arp47432 p050  (Aviva Systems)

Journal: Cells

Article Title: DRAM1 Promotes Lysosomal Delivery of Mycobacterium marinum in Macrophages

doi: 10.3390/cells12060828

Figure Lengend Snippet: DRAM1 colocalises with acidified mycobacteria-containing vesicles. ( A ) DRAM1 (magenta) localisation at the plasma membrane (arrowheads) of non-infected cells. Scale bars: 5 μm. ( B ) DRAM1 (magenta) colocalisation with acidic vesicles stained by LysoTracker (yellow). A region showing a LysoTracker-positive and a LysoTracker-negative DRAM1-stained vesicle is outlined, with a dotted box in the merged image that is enlarged in the inset. Scale bars: 5 μm. ( C ) Representative examples of DRAM1 (magenta) colocalisation patterns with Mm (cyan), referred to as membrane, punctate, and luminal patterns. An example of DRAM1-negative Mm (none) is also shown. Acidification of Mm -containing vesicles was assessed by LysoTracker staining (yellow). Scale bars: 2 μm. ( D ) Frequency of Mm colocalisation with DRAM1 or LysoTracker over time. ( E ) Frequency of Mm colocalisation with DRAM1 in punctate, membrane, or luminal patterns over time. ( F ) Frequency of LysoTracker colocalisation with DRAM1-negative Mm and DRAM1-positive membrane, punctate, and luminal Mm patterns over time. Data were accumulated from two independent experiments with the following total numbers of Mm observations per time point: 71 for t = 0, 73 for t = 30, 105 for t = 60, 98 for t = 90, 102 for t = 120 and 111 for t = 150 min.

Article Snippet: Membranes were blocked with 3% BSA in 1× Tris-buffered saline (TBS) solution, with 0.1% Tween-20 (TBST), before incubating with primary and secondary antibodies: polyclonal anti-rabbit DRAM1 (1:1000) (Aviva systems biology, San Diego, CA, USA, ARP47432- P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), and LAMP1 (1:1000) (Abcam, ab24170), as well as anti-rabbit IgG, and HRP-linked antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Clinical Proteomics, Membrane, Infection, Staining

Journal: Cells

Article Title: DRAM1 Promotes Lysosomal Delivery of Mycobacterium marinum in Macrophages

doi: 10.3390/cells12060828

Figure Lengend Snippet: Acidified mycobacteria-containing vesicles are labelled by LC3. ( A ) Representative examples of LC3 (magenta) colocalisation patterns with Mm (cyan), referred to as punctate and luminal patterns. An example of LC3-negative Mm (none) is also shown. Acidification of Mm -containing vesicles was assessed by LysoTracker staining (yellow). Scale bars: 2 μm. ( B ) Representative examples of the double colocalisation of LC3 (magenta) and DRAM1 (grey) with Mm (cyan). Scale bars: 2 μm. ( C ) Frequency of Mm colocalisation with LC3 or LysoTracker over time. ( D ) Frequency of Mm colocalisation with LC3 in punctate or luminal patterns over time. ( E ) Frequency of LysoTracker colocalisation with LC3-negative Mm and LC3-positive punctate and luminal Mm patterns over time. Data were accumulated from two independent experiments with the following total numbers of Mm observations per time point: 99 for t = 0, 129 for t = 30, 96 for t = 60, 124 for t = 90, 133 for t = 120 and 127 for t = 150 min.

Article Snippet: Membranes were blocked with 3% BSA in 1× Tris-buffered saline (TBS) solution, with 0.1% Tween-20 (TBST), before incubating with primary and secondary antibodies: polyclonal anti-rabbit DRAM1 (1:1000) (Aviva systems biology, San Diego, CA, USA, ARP47432- P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), and LAMP1 (1:1000) (Abcam, ab24170), as well as anti-rabbit IgG, and HRP-linked antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Staining

Journal: Cells

Article Title: DRAM1 Promotes Lysosomal Delivery of Mycobacterium marinum in Macrophages

doi: 10.3390/cells12060828

Figure Lengend Snippet: DRAM1 knockdown reduces the acidification of Mm-containing vesicles. ( A ) Representative c with Mm (cyan) in control (Ctrl) and DRAM1 knockdown cell lines (KD1-3), at 120 min post infection. The arrowheads indicate the colocalisation of DRAM1, Mm, and LysoTracker. Scale bars: 2 μm. ( B ) Percentage of colocalisation of Mm with DRAM1 or LysoTracker in DRAM1 knockdown and control cell lines. ( C ) Percentage of colocalisation of Mm with DRAM1 in punctate, membrane, and luminal patterns in DRAM1 knockdown and control cell lines. ( D ) Percentage of colocalisation of LysoTracker with DRAM1-negative Mm (none) and DRAM1-positive punctate, membrane and luminal Mm patterns, in DRAM1 knockdown and control cell lines. Bar graphs show the data from three independent experiments, where the mean of each replicate is indicated with a colored symbol. In total, 18 ROIs were analysed per control or DRAM1 knockdown (KD1-3) group, with the following total numbers of Mm observations per group: Ctrl = 591, KD1 = 632, KD = 525, and KD3 = 567. Statistical significance was assessed by beta-binomial logistic regression with Dunnett’s multiple test correction. Error bars represent the standard deviation of the three independent replications of the experiment. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Membranes were blocked with 3% BSA in 1× Tris-buffered saline (TBS) solution, with 0.1% Tween-20 (TBST), before incubating with primary and secondary antibodies: polyclonal anti-rabbit DRAM1 (1:1000) (Aviva systems biology, San Diego, CA, USA, ARP47432- P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), and LAMP1 (1:1000) (Abcam, ab24170), as well as anti-rabbit IgG, and HRP-linked antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Control, Infection, Membrane, Standard Deviation

Journal: Cells

Article Title: DRAM1 Promotes Lysosomal Delivery of Mycobacterium marinum in Macrophages

doi: 10.3390/cells12060828

Figure Lengend Snippet: DRAM1 knockdown reduces LC3 trafficking to Mm . ( A ) Representative examples of LC3 (magenta) and LysoTracker (yellow) colocalisation with Mm (cyan) in DRAM1 knockdown (KD1-3) and control (Ctrl) cell lines at 120 min post infection. The arrowheads indicate the colocalisation of LC3, Mm and LysoTracker. Scale bars: 2 μm. ( B ) Percentage of colocalisation of Mm with LC3 or LysoTracker in DRAM1 knockdown and control cell lines. ( C ) Percentage of colocalisation of Mm with LC3 in punctate and luminal patterns, in DRAM1 knockdown and control cell lines. ( D ) Percentage of colocalisation of LysoTracker with LC3-negative Mm and LC3-positive punctate and luminal Mm patterns, in DRAM1 knockdown and control cell lines. Bar graphs show the data from three independent experiments, where the mean of each replicate is indicated with a colored symbol. In total, 18 ROIs were analysed per control or DRAM1 knockdown (KD1-3) group, with the following total numbers of Mm observations per group: Ctrl = 527, KD1 = 551, KD2 = 516, KD3 = 537. Statistical significance was assessed by beta-binomial logistic regression with Dunnett’s multiple test correction. Error bars represent the standard deviation of the three independent replications of the experiment. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Membranes were blocked with 3% BSA in 1× Tris-buffered saline (TBS) solution, with 0.1% Tween-20 (TBST), before incubating with primary and secondary antibodies: polyclonal anti-rabbit DRAM1 (1:1000) (Aviva systems biology, San Diego, CA, USA, ARP47432- P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), and LAMP1 (1:1000) (Abcam, ab24170), as well as anti-rabbit IgG, and HRP-linked antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Control, Infection, Standard Deviation

Journal: Cells

Article Title: DRAM1 Promotes Lysosomal Delivery of Mycobacterium marinum in Macrophages

doi: 10.3390/cells12060828

Figure Lengend Snippet: DRAM1 knockdown reduces LAMP1 staining of Mm -containing vesicles. ( A ) Representative examples of LAMP1 (magenta) colocalisation patterns with Mm (cyan), referred to as membrane, punctate, and luminal patterns, at 120 min post infection. Acidification of Mm -containing vesicles was assessed by LysoTracker staining (yellow). Scale bars: 2 μm. ( B ) Representative examples of LAMP1 (magenta) colocalisation with Mm (cyan), and of the acidification of Mm -containing vesicles, assessed by LysoTracker staining (yellow) in DRAM1 knockdown (KD1-3), as well as control cell lines, at 120 min post infection. The arrowheads indicate colocalisation of LAMP1, Mm and LysoTracker. Scale bars: 2 μm. ( C ) Percentage of colocalisation of Mm with LAMP1 or LysoTracker in DRAM1 knockdown and control cell lines. ( D ) Percentage of colocalisation of Mm with LAMP1 in punctate, membrane, and luminal patterns, in DRAM1 knockdown and control cell lines. ( E ) Percentage of colocalisation of LysoTracker with LAMP1-negative Mm and LAMP1-positive Mm punctate, membrane, and luminal Mm patterns, in DRAM1 knockdown and control cell lines. Bar graphs show the data from three independent experiments, where the mean of each replicate is indicated with a colored symbol. In total, 18 ROIs were analysed per control or DRAM1 knockdown (KD1-3) group, with the following total numbers of Mm observations per group: Ctrl = 479, KD1 = 473, KD2 = 469, and KD3 = 502. Significance was assessed by beta-binomial logistic regression with Dunnett’s multiple test correction. Error bars represent the standard deviation of the three independent replications of the experiment (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Membranes were blocked with 3% BSA in 1× Tris-buffered saline (TBS) solution, with 0.1% Tween-20 (TBST), before incubating with primary and secondary antibodies: polyclonal anti-rabbit DRAM1 (1:1000) (Aviva systems biology, San Diego, CA, USA, ARP47432- P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), and LAMP1 (1:1000) (Abcam, ab24170), as well as anti-rabbit IgG, and HRP-linked antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Staining, Membrane, Infection, Control, Standard Deviation

Journal: Cells

Article Title: DRAM1 Promotes Lysosomal Delivery of Mycobacterium marinum in Macrophages

doi: 10.3390/cells12060828

Figure Lengend Snippet: DRAM1 knockdown increases the susceptibility of macrophages to Mm infection. ( A ) Representative examples of infected cells in DRAM1 knockdown and control cell lines at 7 h post infection. Scale bars: 20 μm ( B ) Percentage of infected cells in DRAM1 knockdown (KD1-3) and control cells at 7 h post infection. ( C ) CFU/mL in DRAM1 knockdown cells normalised to the control cell lines at 7 h post infection. ( D ) Representative examples of SYTOX Green staining of Mm - infected DRAM1 knockdown and control cells at 7 h post infection. Scale bars: 20 μm. ( E ) Percentage of SYTOX green-positive infected cells in DRAM1 knockdown and control cell lines at 7 h post infection. Bar graphs ( B , C , E ) show the data from three independent experiments, where the mean of each replicate is indicated with a colored symbol. All replicates in ( B , C ) were performed with 3 knockdown lines (KD1-3), while the data in ( E ) are based on 3 replicates for KD2 and KD3 and 1 replicate for KD1. Statistical significance was assessed by one-way ANOVA and pairwise comparison with Dunnett’s correction. (* p < 0.05; ** p < 0.01; **** p < 0.0001).

Article Snippet: Membranes were blocked with 3% BSA in 1× Tris-buffered saline (TBS) solution, with 0.1% Tween-20 (TBST), before incubating with primary and secondary antibodies: polyclonal anti-rabbit DRAM1 (1:1000) (Aviva systems biology, San Diego, CA, USA, ARP47432- P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), and LAMP1 (1:1000) (Abcam, ab24170), as well as anti-rabbit IgG, and HRP-linked antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Infection, Control, Staining, Comparison

Journal: bioRxiv

Article Title: DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages

doi: 10.1101/2022.10.25.513660

Figure Lengend Snippet: DRAM1 is predominantly localised at the plasma membrane and in acidic vesicles of RAW 267.4 macrophages. (A) DRAM1 immunofluorescence (magenta) with the nucleus stained by DAPI (blue). Scale bars: 5 μm (B) DRAM1 (magenta) colocalisation with acidic vesicles stained by LysoTracker (yellow). The nucleus is stained by DAPI (blue). A region showing a LysoTracker-positive and a LysoTracker-negative DRAM1-stained vesicle is outlined with a dotted box in the merged image and enlarged in the inset. Scale bars: 5 μm.

Article Snippet: Membranes were blocked with 3% BSA in lx Tris buffered saline (TBS) solution with 0.1% Tween-20 (TBST) before incubating with primary and secondary antibodies: polyclonal rabbit anti DRAM1 (1:1000) (Aviva systems biology, ARP47432-P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), LAMP1 (1:1000) (Abcam, ab24170), Antirabbit IgG, HRP-Linked Antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages

doi: 10.1101/2022.10.25.513660

Figure Lengend Snippet: DRAM1 colocalises with acidified mycobacteria-containing vesicles. (A) Representative examples of DRAM1 (magenta) colocalisation patterns with Mm (cyan), referred to as membrane, punctate, and luminal patterns. An example of DRAM1-negative Mm (none) is also shown. Acidification of Mm-containing vesicles is assessed by LysoTracker staining (yellow). Scale bars: 2 μm (B) Representative example of DRAM1 (magenta) colocalisation with LysoTracker (yellow) and Mm (cyan) at 120 min post infection, when luminal colocalisation patterns are most frequent. Scale bars: 5 μm (C) Frequency of Mm colocalisation with DRAM1 or LysoTracker over time. (D) Frequency of Mm colocalisation with DRAM1 in punctate, membrane or luminal patterns over time. (E) Frequency of LysoTracker colocalisation with DRAM1-negative Mm and DRAM1-positive membrane, punctate and luminal Mm patterns over time. Data are accumulated from two independent experiments (n0=71, n30=73, n60=105, n90=98, n120=102, n150=111 bacterial clusters).

Article Snippet: Membranes were blocked with 3% BSA in lx Tris buffered saline (TBS) solution with 0.1% Tween-20 (TBST) before incubating with primary and secondary antibodies: polyclonal rabbit anti DRAM1 (1:1000) (Aviva systems biology, ARP47432-P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), LAMP1 (1:1000) (Abcam, ab24170), Antirabbit IgG, HRP-Linked Antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Membrane, Staining, Infection

Journal: bioRxiv

Article Title: DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages

doi: 10.1101/2022.10.25.513660

Figure Lengend Snippet: Acidified mycobacteria-containing vesicles are labelled by LC3. (A) Representative examples of LC3 (magenta) colocalisation patterns with Mm (cyan), referred to as punctate and luminal patterns. An example of LC3-negative Mm (none) is also shown. Acidification of Mm-containing vesicles is assessed by LysoTracker staining (yellow). Scale bars: 2 μm (B) Representative example of LC3 (magenta) colocalisation with LysoTracker (yellow) and Mm (cyan) at 120 min post infection, when luminal colocalisation patterns are most frequent. Scale bars: 5 μm (C) Representative examples of double colocalisation of LC3 (magenta) and DRAM1 (grey) with Mm (cyan),. Scale bars: 2 μm (D) Frequency of Mm colocalisation with LC3 or LysoTracker over time. (E) Frequency of Mm colocalisation with LC3 in punctate or luminal patterns over time. (F) Frequency of LysoTracker colocalisation with LC3-negative Mm and LC3-positive punctate and luminal Mm patterns over time. Data are accumulated from two independent experiments (n0=99, n30=129, n60=96, n90=124, n120=133, n150=137 bacterial clusters).

Article Snippet: Membranes were blocked with 3% BSA in lx Tris buffered saline (TBS) solution with 0.1% Tween-20 (TBST) before incubating with primary and secondary antibodies: polyclonal rabbit anti DRAM1 (1:1000) (Aviva systems biology, ARP47432-P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), LAMP1 (1:1000) (Abcam, ab24170), Antirabbit IgG, HRP-Linked Antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Staining, Infection

Journal: bioRxiv

Article Title: DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages

doi: 10.1101/2022.10.25.513660

Figure Lengend Snippet: Confirmation of DRAM1 knockdown in RAW 264.7 macrophages. (A) qPCR analysis of Dram1 expression levels in control and knockdown cell lines (KD1-3). Statistical significance is assessed by logistic regression and pairwise comparison with Turkey correction. (**p<0.01) (B). Western blot analysis of DRAM1 protein levels in control and knockdown cells. Each cell line is analyzed in duplicate. DRAM1 protein levels relative to the control and normalized to GAPDH are indicated.

Article Snippet: Membranes were blocked with 3% BSA in lx Tris buffered saline (TBS) solution with 0.1% Tween-20 (TBST) before incubating with primary and secondary antibodies: polyclonal rabbit anti DRAM1 (1:1000) (Aviva systems biology, ARP47432-P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), LAMP1 (1:1000) (Abcam, ab24170), Antirabbit IgG, HRP-Linked Antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Expressing, Control, Comparison, Western Blot

Journal: bioRxiv

Article Title: DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages

doi: 10.1101/2022.10.25.513660

Figure Lengend Snippet: DRAM1 knockdown reduces acidification of Mm-containing vesicles. (A) Representative examples of DRAM1 (magenta) and LysoTracker (yellow) colocalisation with Mm (cyan) n control (Ctrl) and DRAM1 knockdown cell lines (KD1-3) at 120 min post infection Scale bars: 5 μm (B) Percentage of colocalisation of Mm with DRAM1 or LysoTracker in DRAM1 knockdown and control cell lines. (C) Percentage of colocalisation of Mm with DRAM1 in punctate, membrane, and luminal patterns in DRAM1 knockdown and control cell lines. (D) Percentage of colocalisation of LysoTracker with DRAM1-negative Mm (none) and DRAM1-positive punctate, membrane and luminal Mm patterns in DRAM1 knockdown and control cell lines. Data are accumulated from two independent experiments (nCtrl=117, nKD1=107, nKD2=92, nKD3=93 bacterial clusters). Statistical significance is assessed by logistic regression and pairwise comparison with Turkey correction. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001)

Article Snippet: Membranes were blocked with 3% BSA in lx Tris buffered saline (TBS) solution with 0.1% Tween-20 (TBST) before incubating with primary and secondary antibodies: polyclonal rabbit anti DRAM1 (1:1000) (Aviva systems biology, ARP47432-P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), LAMP1 (1:1000) (Abcam, ab24170), Antirabbit IgG, HRP-Linked Antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Control, Infection, Membrane, Comparison

Journal: bioRxiv

Article Title: DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages

doi: 10.1101/2022.10.25.513660

Figure Lengend Snippet: DRAM1 knockdown does not affect overall levels of LysoTracker, LC3 and LAMP1. Data are accumulated from three independent experiments with 12 images each and particles/intensity analysis are normalized by number of cells. (A,D,G) Representative examples of LysoTracker (A), LC3 (D) and LAMP1 (G) staining in non-infected DRAM1 knockdown (KD1-3) and control cells. Scale bars: 5 μm (B,C,E,F,H,I) Relative quantification of LysoTracker (B,C), LC3 (E,F) and LAMP1 (H,I) particles and signal intensity in noninfected DRAM1 knockdown and control cells.(J,K) Western blot analysis of LC3 (J) and LAMP1 (K) protein level relative to GAPDH in noninfected DRAM1 knockdown and control cells.

Article Snippet: Membranes were blocked with 3% BSA in lx Tris buffered saline (TBS) solution with 0.1% Tween-20 (TBST) before incubating with primary and secondary antibodies: polyclonal rabbit anti DRAM1 (1:1000) (Aviva systems biology, ARP47432-P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), LAMP1 (1:1000) (Abcam, ab24170), Antirabbit IgG, HRP-Linked Antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Staining, Infection, Control, Quantitative Proteomics, Western Blot

Journal: bioRxiv

Article Title: DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages

doi: 10.1101/2022.10.25.513660

Figure Lengend Snippet: DRAM1 knockdown reduces LC3 trafficking to Mm. (A) Representative examples of LC3 (magenta) and LysoTracker (yellow) colocalisation with Mm (cyan) in DRAM1 knockdown (KD1-3) and control (Ctrl) cell lines at 120 min post infection. Scale bars: 5 μm (B) Percentage of colocalisation of Mm with LC3 or LysoTracker in DRAM1 knockdown and control cell lines. (C) Percentage of colocalisation of Mm with LC3 in punctate and luminal patterns in DRAM1 knockdown and control cell lines. (D) Percentage of colocalisation of LysoTracker with LC3-negative Mm and LC3-positive punctate and luminal Mm patterns in DRAM1 knockdown and control cell lines. Data are accumulated from two independent experiments (nCtrl=210, nKD1=184, nKD2=128, nKD3=120 bacterial clusters). Statistical significance is assessed by logistic regression and pairwise comparison with Turkey correction. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001)

Article Snippet: Membranes were blocked with 3% BSA in lx Tris buffered saline (TBS) solution with 0.1% Tween-20 (TBST) before incubating with primary and secondary antibodies: polyclonal rabbit anti DRAM1 (1:1000) (Aviva systems biology, ARP47432-P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), LAMP1 (1:1000) (Abcam, ab24170), Antirabbit IgG, HRP-Linked Antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Control, Infection, Comparison

Journal: bioRxiv

Article Title: DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages

doi: 10.1101/2022.10.25.513660

Figure Lengend Snippet: DRAM1 knockdown reduces fusion between lysosomes and Mm-containing vesicles. (A) Representative examples of LAMP1 (magenta) colocalisation patterns with Mm (cyan), referred to as membrane, punctate, and luminal patterns, at 120 min post infection. Acidification of Mm-containing vesicles is assessed by LysoTracker staining (yellow). Scale bars: 2 μm (B) Representative examples of LAMP1 (magenta) colocalisation with Mm (cyan), and with acidification of Mm-containing vesicles assessed by LysoTracker staining (yellow) in DRAM1 knockdown (KD1-3) and control cell lines at 120 min post infection. Scale bars: 5 μm (C) Percentage of colocalisation of Mm with LAMP1 or LysoTracker in DRAM1 knockdown and control cell lines. (D) Percentage of colocalisation of Mm with LAMP1 in punctate, membrane, and luminal patterns in DRAM1 knockdown and control cell lines. (E) Percentage of colocalisation of LysoTracker with LAMP1-negative Mm and LAMP1-positive Mm punctate, membrane, and luminal Mm patterns in DRAM1 knockdown and control cell lines. Data are accumulated from two independent experiments (nCtrl=156, nKD1=126, nKD2=153, nKD3=118 bacterial clusters). Statistical significance is assessed by logistic regression and pairwise comparison with Turkey correction. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001)

Article Snippet: Membranes were blocked with 3% BSA in lx Tris buffered saline (TBS) solution with 0.1% Tween-20 (TBST) before incubating with primary and secondary antibodies: polyclonal rabbit anti DRAM1 (1:1000) (Aviva systems biology, ARP47432-P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), LAMP1 (1:1000) (Abcam, ab24170), Antirabbit IgG, HRP-Linked Antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Membrane, Infection, Staining, Control, Comparison

Journal: bioRxiv

Article Title: DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages

doi: 10.1101/2022.10.25.513660

Figure Lengend Snippet: DRAM1 knockdown increases the susceptibility of macrophages to Mm infection. (A,C) Representative examples of infected cells in DRAM1 knockdown and control cells at 1 h (A) and 7 h (C) post infection. Scale bars: 20 μm (B, D) Percentage of infected cells in DRAM1 knockdown (KD1-3) and control cells at 1 h (B) and 7 h (D) post infection. Data are accumulated from three independent experiments with 12 images each. Statistical significance is assessed by logistic regression and pairwise comparison with Turkey correction. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001)

Article Snippet: Membranes were blocked with 3% BSA in lx Tris buffered saline (TBS) solution with 0.1% Tween-20 (TBST) before incubating with primary and secondary antibodies: polyclonal rabbit anti DRAM1 (1:1000) (Aviva systems biology, ARP47432-P050), GAPDH (1:1000)(D16H11), LC3 (1:1000) (Novus Biologicals, NB100-2331), LAMP1 (1:1000) (Abcam, ab24170), Antirabbit IgG, HRP-Linked Antibody (1:1000) (Cell Signaling, 7074S).

Techniques: Knockdown, Infection, Control, Comparison

Journal: bioRxiv

Article Title: Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

doi: 10.1101/599266

Figure Lengend Snippet: Generation and characterization of the dram1 mutant line. A. Schematic diagram showing the workflow used for the generation of dram1 mutant lines. Target-specific sgRNA and Cas9 mRNA were co-injected into one cell stage embryos (AB/TL, wild type line). Founders were outcrossed to Tg ( CMV:EGFP-map1lc3b ) or wild type fish to obtain F1. After 3-4 months, the F1 was incrossed to obtain homozygous mutant and wild type F2 siblings. dram1 Δ19n/Δ19n were outcrossed with the macrophage marker Tg(mpeg1:mCherryF) umsF001 and after 3-4 months subsequently incrossed to obtain dram1 +/+ , dram1 Δ19/+ , and dram1 Δ19n/Δ19n carrying Tg(mpeg1:mCherryF) umsF001 . B. Sanger sequencing of dram1Δ19n/Δ19n and dram1+/+ from F2 offspring. Red lines indicate CRISPR/Cas9 target sites. The genomic DNA was isolated from fin tissue (>3 months old fish). The dram1Δ19n/Δ19n mutant allele has 21 nucleotides deleted and 2 nucleotides inserted. C. Segregation from dram1 +/Δ19n F1 heterozygous incross. Genotypes of adult fish (>3 months old) were combined from at least three independent breedings and confirmed by PCR and Sanger sequencing. Data were analyzed by Chi Square test. ns, non-significant,*p<0.05,**p<0.01,***p<0.001. D. Segregation from dram1 Δ19n+ /mpeg1:mCherryF F1 heterozygous incross. Genotypes of adult fish (>3 months old) combined from at least three independent breedings were confirmed by PCR and sequencing. Data were analyzed by Chi square test. ns, non-significant,*p<0.05,**p<0.01,***p<0.001 E. Representative confocal micrographs of sections from the tail region showing TUNEL staining performed on dram1 Δ19n/Δ19n and dram1 +/+ larvae at 3dpf. Scale bar, 10 μm.

Article Snippet: Western blot analysis was performed as previously described ( ) Antibodies used were as follows: polyclonal rabbit anti DRAM1 (N-terminal) (1:1000,ARP47432-P050, Aviva systems biology), polyclonal rabbit anti-Optineurin (C-terminal) (1:200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (1:1000, PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1:1000, NB100-2331, lot#AB-3, Novus Biologicals), monoclonal Caspase 3 antibody (1:1000, #9662, Lot#12, Cell Signaling), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1:200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1:1000, 4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1:1000, 7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1:3000, 7076S, Lot#029, Cell Signaling).

Techniques: Mutagenesis, Injection, Marker, Sequencing, CRISPR, Isolation, TUNEL Assay, Staining

Journal: bioRxiv

Article Title: Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

doi: 10.1101/599266

Figure Lengend Snippet: Dram1 deficiency leads to increased susceptibility to Mm infection A. Schematic representation of the zebrafish dram1/Dram1(ENSDARG00000045561/ENSDARP0 0000066996.3) genetic and protein domain architecture and CRISPR/Cas9 target site. Dram1 (240 amino acids) contains six transmembrane domains (indicated with grey boxes and labeled T1-T6 with amino acid numbers indicated above). The gene is depicted with coding exons as grey boxes and introns as solid black lines (introns not drawn to scale). The position of the CRISPR/Cas9 target site and the predicted truncated protein is indicated. B. Confirmation of dram1 mutation by Western blotting analysis. Protein samples were extracted from 4 dpf dram1 Δ19n/Δ19n and dram1 +/+ larvae (>10 larvae/sample). The blots were probed with antibodies against Dram1 and Actin as a loading control. C. Measurements of larval body lengths. dram1 +/+ and dram1 Δ19n/Δ19n larvae (≥10 larvae/group) were imaged by stereo microscopy at 3dpf and body lengths were measured as indicated by the red dotted lines. D. Quantification of TUNEL-positive cells in the indicated region of the tail of dram1 Δ19n/Δ19n and dram1 +/+ larvae at 3 dpf (≥7 larvae/group). E. Representative stereo images of infected dram1 Δ19n/Δ19n and dram1 +/+ larvae at 3 dpi. The arrowhead indicates the accumulation of bacteria in intersegmental veins. F and G. Quantification of bacterial burdens at 3dpi for dram1 mutants, wild type siblings, and unrelated wild types (F) or for dram1 and dram1 Δ19n mRNA injected individuals (G). The data is accumulated from two independent experiments. Each dot represents an individual larva.

Article Snippet: Western blot analysis was performed as previously described ( ) Antibodies used were as follows: polyclonal rabbit anti DRAM1 (N-terminal) (1:1000,ARP47432-P050, Aviva systems biology), polyclonal rabbit anti-Optineurin (C-terminal) (1:200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (1:1000, PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1:1000, NB100-2331, lot#AB-3, Novus Biologicals), monoclonal Caspase 3 antibody (1:1000, #9662, Lot#12, Cell Signaling), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1:200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1:1000, 4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1:1000, 7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1:3000, 7076S, Lot#029, Cell Signaling).

Techniques: Infection, CRISPR, Labeling, Mutagenesis, Western Blot, Microscopy, TUNEL Assay, Injection

Journal: bioRxiv

Article Title: Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

doi: 10.1101/599266

Figure Lengend Snippet: Dram1 deficiency affects gene expression of pathways involved in metabolism, innate immunity and lytic cell death during infection A. Bacterial burdens of larvae injected with different CFU doses of Mm and used for RNAseq. The data is accumulated from three independent sample sets at 4 dpi. Each dot represents the bacterial burden of an individual larva. B. Venn diagram of the significantly enriched KEGG pathways in the transcriptome of larvae infected with Mm. The enrichment comparisons were performed on dram1 Δ19n/Δ19n 150 CFU versus dram1 Δ19n/Δ19n PBS, dram1 Δ19n/Δ19n 300 CFU versus dram1 Δ19n/Δ19n , and dram1 +/+ 300 CFU versus dram1 +/+ PBS. C. Visualization of the lytic cellular death signaling pathway transcriptome shows different responses in the transcriptome of infected dram1 Δ19n/Δ19n and dram1 +/+ . The pathway was adapted from the KEGG pathway necroptosis. In the visualization, the gene expression in the comparison dram1 Δ19n/Δ19n 150 CFU versus dram1 Δ19n/Δ19n PBS, dram1 Δ19n/Δ19n 300 CFU versus dram1 Δ19n/Δ19n , and dram1 +/+ 300 CFU versus dram1 +/+ PBS are depicted by colour gradient (yellow, upregulated, blue downregulated). The expression of all genes of the pathway present in the RNA sequencing was plotted independently of their significance. While the effector genes of the apoptosis pathway did not show high expression changes, the genes from lytic cell death forms, including pyroptosis, showed high expression.

Article Snippet: Western blot analysis was performed as previously described ( ) Antibodies used were as follows: polyclonal rabbit anti DRAM1 (N-terminal) (1:1000,ARP47432-P050, Aviva systems biology), polyclonal rabbit anti-Optineurin (C-terminal) (1:200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (1:1000, PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1:1000, NB100-2331, lot#AB-3, Novus Biologicals), monoclonal Caspase 3 antibody (1:1000, #9662, Lot#12, Cell Signaling), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1:200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1:1000, 4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1:1000, 7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1:3000, 7076S, Lot#029, Cell Signaling).

Techniques: Expressing, Infection, Injection, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

doi: 10.1101/599266

Figure Lengend Snippet: Transcriptome analysis of uninfected and infected dram1 mutants A. Experimental design to obtain samples for RNA deep sequencing. Mycobacterium marinum strain M (Mm) fluorescently labeled with mCherry was microinjected into the blood island of embryos at 28 hpf at an injection dosage of 300 CFU of 150 CFU. Control groups were injected with PBS. B. Principal component analysis of the gene expression data obtained by RNA sequencing. The RNA sequencing samples clustered according to their condition, as pictured by the dashed ellipses grouping the samples. The data sets of one family of dram1 Δ19n/Δ19n (Mm infected and uninfected) diverged from the rest of the libraries (data points outside the dashed ellipses) and were discarded from the analysis. C. General profile of differential gene expression between the different conditions. The number of genes upregulated are coloured in yellow while downregulated in blue, with indication of the fold-change by colour intensity. D. Venn diagram of the differentially expressed genes common and different between the dram1 Δ19n/Δ19n and dram1 +/+ ,dram1 Δ19n/Δ19n 150 CFU and dram1 Δ19n/Δ19n PBS, dram1 Δ19n/Δ19n 300 CFU and dram1 Δ19n/Δ19n , dram1 +/+ 300 CFU and dram1 +/+ PBS comparisons.

Article Snippet: Western blot analysis was performed as previously described ( ) Antibodies used were as follows: polyclonal rabbit anti DRAM1 (N-terminal) (1:1000,ARP47432-P050, Aviva systems biology), polyclonal rabbit anti-Optineurin (C-terminal) (1:200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (1:1000, PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1:1000, NB100-2331, lot#AB-3, Novus Biologicals), monoclonal Caspase 3 antibody (1:1000, #9662, Lot#12, Cell Signaling), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1:200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1:1000, 4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1:1000, 7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1:3000, 7076S, Lot#029, Cell Signaling).

Techniques: Infection, Sequencing, Labeling, Injection, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

doi: 10.1101/599266

Figure Lengend Snippet: Effect of dram1 mutation on TLR signaling KEGG pathway of TLR signaling showing differential gene expression in infected dram1 Δ19n/Δ19n and dram1 +/+ . The three data sets used for comparison are shown in the legend of the figure. The expression fold change of the genes is depicted by colour (yellow, upregulated, blue downregulated).

Article Snippet: Western blot analysis was performed as previously described ( ) Antibodies used were as follows: polyclonal rabbit anti DRAM1 (N-terminal) (1:1000,ARP47432-P050, Aviva systems biology), polyclonal rabbit anti-Optineurin (C-terminal) (1:200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (1:1000, PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1:1000, NB100-2331, lot#AB-3, Novus Biologicals), monoclonal Caspase 3 antibody (1:1000, #9662, Lot#12, Cell Signaling), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1:200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1:1000, 4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1:1000, 7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1:3000, 7076S, Lot#029, Cell Signaling).

Techniques: Mutagenesis, Expressing, Infection

Journal: bioRxiv

Article Title: Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

doi: 10.1101/599266

Figure Lengend Snippet: Dram1 is required for GFP-Lc3 targeting to Mm clusters A-B. Representative confocal micrographs and quantification of GFP-Lc3 puncta in dram1 Δ19n/Δ19n and dram1 +/+ larvae in an unstimulated situation (basal autophagy, A) and following BafA1 treatment (B). Each larva was imaged at a pre-defined region of the tail fin as indicated by the red boxed area in the schematic fish drawing above the graphs. (≥11 larvae/group). Results are accumulated from two independent experiments, each dot represents an individual larva. ns, non-significant *p<0.05,**P<0.01,***p<0.001. Scale bars, 10 μm. C-E. Western blot analysis of autophagy. Protein samples were obtained from 4 dpf dram1 Δ19n/Δ19n and dram1 +/+ larvae (>10 larvae/sample). Lc3 (C and E), or p62 and Optineurin (D) protein levels were detected in in absence or presence of BafA1 (C and D) or in the presence or absence of Mm (E). Actin was used as a loading control. WB were repeated three (C and D) or two (E) times with protein extracts derived from independent experiments. The Lc3II/Actin or p62/Actin and Optineurin/Actin ratio is indicated below the blots. F-G. Representative confocal micrographs and quantification of GFP-Lc3 co-localization with Mm clusters in infected dram1 Δ19n/Δ19n and dram1 +/+ larvae. The top image shows the entire region of imaging as indicated in the schematic. The bottom images show GFP-Lc3 colocalization of Mm clusters in dram1 Δ19n/Δ19n and dram1 +/+ larvae. The arrowheads indicate GFP-Lc3-positive Mm clusters. The data is accumulated from two independent experiments, each dot represents an individual larva (≥15 larvae/group). Scale bars, 10 μm.

Article Snippet: Western blot analysis was performed as previously described ( ) Antibodies used were as follows: polyclonal rabbit anti DRAM1 (N-terminal) (1:1000,ARP47432-P050, Aviva systems biology), polyclonal rabbit anti-Optineurin (C-terminal) (1:200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (1:1000, PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1:1000, NB100-2331, lot#AB-3, Novus Biologicals), monoclonal Caspase 3 antibody (1:1000, #9662, Lot#12, Cell Signaling), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1:200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1:1000, 4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1:1000, 7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1:3000, 7076S, Lot#029, Cell Signaling).

Techniques: Western Blot, Derivative Assay, Infection, Imaging

Journal: bioRxiv

Article Title: Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

doi: 10.1101/599266

Figure Lengend Snippet: Dram1 deficiency does not affect the capability of macrophages to phagocytose Mm A. Representative stereo images of the whole tail of dram1 Δ19n/Δ19n and dram1 +/+ larvae following an immunohistochemical peroxidase activity detection protocol. The number of neutrophils in this region was quantified per individual larva (≥18 larvae/group). Each data point represents an individual larva. Results are accumulated from two independent experiments. ns, non-significant *p<0.05,**P<0.01,***p<0.001. B. Representative stereo micrographs of macrophages in the whole tail region and quantification of the number of macrophages in this region. 3 dpf dram1 Δ19n/Δ19n and dram1 +/+ / mpeg1:mCherryF larvae were obtained from incrossed dram1 +/Δ19n animals and the number of macrophages for each larva were counted before knowing the genotype. Genotyping was performed by PCR and Sanger sequencing (≥28 larvae/ group). Results are accumulated from two independent experiments, each dot represents an individual larva. ns, non-significant, *p<0.05,**P<0.01,***p<0.001 C. Representative confocal micrographs of the yolk of infected dram1 Δ19n/Δ19n and dram1 +/+ embryos in mpeg1:mCherryF background at 1 hour post infection (hpi). Scale bars, 10 μm. D. Quantification of phagocytosis of Mm by macrophages at 1 hpi. dram1 Δ19n/Δ19n and dram1 +/+ embryos in mpeg1:mCherryF background were infected Mm at 30 hpf and fixed at 1 hpi. Each dot represents the percentage of macrophages that have phagocytosed Mm clusters in an individual larva (≥16 larvae/ group). Results are accumulated from two independent experiments,. ns, nonsignificant, *p<0.05,**P<0.01,***p<0.001. E. Mm bacterial burden for dram1 Δ19n/Δ19n and dram1 +/+ at 1 and 2 dpi. Each dot represents an individual infected larva.

Article Snippet: Western blot analysis was performed as previously described ( ) Antibodies used were as follows: polyclonal rabbit anti DRAM1 (N-terminal) (1:1000,ARP47432-P050, Aviva systems biology), polyclonal rabbit anti-Optineurin (C-terminal) (1:200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (1:1000, PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1:1000, NB100-2331, lot#AB-3, Novus Biologicals), monoclonal Caspase 3 antibody (1:1000, #9662, Lot#12, Cell Signaling), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1:200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1:1000, 4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1:1000, 7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1:3000, 7076S, Lot#029, Cell Signaling).

Techniques: Immunohistochemical staining, Activity Assay, Sequencing, Infection

Journal: bioRxiv

Article Title: Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

doi: 10.1101/599266

Figure Lengend Snippet: Macrophages fail to restrict Mm infection in Dram1-deficient larvae A. Representative confocal images of LysoTracker staining performed on dram1 Δ19n/Δ19n and dram1 +/+ embryos at 1 dpi. The arrowheads indicate Lysotracker-positive (LysoTracker + ) Mm clusters. Scale bars, 10 μm. B. The percentage of LysoTracker + Mm clusters was determined in infected embryos (≥15 embryos/group) at 1 dpi. Each dot represents the percentage of Mm clusters that are LysoTracker + in an individual infected larva. Results are accumulated from two independent experiments. ns, non-significant *p<0.05,**P<0.01,***p<0.001. C and D: Representative confocal images of dram1 Δ19n/Δ19n and dram1 +/+ embryos/larvae in mpeg1:mCherryF background, infected as described in , at 1 dpi (C) and 2 dpi (D). The entire CHT region of fixed embryos or larvae was imaged. The arrowheads indicate extracellular Mm clusters and stars (*) indicate remnants from dead macrophages. Scale bars, 10 μm. E and F: Percentage of Mm clusters restricted inside macrophages at 1 dpi (E) and 2 dpi (F) (≥10 embryos/group). Each dot represents the percentage of intracellular Mm clusters in an individual embryo. Results are accumulated from two independent experiments, ns, non-significant *p<0.05,**P<0.01,***p<0.001

Article Snippet: Western blot analysis was performed as previously described ( ) Antibodies used were as follows: polyclonal rabbit anti DRAM1 (N-terminal) (1:1000,ARP47432-P050, Aviva systems biology), polyclonal rabbit anti-Optineurin (C-terminal) (1:200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (1:1000, PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1:1000, NB100-2331, lot#AB-3, Novus Biologicals), monoclonal Caspase 3 antibody (1:1000, #9662, Lot#12, Cell Signaling), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1:200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1:1000, 4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1:1000, 7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1:3000, 7076S, Lot#029, Cell Signaling).

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

doi: 10.1101/599266

Figure Lengend Snippet: Dram1 deficiency results in increased pyroptotic cell death A. Representative confocal images of TUNEL staining in dram1 Δ19n/Δ19n and dram1 +/+ larvae at 2 dpi. The entire CHT region of 2 dpi fixed dram1 Δ19n/Δ19n and dram1 +/+ larvae was imaged. The arrowheads indicate the cells positive for TUNEL staining (TUNEL + ). Scale bars, 10 μm. B. Quantification of the percentage of Mm clusters TUNEL + in dram1 Δ19n/Δ19n and dram1 +/+ larvae. Each dot represents the percentage of Mm clusters TUNEL + in the CHT region of an individual infected larva (≥ 12 larvae/group). Results are accumulated from two independent experiments, ns, non-significant,*p<0.05,**P<0.01,***p<0.001 C. Detection of pro-Caspase 3 and cleaved Caspase 3 protein in dram1 Δ19n/Δ19n and dram1 +/+ embryos. Protein samples were extracted from 4 dpf infected and uninfected dram1 Δ19n/Δ19n and dram1 +/+ larvae (>10 larvae/sample). The western blots were probed with antibodies against Caspase 3 and Actin as a loading control. Data is representative of two independent experiments. D. Detection of Caspase activity (YVAD-AFC) in dram1 Δ19n/Δ19n and dram1 +/+ embryos. Protein samples were obtained from 2 dpf control and infected dram1 Δ19n/Δ19n and dram1 +/+ embryos in GFP-Lc3 background (35 embryos/sample). The data is accumulated from two independent experiments, each dot represents an individual larva. ns, non-significant *p<0.05,**P<0.01,***p<0.001 E. Mm bacterial burden at 2 dpi following knockdown of caspa in dram1 Δ19n/Δ19n and dram1 +/+ embryos. The data is accumulated from two independent experiments. F. Mm bacterial burden at 2 dpi following knockdown of gsmdeb in dram1 Δ19n/Δ19n and dram1 +/+ embryos. The data is accumulated from independent experiments, each dot represents an individual larva. ns, non-significant *p<0.05,**P<0.01,***p<0.001

Article Snippet: Western blot analysis was performed as previously described ( ) Antibodies used were as follows: polyclonal rabbit anti DRAM1 (N-terminal) (1:1000,ARP47432-P050, Aviva systems biology), polyclonal rabbit anti-Optineurin (C-terminal) (1:200, lot#100000; Cayman Chemical), polyclonal rabbit anti-p62 (C-terminal) (1:1000, PM045, lot#019, MBL), polyclonal rabbit anti Lc3 (1:1000, NB100-2331, lot#AB-3, Novus Biologicals), monoclonal Caspase 3 antibody (1:1000, #9662, Lot#12, Cell Signaling), Anti mono-and polyubiquitinated conjugates mouse monoclonal antibody (1:200; BML-PW8810-0100, lot#01031445, Enzo life Sciences), Polyclonal actin antibody (1:1000, 4968S, lot#3, Cell Signaling), Anti-rabbit IgG, HRP-Linked Antibody (1:1000, 7074S, Lot#0026, Cell Signaling), Anti-mouse IgG, HRP-linked Antibody (1:3000, 7076S, Lot#029, Cell Signaling).

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