Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Tissue fungal burden and galectin-3 concentrations in adult mice with disseminated candidiasis. Mice (n=7) were infected via tail-vein injection with C. albicans and euthanized at 48 h after injection. Panel A: Tissue fungal burden. Panel B: Mean gal3 concentration in tissue homogenates compared to animals receiving saline (n=10). Error bars represent standard deviation. No differences in tissue gal3 concentration were detected by ANOVA with inter-group comparisons by the Student-Newman-Keuls Method.

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Infection, Injection, Concentration Assay, Saline, Standard Deviation

Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Tissue fungal burden and galectin-3 concentrations in neonatal mice with disseminated candidiasis. Two-day-old mouse pups (n=14) were infected via intraperitoneal injection with C. albicans and euthanized at 24 h after injection. Panel A: Tissue fungal burden. Panel B: Mean gal3 concentration in tissue homogenates compared to pups receiving saline (n=14). Error bars represent standard deviation. Mean gal3 concentration was reduced in the spleen of infected pups relative to controls based on ANOVA with inter-group comparisons by the Student-Newman-Keuls Method (p=0.04).

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Infection, Injection, Concentration Assay, Saline, Standard Deviation

Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Survival curve of neonatal mice with disseminated candidiasis after pretreatment with recombinant galectin-3. Two-day-old mouse pups were given intraperitoneal injections of either saline or recombinant gal3, 2 h prior to infection with C. albicans . Uninfected pups receiving gal3 only were included as a control. Pretreatment with gal3 reduced mortality in infected compared to saline treated pups based on log-rank test (p=0.02).

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Recombinant, Saline, Infection, Control

Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Tissue fungal burden in neonatal mice with disseminated candidiasis pretreated with galectin-3. Two-day-old mouse pups were given intraperitoneal injections of either saline or recombinant gal3, 2 h prior to infection with C. albicans (n=10 pups per group). Tissues were collected at the time of death or at 72 h in surviving animals. Fungal burden is depicted with the bars representing median values. P values were derived based on analysis using a negative binomial model to account for the variability in these data which are not normally distributed.

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Saline, Recombinant, Infection, Derivative Assay

Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Tissue fungal burden in neonatal mice with disseminated candidiasis at early time points following infection and pretreatment with galectin-3. Two-day-old mouse pups were given intraperitoneal injections of either saline or recombinant gal3, 2 h prior to infection with C. albicans (n=5 pups per group). Pups were euthanized and tissues were collected at 24 and 36 h following infection. Fungal burden is depicted with the bars representing median values. P values were derived based on analysis using a negative binomial model to account for the variability in these data which are not normally distributed. NS – not significant.

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Infection, Saline, Recombinant, Derivative Assay

Journal: BMC Women's Health

Article Title: The relationship between serum galectin-3 level and echocardiographic findings in patients with polycystic ovary syndrome

doi: 10.1186/s12905-026-04309-1

Figure Lengend Snippet: Scatter plot diagram between galectin-3 and global longitidunal strain

Article Snippet: Serum galectin-3 concentrations were calculated from a standard curve generated using the recombinant galectin-3 standards provided in the kit.

Techniques:

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Scheme depicting proximity biotinylation of proteins in response to recruitment of LGALS1, LGALS3, and LGALS8 proteins to damaged lysosomes. ( B ) Experimental workflow for galectin proximity biotinylation. APEX2-tagged LGALS1, LGALS3, and LGALS8 expressed in HeLa cells were subjected to proximity biotinylation 60 min post-LLOMe treatment using 10-plex TMT. ( C ) Volcano plot for LLOMe (60 min)-treated cells versus untreated cells (Log 2 FC versus −Log 10 p-value) for APEX-LGALS8-based proximity biotinylation based on the TMT experiment in (B). Specific categories of proteins are indicated by colored circles. ( D ) Volcano plot for LLOMe (60 min)-treated cells versus untreated cells (Log 2 FC versus −Log 10 p-value) for APEX-LGALS3-based proximity biotinylation based on the TMT experiment in B. Specific categories of proteins are indicated by colored circles. ( E ) Volcano plot for LLOMe (60 min)-treated cells versus untreated cells (Log 2 FC versus −Log 10 p-value) for APEX-LGALS1-based proximity biotinylation based on the TMT experiment in B. Specific categories of proteins are indicated by colored circles. ( F ) Log 2 FC for individual proteins localized to the lysosomal compartment found to be enriched in biotinylated proteins from cells expressing the indicated APEX2-galectin protein. Mean and standard deviation are calculated from two untreated and two treated biological replicates. ( G ) GO: process enrichment categories for APEX2-LGALS8. Figure 3—source data 1. Log 2 FCs for lysosomal proteins from APEX2-LGALS1, 3, and 8 for . Figure 3—source data 2. GO enrichments for APEX2-LGALS8 for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Expressing, Standard Deviation

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Summary of proteins in proximity to galectins and integration with associations found with APEX2-ATG8 (bold) and Lyso-IP (underline). Other functional classes are indicated. ( B ) Localization of LGALS3 with LAMP1 and MAP1LC3B in response to lysosomal damage. Cells were treated with LLOMe for 1 hr and the LLOMe washed out for 4 hr prior to immunofluorescence using the indicated antibodies and imaging by confocal microscopy. Scale bars 10 μm. Zoom-in panels, 10 μm × 10 μm. ( C ) Cells were left untreated (Untreated), treated with LLOMe for 1 hr and either fixed (LLOMe 1 hr) or the LLOMe was washed out for 4 hr prior to fixation (LLOMe 1 hr+ washout 4 hr). Immunofluorescence was done using α-OPTN/α-LAMP1 and imaging by confocal microscopy. Scale bars 10 μm. Zoom-in panels, 10 μm × 10 μm. ( D ) Cells were treated as in (C). Immunofluorescence was done using α-TAX1BP1/α-LAMP1 and imaging by confocal microscopy. Scale bars 10 μm. Zoom-in panels, 10 μm × 10 μm. ( E ) Cells were treated as in (C). Immunofluorescence was done using α-CALCOCO2/α-LAMP1 and imaging by confocal microscopy. Scale bars 10 μm. Zoom-in panels, 10 μm × 10 μm. ( F ) Quantification of OPTN localization at LAMP1 lysosomes using Mander’s overlap coefficient (MOC). 23 (0 hr), 19 (1 hr), and 22 (4 hr washout) cells were analyzed for MOC. ***p < 0.001. + marks the mean and the line marks the median. The plot represents merged data from three biological replicates for each condition. ( G ) Quantification of TAX1BP1 localization at LAMP1 lysosomes using Mander’s overlap coefficient (MOC). 20 (0 hr), 17 (1 hr), and 22 (4 hr washout) cells were analyzed for MOC. ***p < 0.001. + marks the mean and the line marks the median. The plot represents merged data from three biological replicates for each condition. ( H ) Quantification of CALCOCO2 localization at LAMP1 lysosomes using MOC. 18 (0 hr), 20 (1 hr), and 21 (4 hr washout) cells were analyzed for MOC. **p < 0.01 and ***p < 0.001. + marks the mean and the line marks the median. The plot represents merged data from three biological replicates for each condition. Figure 4—source data 1. Mander’s overlap coefficient (MOC) values for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Functional Assay, Immunofluorescence, Imaging, Confocal Microscopy

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Scheme depicting measurement of lysophagic flux using Lyso-Keima (Keima-LGALS3). Cells stably expressing Keima-LGALS3 are treated with LLOMe (1 hr), and the Keima-LGALS3 is recruited from the cytosol to damaged lysosomes, representing the initial recruitment step (green dot). After removing LLOMe (washout), damaged lysosomes undergo autophagy-dependent trafficking to a healthy lysosome, leading to a red-shift in Keima fluorescence (red dots) due to the acidic environment of the lysosome. Cells can be analyzed by imaging, flow cytometry or SDS–PAGE for processed Keima. ( B ) Keima-LGALS3 in untreated HeLa cells or in cells that were treated with LLOMe for 1 hr and the LLOMe washed out for 4 or 12 hr and imaged using excitation at 442 or 561 nm. Scale bar 10 μm. Zoom-in panels, 10 μm × 20 μm. ( C ) Keima-LGALS3 HeLa cells were either left untreated or treated for 1 hr followed by washout (12 hr) with or without prior addition of TBK1i or BafA. Cells were imaged using excitation at 442 or 561 nm. A ratio of the 561 nm/442 nm images was taken and puncta were identified from this 561 nm/442 nm image. Scale bar 10 μm. Zoom-in panels, 10 μm × 20 μm. ( D ) Quantification of Keima-positive lysosomes. 69 (untreated), 83 (BafA), and 66 (TBKi) cells were analyzed ****p < 0.0001. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. ( E ) Triplicate HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout (12 hr) with or without addition of BafA. Cells were then subjected to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample. ****p < 0.0001. The plot represents mean and standard deviation from three biological replicates. ( F ) Triplicate HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout (12 hr) with or without prior addition of TBK1i, ULK1i, TAK243, and p97i. Cells were then subjected to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample. ****p < 0.0001. The plot represents mean and standard deviation from three biological replicates. ( G ) HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout followed by harvesting at the indicated times. Lysed cells were then subjected to immunoblotting with the indicated antibodies. ( H ) Cells were left untreated (Untreated), treated with LLOMe for 1 hr and either fixed (LLOMe 1 hr) or the LLOMe was washed out for 4 hr prior to fixation (LLOMe 1 hr + washout 4 hr). Immunofluorescence was done using α-pTBK1/α-LAMP1 and imaging by confocal microscopy. Scale bar = 10 μm. Zoom-in panels, 10 μm × 10 μm. Right: quantification of localization using Mander’s overlap coefficient (MOC). 23 (0 hr), 21 (1 hr), and 18 (4 hr washout) cells were analyzed for MOC. ***p < 0.001. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. ( I ) HeLa cells expressing Keima-LGALS3 were either left untreated or treated with LLOMe for 1 hr and then incubated for four or 12 hr post-washout in the presence or absence of either BafA or TBK1i. Cell lysates were subjected to immunoblotting using the indicated antibodies. ( J ) Triplicate WT, ATG5 −/− , or TBK1 −/− HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout (4 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample within each genotype. The plot represents mean and standard deviation from three biological replicates. Figure 5—source data 1. Quantification of Keima-positive lysosomes. Figure 5—source data 2. 561/488 Keima ratios for . Figure 5—source data 3. 561/488 Keima ratios for . Figure 5—source data 4. Uncropped blots for . Figure 5—source data 5. MOC values for . Figure 5—source data 6. 561/488 Keima ratios for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Stable Transfection, Expressing, Fluorescence, Imaging, Flow Cytometry, SDS Page, Standard Deviation, Western Blot, Immunofluorescence, Confocal Microscopy, Incubation

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Raw flow cytometry data. HeLa cells expressing Keima-LGALS3 were either left untreated (red), or treated for 1 hr followed by washout (12 hr) with (orange) or without (blue) addition of BafA. Cells were then subjected to flow cytometry to measure the 561 nm/488 nm ratio. ( B ) HeLa cells expressing Keima-LGALS3 were treated with LLOMe (1 hr) prior to washout for 4 or 8 hr. In one set of samples, the E1 inhibitor TAK243 at 2 μM was added prior to damage. Cell extracts at the indicated time were subjected to immunoblotting with the indicated antibodies. Figure 5—figure supplement 1—source data 1. Uncropped blots.

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Triplicate WT; ATG5 −/− ; or OPTN −/− ; TAX1BP1 −/− ; CALCOCO2 −/− (TKO) HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample within each genotype. The plot represents mean and standard deviation from three biological replicates. ( B ) Triplicate WT or TKO HeLa cells expressing Keima-LGALS3 were reconstituted with lentivirally expressed EGFP-FLAG-HA, EGFP-CALCOCO2, EGFP-OPTN, or EGFP-TAX1BP1. Cells were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. As a control for lysophagic flux, some samples were also treated with BafA during the washout. All values are normalized to the untreated sample within each genotype. ****p < 0.0001. The plot represents mean and standard deviation from three biological replicates. ( C ) Cells from panel B were lysed and subjected to immunoblotting with the indicated antibodies. ( D ) HeLa cells expressing Keima-LGALS3 (with or without deletion of ATG7, TAX1BP1, OPTN, CALCOCO2, or SQSTM1) were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample within each genotype. ****p < 0.0001. The plot represents mean and standard deviation from three biological replicates. ( E ) HeLa cells (with or without deletion of TAX1BP1, OPTN, CALCOCO2, or SQSTM1) were either left untreated or treated for 1 hr followed by washout (10 hr) prior to immunostaining with α-LAMP1 (green) and α-LGALS3 (magenta). The number of LGALS3 puncta per cell present after washout is plotted (right top panel). The block to lysophagic flux was rescued by expression of EGFP-TAX1BP1 but not EGFP (lower right panel). 41 (WT), 21 (TAX1BP1), 25 (OPTN), 21 (CALCOCO2), and 27 (SQSTM1) cells were analyzed in the upper graph. 29 (WT), 28 (EGFP), and 32 (EGFP-TAX1BP1) cells were analyzed in the bottom graph. ****p < 0.0001. Scale bar 10 μm. Zoom-in panels, 10 μm × 10 μm. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. Figure 6—source data 1. 561/488 Keima ratios for . Figure 6—source data 2. 561/488 Keima ratios for . Figure 6—source data 3. 561/488 Keima ratios for . Figure 6—source data 4. The number of galectin puncta per cell post-washout for . Figure 6—source data 5. Uncropped blots for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Expressing, Flow Cytometry, Standard Deviation, Control, Western Blot, Immunostaining, Blocking Assay

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) RFP-EGFP-LGALS3 is trafficked to lysosomes in iNeurons. ES cells expressing RFP-EGFP-LGALS3 via a PiggyBac vector were converted to iNeurons using inducible NGN2 (see Materials and methods) and imaged for EGFP, RFP, and LAMP1 using α-LAMP1 antibodies. While EGFP signal was diffusely localized in the soma, RFP-positive puncta colocalized with lysosomes based on colocalization with LAMP1 staining, indicating that a subset of the RFP-EGFP-LGALS3 protein is trafficked to the lysosome under basal conditions. Scale bar = 20 μm. iN soma zoom-in panels, 30 μm × 40 μm. ( B ) iNeurons expressing RFP-EGFP-LGALS3 were either left untreated, treated with LLOMe for 1 hr, or treated with LLOMe for 1 hr followed by a 12 hr washout. Cells were imaged for EGFP and RFP and the number of EGFP puncta per cell quantified. Loss of EGFP puncta during the washout period is indicative of lysophagic flux. Scale bar = 20 μm. ( C ) Quantification of EGFP puncta per cell after washout from experiments in panel B. The average EGFP puncta per cell was 0.289 at 0 hr (45 cells), 6.46 at 1 hr LLOMe (55 cells) and 0.652 at 12 hr washout after LLOMe (66 cells). ****p < 0.0001. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. ( D ) iNeurons were subjected to LLOMe treatment and washout as in panel B but treated with or without TBK1i, VPS34i, or BafA during the washout period. Cells were imaged for EGFP and RFP. Scale bar = 20 μm. iN soma zoom-in panels, 30 μm × 40 μm. ( E ) Quantification of EGFP puncta per cell from the experiment in panel D. The average EGFP puncta per cell at 12 hr washout was 0.65 with no inhibitor (66 cells), 5.14 with BafA (49 cells), 11.95 with VPS34i (44 cells), and 5.84 with TBKi (63 cells). ****p < 0.0001, ***p < 0.001. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. ( F ) WT, TAX1BP1 −/− , or TAX1BP1 −/− ; OPTN −/− iNeurons were subjected to LLOMe treatment and washout as in panel B. Cells were imaged for EGFP and RFP. Scale bar = 10 μm. iN soma zoom-in panels, 20 μm × 20 μm. ( G ) Quantification of EGFP puncta per cell from the experiment in panel F. The average EGFP puncta per cell at 12hr washout after LLOMe for wild-type cells was 0.474 (38 cells), for TAX1BP1 −/− cells was 4.36 (62 cells) and for TAX1BP1 −/− ; OPTN −/− cells was 4.03 (39 cells). ****p < 0.0001, **p< 0.01. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. Figure 7—source data 1. The number of GFP puncta per cell for . Figure 7—source data 2. The number of GFP puncta per cell for . Figure 7—source data 3. The number of GFP puncta per cell for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Expressing, Plasmid Preparation, Staining

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) iNeurons stably expressing RFP-EGFP-LGALS3 R186S were either left untreated, treated with LLOMe for 1 hr, or treated with LLOMe for 1 hr followed by a 12hr washout. Cells were imaged for EGFP and RFP. Scale bar = 10 μm. Zoom-in panels, 10 μm × 10 μm. ( B ) Quantification of GFP puncta per cell after washout from experiments in panel A demonstrates the absence of GFP-positive puncta in response to lysosomal damage. + marks the mean and the line is at the median. The plot represents data from one replicate. ( C ) RFP-positive puncta in cells expressing RFP-EGFP-LGALS3 WT or the R186S mutant demonstrates comparable number of puncta. + marks the mean and the line is at the median. The plot represents data from one replicate. ( D ) TAX1BP1 lesion read by Illumina Miseq analysis. ( E ) Extracts from WT or TAX1BP1 −/− ES cells were immunoblotted with the indicated antibodies to demonstrate deletion of TAX1BP1. ( F ) OPTN lesion read by Illumina Miseq analysis. ( G ) Extracts from WT, TAX1BP1 −/− , or TAX1BP1 −/− ; OPTN −/− ES cells were immunoblotted with the indicated antibodies to demonstrate deletion of OPTN and TAX1BP1 . Figure 7—figure supplement 1—source data 1. The number of EGFP puncta per cell for . Figure 7—figure supplement 1—source data 2. Uncropped blots for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Stable Transfection, Expressing, Mutagenesis

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Domain structure of TAX1BP1 showing the location of mutations examined in this study. ( B ) Domain structure of OPTN showing the location of mutations examined in this study. ( C ) HeLa TKO cells expressing Keima-LGALS3 were infected with lentiviruses expressing GFP-tagged WT or mutant TAX1BP1 proteins to obtain stable expression. Cells in biological triplicate were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample within each genotype. The plot represents mean and standard deviation from three biological replicates. ****p < 0.0001 ( D ) Immunoblot of cell extracts from panel C probed with α-TAX1BP1 or α-actin as a loading control. Note that some mutants are highly stabilized, as reported previously . The EGFP-TAX1BP1 CC2Δ mutant is not detected by western blot due to the loss of the epitope-binding site of the antibody, nevertheless is detected by FACS . ( E ) HeLa TKO cells expressing Keima-LGALS3 were infected with lentiviruses expressing GFP-tagged WT or mutant OPTN proteins to obtain stable expression. Cells in biological triplicate were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. The plot represents mean and standard deviation from three biological replicates. ****p < 0.0001. ( F ) Immunoblot of cell extracts from panel E probed with α-GFP or α-actin as a loading control. ( G ) HeLa TKO cells were infected with lentiviruses expressing EGFP-tagged WT or mutant TAX1BP1 proteins to obtain stable expression. Cells were either left untreated or treated for 1 hr with LLOMe followed by washout. Cells were harvested at the indicated times and subjected to immunoblotting with the indicated antibodies. ( H ) HeLa TKO cells were infected with lentiviruses expressing EGFP-tagged WT or mutant OPTN proteins to obtain stable expression. Cells were either left untreated or treated for 1 hr with LLOMe followed by washout (12 hr). Cells were harvested at the indicated times and subjected to immunoblotting with the indicated antibodies. ( I ) Model figure. Lysosomal rupture leads to the parallel recruitment of galectins and unleashes a wave of ubiquitination on the lysosome (Steps 1a and b). In step 2, ubiquitination promotes the recruitment of both OPTN-TBK1 and TAX1BP1-TBK1-RB1CC1 complexes to the damage lysosome, thereby promoting de novo phagophore formation and local TBK1 activation to drive efficient lysophagy (Steps 3–5). Figure 8—source data 1. 561/488 Keima ratios for . Figure 8—source data 2. 561/488 Keima ratios for . Figure 8—source data 3. Uncropped blots for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Expressing, Infection, Mutagenesis, Flow Cytometry, Standard Deviation, Western Blot, Control, Binding Assay, Ubiquitin Proteomics, Activation Assay

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet:

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Immunofluorescence, Magnetic Beads, Peptide Fractionation, Protease Inhibitor, Recombinant, Staining, Plasmid Preparation, Software

Journal: Immunology

Article Title: Galectin‐3 is an amplifier of the interleukin‐1 β ‐mediated inflammatory response in corneal keratinocytes

doi: 10.1111/imm.12899

Figure Lengend Snippet: Exogenous galectin‐3 binds to and is internalized by corneal keratinocytes. (a) A SNAP‐galectin‐3 chimera, SNAP‐rhGal3, was produced by fusing the coding sequence of the SNAP‐tag (181 amino acids) to the N terminus of human galectin‐3 (amino acids 183–432). Increasing amounts of the protein were visualized on a polyacrylamide gel by Coomassie blue staining and fluorescence imaging. CRD, carbohydrate recognition domain; PGR, proline, glycine and tyrosine‐rich domain. (b) Corneal keratinocytes were treated with varying doses of SNAP‐rhGal3, pre‐incubated for 15 min with or without 0·1 m lactose, a competitive inhibitor of galectin‐3, for 6 hr at 37°. The presence of exogenous and endogenous galectin‐3 in cell lysates was determined by immunoblot. Exogenous galectin‐3 was visualized by fluorescence imaging. (c) Corneal keratinocytes were treated with or without 100 μg/ml SNAP‐rhGal3 for 6 hr at 37°. Thereafter, cells were permeabilized with digitonin for 10 min and the amount of galectin‐3 or caveolin‐1 leaked into the supernatant (cytosol), or remaining in the cells (homogenate), was analysed by immunoblot. NT, non‐treated. (d) Corneal keratinocytes were incubated with 100 μg/ml of SNAP‐rhGal3 for 0, 2 or 6 hr. Fluorescence microscopy images show surface localization (red) and internalization (green) of SNAP‐rhGal3. Nuclei were counterstained using DAPI (blue). Scale bar, 50 μm. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Article Snippet: Antibodies to human galectin‐3 (1 : 2000; sc20157), caveolin‐1 (1 : 2000; sc894) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; 1 : 2000; sc25778) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Produced, Sequencing, Staining, Fluorescence, Imaging, Incubation, Western Blot, Microscopy

Journal: Immunology

Article Title: Galectin‐3 is an amplifier of the interleukin‐1 β ‐mediated inflammatory response in corneal keratinocytes

doi: 10.1111/imm.12899

Figure Lengend Snippet: Exogenous galectin‐3 amplifies the response to interleukin‐1β (IL‐1β). (a) Immunofluorescence microscopy demonstrating the presence of IL‐1 receptor 1 (IL1R1) (green) in healthy human corneal epithelial tissue. Nuclei were counterstained using DAPI (blue). Scale bar, 100 μm. (b and c) Stratified cultures of human corneal keratinocytes were incubated with IL‐1β (10 ng/ml) or recombinant human galectin‐3 (100 μg/ml) alone or in combination for 6 and 24 hr at 37°. Levels of IL‐8 and IL‐6 in the cell culture media, and IL1R1 in cell lysates, were determined by immunoblot. NT, non‐treated. Results in (b) and (c) represent six independent experiments. The box and whisker plots show the 25th and 75th centiles (box), the median and the minimum and maximum data values (whiskers). Significance was determined using repeated measures one‐way analysis of variance with Dunnett's post hoc multiple comparison test. *P < 0·05, **P < 0·01; ns, non‐significant. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Article Snippet: Antibodies to human galectin‐3 (1 : 2000; sc20157), caveolin‐1 (1 : 2000; sc894) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; 1 : 2000; sc25778) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Immunofluorescence, Microscopy, Incubation, Recombinant, Cell Culture, Western Blot, Whisker Assay

Journal: Immunology

Article Title: Galectin‐3 is an amplifier of the interleukin‐1 β ‐mediated inflammatory response in corneal keratinocytes

doi: 10.1111/imm.12899

Figure Lengend Snippet: The galectin‐3 pro‐inflammatory activity is established through the N and C termini. (a) A truncated form of galectin‐3 lacking the first 62 amino acids in the N‐terminal domain (rhG3C) was obtained by site‐directed mutagenesis as previously described.15 Stratified cultures of human corneal keratinocytes were incubated with interleukin‐1β (IL‐1β) (10 ng/ml) alone or in combination with 100 μg/ml exogenous full‐length galectin‐3 (rhG3) or rhG3C for 24 hr at 37°. Levels of IL‐8 in the cell‐culture media were determined by immunoblot. CRD, carbohydrate recognition domain; PGR, proline, glycine and tyrosine‐rich domain. NT, not treated. (b) The 1H‐NMR and 13C‐NMR spectra of the galectin inhibitor (GI) are described in the Supplementary material (Figure S1). Galectin affinities were determined in a competitive fluorescence anisotropy assay. Stratified cultures of human corneal keratinocytes were incubated with 1 μm GI alone or in combination with IL‐1β and rhG3 for 24 hr at 37°. Levels of IL‐8 in the cell‐culture media were determined by immunoblot. aN, N‐terminal domain; bC, C‐terminal domain. (c) By immunoblot, addition of exogenous full‐length galectin‐3 had no effect on the levels of IL‐1β in the cell culture media. (d) Cell extracts were collected from stratified keratinocyte cell cultures and subjected to galectin‐3 affinity chromatography. The column was eluted with a non‐competing disaccharide, sucrose, before elution with the competing sugar lactose. FT, flow through. Results in (a) represent three independent experiments performed at least in duplicate, whereas results in (b) represent two independent experiments performed in triplicate. Dissociation constants (K d) in (b) were determined in at least two independent experiments each including at least four data‐points. The box and whisker plots show the 25th and 75th centiles (box), the median and the minimum and maximum data values (whiskers). Results in (c) represent seven independent experiments and data are reported as mean ± standard deviation. Significance was determined using ordinary one‐way analysis of variance with Tukey's post hoc multiple comparison test (a and b), and paired t test (c). *P < 0·05, ****P < 0·0001; ns, non‐significant.

Article Snippet: Antibodies to human galectin‐3 (1 : 2000; sc20157), caveolin‐1 (1 : 2000; sc894) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; 1 : 2000; sc25778) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Activity Assay, Mutagenesis, Incubation, Cell Culture, Western Blot, Fluorescence, Affinity Chromatography, Whisker Assay, Standard Deviation

Journal: Immunology

Article Title: Galectin‐3 is an amplifier of the interleukin‐1 β ‐mediated inflammatory response in corneal keratinocytes

doi: 10.1111/imm.12899

Figure Lengend Snippet: Suppression of endogenous galectin‐3 impairs the interleukin‐1β (IL‐1β) response in a p38 mitogen‐activated protein kinase (MAPK) ‐independent manner. (a) Stratified human corneal keratinocytes were transfected with galectin‐3 small interfering RNA (siRNA) (Gal3KD) or control siRNA (Scr) using Lipofectamine 2000. The cultures were then incubated with IL‐1β (10 ng/ml) for 0, 1, 2 and 6 hr at 37°. Levels of IL‐8 and IL‐6 in the cell culture media, and galectin‐3 in cell lysates, were determined by immunoblot. (b) Real‐time quantitative PCR analysis of IL‐8 and IL‐6 gene expression in stratified human corneal keratinocytes transfected with galectin‐3 or control siRNA. The cultures were incubated with IL‐1β (10 ng/ml) for 0, 1, 2 and 6 hr at 37°. (c) Cells were treated with IL‐1β (10 ng/ml) for 0, 5, 15 and 30 min at 37°. Phosphorylated p38 MAPK (pp38) and total p38 MAPK (p38) were measured by immunoblot. (d) Cells transfected with galectin‐3 or control siRNA were incubated with or without 10 ng/ml IL‐1β for 6 hr at 37°. Levels of IL1R1 in cell lysates were determined by immunoblot. Cell surface labelling of IL1R1 and galectin‐3 was carried out using biotinylation as described in Materials and methods. Results in (a) and (b) represent four independent experiments and data are reported as mean ± standard deviation. Significance was determined using two‐way analysis of variance with Sidak's post hoc multiple comparison test. ****P < 0·0001; ns, non‐significant.

Article Snippet: Antibodies to human galectin‐3 (1 : 2000; sc20157), caveolin‐1 (1 : 2000; sc894) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; 1 : 2000; sc25778) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Transfection, Small Interfering RNA, Incubation, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

Journal: PLOS Neglected Tropical Diseases

Article Title: Distinct features of the host-parasite interactions between nonadherent and adherent Trichomonas vaginalis isolates

doi: 10.1371/journal.pntd.0011016

Figure Lengend Snippet: A and B. Surface expression or secretion of galectins. CFSE-labeled TH17 trophozoites (green) were co-incubated with h VECs (moi 1:3) were sampled at intervals for the detection of galectin-1 (red) and galectin-3 (cyan) by IFA (A), while these galectins in the conditioned medium were detected by ELISA (B). PI, post-infection. Adherent trophozoites in A are indicated by white triangles. C, D, and E. T1 or TH17 trophozoites were incubated in fresh or conditioned KSFM (D) from h VECs cultures for 30 min. Cells were fixed for detection of galectin-1 or -3 by IFA without permeation for (C) or with permeation for (D and E) before immune detection. E. IFA co-staining with anti-galectin-3 antibody and LysoTracker. F and G. T1 or TH17 trophozoites were incubated with FITC-conjugated recombinant galectin-1 or galectin-3 and analyzed by flow cytometry (F) or fluorescence microscopy (G). The bar graph is shown by mean ± SD. All assays were repeated three times and the representative data are shown here. Differences were statistically analyzed by Student’s t-test, with P <0.01(**) and P <0.05(*).

Article Snippet: In brief, standards and serial-diluted supernatants recovered from human cultures challenged with T . vaginalis were incubated in 96-well plates precoated with 50 μl of goat anti-human galectin-1 (1 μg/ml, R&D Systems AF1152) or rabbit anti-human galectin-3 antibody (1 μg/ml, GeneTex) at room temperature for 1 hr.

Techniques: Expressing, Labeling, Incubation, Enzyme-linked Immunosorbent Assay, Infection, Staining, Recombinant, Flow Cytometry, Fluorescence, Microscopy

Journal: PLOS Neglected Tropical Diseases

Article Title: Distinct features of the host-parasite interactions between nonadherent and adherent Trichomonas vaginalis isolates

doi: 10.1371/journal.pntd.0011016

Figure Lengend Snippet: On infection, T . vaginalis may trigger the surface expression and secretion of galectins from h VECs. The extracellular galectins may initially bind on the parasite surface, then galectin-3 is internalized and enclosed in the lysosomes inside the trophozoites of adherent isolate. The extracellular galectins bound to the parasite might trigger the aggregation of trophozoites on h VECs, as this is diminished in the presence of lactose. In the adherent isolate, the predominant flagellate-amoeboid transition, as well as cytoadherence, were simultaneously suppressed by CytD, speculating that actin cytoskeleton-based behavior may mediate cytoadherence. When axostyle microtubule assembly was disturbed by TPI, the axostyle anchoring was abolished with the reduced cytoadherence, suggesting that axostyle anchoring might be an early event required for cytoadherence. For T . vaginalis , it is speculated that the cytoskeleton and cell surface adhesion molecules may coordinate their cytoadherence, so these factors should be considered in the study of host-parasite interactions and provide new insights into the host colonization of T . vaginalis .

Article Snippet: In brief, standards and serial-diluted supernatants recovered from human cultures challenged with T . vaginalis were incubated in 96-well plates precoated with 50 μl of goat anti-human galectin-1 (1 μg/ml, R&D Systems AF1152) or rabbit anti-human galectin-3 antibody (1 μg/ml, GeneTex) at room temperature for 1 hr.

Techniques: Infection, Expressing

Journal: PLoS Pathogens

Article Title: A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica

doi: 10.1371/journal.ppat.1004381

Figure Lengend Snippet: (A–D) ELISA quantification in compartments S1, S2 and S3 of the 3D-liver model (Model) or the setup without LSEC (no LSEC), in the absence or the presence of E. histolytica (− or + Eh). Samples used for ELISA assays were prepared as for . (A) Amounts of IL-6 at 3 h, (B) IL-1β, (C) galectin-1 and (D) galectin-3 at 6 h of incubation with fresh serum-free medium containing or not virulent E. histolytica . Graphs with standard deviation and statistical evaluation by One way ANOVA, p<0.01*, <0.001**, <0.0001*** for 3–5 independent experiments. None of the cytokines was detected in the COL-I matrix control without hepatic cells. n/d = not detected. (E, F) Summary of the results from and , for the setup without LSEC (E) and the 3D-liver model (F), showing the complexity of the pro-inflammatory response induced by E. histolytica in the 3D-liver model.

Article Snippet: For quantitative ELISA-like assays, trophozoites were harvested, allowed to adhere to wells of a 96 well plate (6×10 4 /well), incubated for 25 min with recombinant galectin-1, galectin-3 or BSA, gently washed and then incubated with biotinylated anti-galectin-1 or -3 antibodies (R&D systems).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Standard Deviation

Journal: PLoS Pathogens

Article Title: A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica

doi: 10.1371/journal.ppat.1004381

Figure Lengend Snippet: Bacterially expressed purified human galectin-1 and -3, and BSA as a control, were used for the incubations with the amoebae. Galectin-1 (A) or -3 (B) binding to the trophozoite surface, visualized by immunofluorescence with human galectin-specific antibodies. Quantification of galectin-1 (C) or -3 (D) binding to immobilized trophozoites by ELISA-like assays. (E) Trophozoite adhesion to immobilized galectin-1 or -3 and competition with lactose (lac).

Article Snippet: For quantitative ELISA-like assays, trophozoites were harvested, allowed to adhere to wells of a 96 well plate (6×10 4 /well), incubated for 25 min with recombinant galectin-1, galectin-3 or BSA, gently washed and then incubated with biotinylated anti-galectin-1 or -3 antibodies (R&D systems).

Techniques: Purification, Binding Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica

doi: 10.1371/journal.ppat.1004381

Figure Lengend Snippet: Immunofluorescence localization of cell surface-associated (A) galectin-3 on Huh-7 and (C) galectin-1 on LSEC. Amoeba adhesion to (B) Huh-7 or (D) LSEC 2D cultures Control (CTL), corresponding to incubations without proteins or sugars added, was set to 100%. (E) LSEC galectin-1 surface label 72 h after transfection with galectin-1 specific siRNA. (F) Amoeba binding to LSEC transfected with galectin-1 specific or unrelated control siRNA. Incubations and quantification of trophozoite binding as for B and D. Graphs with standard deviation and statistical evaluation by One way ANOVA, p<0.0001*** for 3 independent experiments.

Article Snippet: For quantitative ELISA-like assays, trophozoites were harvested, allowed to adhere to wells of a 96 well plate (6×10 4 /well), incubated for 25 min with recombinant galectin-1, galectin-3 or BSA, gently washed and then incubated with biotinylated anti-galectin-1 or -3 antibodies (R&D systems).

Techniques: Immunofluorescence, Transfection, Binding Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica

doi: 10.1371/journal.ppat.1004381

Figure Lengend Snippet: Cytokine quantification by ELISA assays in fractions S1 (A–C) or S3 (D) after 6 h of incubation of 3D-liver models with bacterially expressed purified human galectin-1 or -3 (1 µg/ml) or E. histolytica . Control (CTL) corresponds to no protein added. Incubations with amoebae were performed in the presence of lactose or in its absence (CTL). Graphs with standard deviation, statistical evaluation by One way ANOVA, p<0.05*, p<0.001**, p<0.0001*** for (A–C) and values significantly different from 0 for galectin-3 p = 0.0068, CTL p = 0,0049 (D), for 3 independent experiments. n/d = not detected.

Article Snippet: For quantitative ELISA-like assays, trophozoites were harvested, allowed to adhere to wells of a 96 well plate (6×10 4 /well), incubated for 25 min with recombinant galectin-1, galectin-3 or BSA, gently washed and then incubated with biotinylated anti-galectin-1 or -3 antibodies (R&D systems).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Purification, Standard Deviation

Journal: PLoS Pathogens

Article Title: A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica

doi: 10.1371/journal.ppat.1004381

Figure Lengend Snippet: Schematic presentation of the E. histolytica invasion mechanisms at the 3D-liver model: the early LSEC adhesion interactions and crossing as well as the induction of the pro-inflammatory cytokine release into the different compartments of the 3D-liver model. The summary highlights the involvement of amoebic Gal/GalNAc lectin in the interactions with the human cells, and the dual role of human galectin-1 and 3. First, surface-associated galectins promotes amoeba adhesion during the early stages, and second, released galectins participate in the induction of the inflammatory response later in the invasion process.

Article Snippet: For quantitative ELISA-like assays, trophozoites were harvested, allowed to adhere to wells of a 96 well plate (6×10 4 /well), incubated for 25 min with recombinant galectin-1, galectin-3 or BSA, gently washed and then incubated with biotinylated anti-galectin-1 or -3 antibodies (R&D systems).

Techniques:

Journal: Glia

Article Title: ApoD, a glia‐derived apolipoprotein, is required for peripheral nerve functional integrity and a timely response to injury

doi: 10.1002/glia.21010

Figure Lengend Snippet: Quantitative RT‐PCR expression profiles of intact nerves of different genotypes and age groups. A : Profile of genes that change their expression in intact 15 weeks nerves of ApoD‐KO and HApoD‐Tg mice. WT intact nerve expression is used as the calibrator for each gene ( n = 6/group). B, C : Immunoblot analysis of Il6 in total protein preparations of 33 weeks old intact WT and ApoD‐KO nerves. Protein levels were quantified by band densitometry normalized to β‐actin signal. Il6 expression shows a 1.4 fold increase in ApoD‐KO nerves with respect to WT. D : Gene profiles in 40‐week‐old intact nerves. Left graph shows the age effect in the profile of WT gene expression (WT 40‐week and 15‐week nerves are compared). Right graph shows the gene profile of 40‐week intact nerves from ApoD‐KO mice using the 40‐week WT intact nerve as calibrator. Gray and black boxes point to genes turned on and off respectively. Underlined genes are ApoD‐dependent in both age groups. n = 8/group. Only statistically supported changes (Mann‐Whitney's U‐test) with Log 2 −ΔΔ Ct ≥ ±0.3 (twofold increase) are shown.

Article Snippet: Immunoblot analysis was carried out with 15 μg of total protein/lane using standard procedures with the following primary antibodies: Goat anti‐human Il6 (R&D), rat anti‐Galectin‐3 (ATCC), and rabbit anti‐GAP43 (Abcam).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, MANN-WHITNEY