 µm. Right: Fold change of Mtb growth in HMDM CTRL (green), PEX3 nf (orange), and CYBB nf (light blue) over 98 h of infection. The top graph shows the fold change of Mtb WT and the bottom graph the fold change of Mtb ΔRD1. Data representative from one out of two independent experiments ( n = 3 independent wells per replicate). Significance was determined only for the last time point (98 hpi) by two-way ANOVA with Dunnett’s post-doc test. P value 0.033 (*).

    Article Snippet: For the PEX3_Turbo, the PEX3 sequence was amplified by PCR with the PEX3_cDNA_F_infusion and PEX3_cDNA_R_TURBO_infusion as primers ( ) and the PEX3 cDNA ORF (HG14106-UT; SinoBiological) as template.

    Techniques: Activity Assay, Western Blot, Immunofluorescence, Staining, Infection