Journal: Cell Host & Microbe

Article Title: Complement C4 Prevents Viral Infection through Capsid Inactivation

doi: 10.1016/j.chom.2019.02.016

Figure Lengend Snippet: C1 and C4 Prevent Adenoviral Protein VI Exposure and Endosomal Escape (A) IP of Ad5 with 9C12 after Ad5 was incubated at the indicated temperatures for 30 min. WB: anti-adenovirus. (B) IP after Ad5 and 9C12 were complexed with C1 or C1/C4 and then incubated at 37°C or 49°C for 30 min. WB: anti-adenovirus. (C and D) HeLa TRIM21 KO cells were infected for 30 min in the presence of 9C12 or 9C12+ complement. Error bars depict the mean ± SEM of the indicated number of cells (n) acquired in three independent experiments. Scale bar, 5 μm. (C) Left: Ad5 staining is displayed in green; protein VI staining is depicted in red. Right: quantification of protein VI puncta per cell in the indicated conditions. (D) Left: Ad5 staining is displayed in green; Galectin-3 staining is depicted in red. Right: quantification of Galectin-3 puncta per cell in the indicated conditions. Original western blots are included in Figure S6 .

Article Snippet: Z-stacks with 1 μm thick optical slices were acquired and protein VI and Galectin 3 puncta were quantified using NIS Elements 4.30 (Nikon).

Techniques: Incubation, Infection, Staining, Western Blot

Journal: Autophagy

Article Title: LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons

doi: 10.1080/15548627.2021.1967615

Figure Lengend Snippet: DSP-SNCA is secreted in response to lysosomal acidification inhibitors and exogenously added SNCA fibrils. (A) A cartoon depiction of SNCA linked to complementing halves A and B of the DSP construct. (B) Relative light units (RLUs) were measured from the RLuc signal in the cultured media from the stated cell lines 24 h after treatment with vehicle (0.1% DMSO) or Baf-A1 indicating the levels of complemented DSP-SNCA. (C) RLU signal detected in the cultured media from SH-SY5Y DSP-SNCA cells 24 h after control or SNCA fibril treatment. Statistical significance determined by a paired t -test. Data shows the mean value from at least three independent experiments where each data point indicates a biological replicate. Data are expressed as M ± SE . For all statistical tests *, **, ***, ****, p < 0.05, 0.01, 0.001, and 0.0001, respectively. (D) Z-stacks of untreated or fluorescently labeled SNCA fibril-treated HeLa DSP-SNCA and mCherry galectin-expressing cells. (E) The white arrow point to colocalized DSP-SNCA + and mCherry LGALS3 + events in untreated cells and colocalized DSP-SNCA + , mCherry LGALS3 + , and SNCA fibril + events in the SNCA fibril-treated cells. (F) A representative z-stack of dually transduced DSP-SNCA SH-SY5Y cells from untreated and SNCA fibril-treated cells probed for LGALS3. Cell boundaries are shown as a white border. White arrows point to DSP-SNCA + , LGALS3 + double-positive events in untreated and DSP-SNCA + , LGALS3 + , SNCA fibril + triple-positive events in the SNCA fibril-treated conditions. (F) Extracellular colocalization of DSP-SNCA + and mCherry LGALS3 + events observed in untreated cells.

Article Snippet: Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions.

Techniques: Construct, Cell Culture, Control, Labeling, Expressing

Journal: Autophagy

Article Title: LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons

doi: 10.1080/15548627.2021.1967615

Figure Lengend Snippet: Secreted LGALS3 and DSP-SNCA colocalize with exogenously added SNCA fibrils. (A) A representative z-stack from a constructed z-stack MIP obtained from the cultured media of untreated HeLa DSP-SNCA and those treated with SNCA fibrils. Representative MIPs of z-stack images for cultured media obtained from untreated HeLa DSP-SNCA processed for immunofluorescence without the primary antibody (2° Ab treatment) are also given. The white arrows point to DSP-SNCA + , LGALS3 + double-positive events in untreated and DSP-SNCA + , LGALS3 + , SNCA Fibril + triple-positive events in the SNCA fibril treated conditions. (B) Demonstration of the Spots masking algorithm built around the DSP-SNCA channel from a z-stack MIP showing specificity for the desired channel. (C) Representative colocalization plots of the maximum fluorescence intensities of the LGALS3 (y-axis) and SNCA fibril (x-axis) signal found within each DSP-SNCA masking algorithm Spot pooled from a total of 15 images from a single treatment conditions’ coverslip. Each data point represents a single recognized DSP-SNCA + spot within an image. Quadrant numbers indicate the percent colocalization of DSP-SNCA + where the relative background signal was determined based on the LGALS3 and SNCA fibril maximum intensity signal measured in the 2° antibody control condition. The percent single DSP-SNCA + positive, DSP-SNCA + SNCA fibril + double positive, DSP-SNCA + LGALS3 + double positive, and DSP-SNCA + SNCA fibril + LGALS3 + triple positive are given in the bottom left, bottom right, top left, and top right quadrants, respectively. (D) Summarized average colocalization of DSP-SNCA + masking algorithm Spots from four independent experiments. DSP-SNCA + Spots that are single positive (green), DSP-SNCA + SNCA fibril + double positive (cyan), DSP-SNCA + LGALS3 + double positive (yellow), and DSP-SNCA + SNCA fibril + LGALS3 + triple positive (white). (E) Representative colocalization plots of the maximum fluorescence intensities of the DSP-SNCA (y-axis) and SNCA fibril (x-axis) signal found within each LGALS3 masking algorithm Spot from the same images set of images. Colocalization plots are from a single image. The percent single LGALS3 + positive, LGALS3 + SNCA fibril + double positive, LGALS3 + DSP-SNCA + double positive, and LGALS3 + SNCA fibril + DSP-SNCA + triple positive are given in the bottom left, bottom right, top left, and top right quadrants, respectively. (F) Summarized average colocalization of LGALS3 + masking algorithm Spots from four independent experiments. LGALS3 + Spots that are single positive (red), LGALS3 + SNCA fibril + double positive (magenta), LGALS3 + DSP-SNCA + double positive (yellow), and LGALS3 + DSP-SNCA + SNCA fibril + triple positive (white). Summarized average colocalization of (D) DSP-SNCA or (F) LGALS3 masking algorithm Spots from four independent experiments. The degree of colocalization of DSP-SNCA or LGALS3 with the respective other channels was determined based on the background signal measured in the 2° Ab control. Data are expressed as M ± SE ( n = 4 coverslips, where M was determined from 15 images).

Article Snippet: Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions.

Techniques: Construct, Cell Culture, Immunofluorescence, Fluorescence, Control

Journal: Autophagy

Article Title: LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons

doi: 10.1080/15548627.2021.1967615

Figure Lengend Snippet: LGALS3 inhibition affects the composition of secreted extracellular vesicles from mDA neurons. (A) TEM from concentrated WT mDA culture media following immunogold labeling. Representative image shows SNCA + (5 nm), CD63 + (10 nm), and LGALS3 + (15 nm) triple positive EVs. (B) NTA analysis of EVs from WT mDA neurons following differential ultracentrifugation. EV sizes binned in 1 nm increments with a bin center of 0.5 nm. NTA of resuspended mDA EVs were performed in technical quintuplicates, denoted as different shades of green, blue, or red for 3 independent samples. Each sample was from distinct mDA differentiation. (C) Representative z-stack from a constructed z-stack MIP obtained from concentrated EVs from mDA neuron cultured media. Concentrated EVs were stained for LGALS3, SNCA, and combined pool of mouse anti-TSPAN/tetraspanin antibodies against CD9, CD63, and CD81. Processed immunofluorescence with only the LGALS3 primary antibody and the secondary antibodies against all three channels (2° antibody control) was also performed to determine the background level of fluorescence. (D) Summarized average colocalization of LGALS3 + masking algorithm Spots from three independent experiments. LGALS3 + Spots that are single positive (green), LGALS3 + SNCA + double positive (yellow), LGALS3 + TSPAN + double positive (cyan), and LGALS3 + SNCA + TSPAN + triple positive (white). (E) Representative non-reducing SDS-PAGE of concentrated EVs and the corresponding lysates from mDA neurons treated with vehicle, TD139, SNCA fibrils, or TD139 + SNCA fibrils. (F) Relative SDS-PAGE band intensities of (E) in the EVs normalized to cell lysate GAPDH intensity. Data are expressed as M ± SE . Intensity data (C-D) ( n = 3 independent experiments, 15 images per experiment, pooled colocalization data from each image among experiments). E, pooled cultured media from n = 3. F, n = 3 derived from the same samples as D.

Article Snippet: Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions.

Techniques: Inhibition, Labeling, Construct, Cell Culture, Staining, Immunofluorescence, Control, Fluorescence, SDS Page, Derivative Assay

Journal: Autophagy

Article Title: LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons

doi: 10.1080/15548627.2021.1967615

Figure Lengend Snippet: Lysosomal damage as a consequence of SNCA fibril or LLOME treatment elicits an autophagic response in mDA neurons. (A, D) Representative western blot of LAMP1, p-S757 ULK1, t-ULK1, p-T389 RPS6KB1, t-RPS6KB1, LGALS3, LC3-I, LC3-II, and GAPDH for mDA neurons treated with vehicle (0.1% DMSO), rapamycin, LLOME, or SNCA fibrils. (B, C) The quantification of p-T389 RPS6KB1, t-RPS6KB1, p-S757 ULK1, and t-ULK1. (E, F) Quantification of LAMP1, LGALS3, and LC3-II for LLOME (E) and SNCA fibrils treatment (F). The different symbols correspond to paired replicates (B, C). Cells were treated with SNCA fibrils, rapamycin, or vehicle for 24 h; cells were treated with LLOME for 4 h. (G, H) Secreted SNCA concentrations present in unlysed culture media following treatment with the early autophagy inhibitors Wor or 3-MA (G), the late-autophagy inhibitor Baf-A1 (H) or vehicle (0.1% DMSO) for 24 h as measured by SNCA sandwich ELISA. (I) A representative western blot demonstrating KD of ATG7 from mDA neurons and SNCA from unlysed, cultured media by ELISA. Cultured media was collected from 48 to 72 h post-transfection. Data are expressed as M ± SE (B, n = 4; E-F, n = 4; G, n = 5; H, n = 6; I, n = 5). Statistical significance was determined following natural log transformation and one-way ANOVA with Tukey’s post hoc tests. (G-I) Statistical significance was determined by paired two-tailed t -tests. For all statistical tests *, **, ***, ****, p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Article Snippet: Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions.

Techniques: Western Blot, Sandwich ELISA, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Transformation Assay, Two Tailed Test

Journal: Autophagy

Article Title: LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons

doi: 10.1080/15548627.2021.1967615

Figure Lengend Snippet: Neuronal LGALS3 influences SNCA secretion and is reciprocally secreted during SNCA fibrils treatment. (A) Relative fold difference in SNCA from mDA cultured media treated with TD139 (5 μM) or vehicle (0.1% DMSO) treated control after 24 h measured by ELISA. (B) Mean fold difference in SNCA in human mDA neuron culture media 48–72 h post siRNA transfection, relative depletion of LGALS3, and representative KD. (B) Values were determined by ELISA and normalized to the mean of the control group (i.e. mean control). (C) A plot of the normalized to mean control data in (A) combined with (Fig. S4(A)) to illustrate the linear relationship between relative LGALS3 depletion (x-axis) and mean fold SNCA secretion (y-axis). (D) RLUs of RLuc activity of cultured media from CRISPR-Cas9-mediated KO of LGALS3 or control in SH-SY5Y DSP-SNCA cell-lines over a 24-h period, one week after selection, and demonstration of KO. (E) RLUs of RLuc activity from control or LGALS3 CRISPR-Cas9 mediated KO SH-SY5Y DSP-SNCA cell lysates after 24 h ( n = 3). (F) Level of SNCA in the cultured media of mDA neuronal cultures at 48 h post-transduction measured by ELISA. (G) SNCA concentration present in empty vector control or FLuc LGALS3 transduced mDA neurons cell lysates after 96 h measured by ELISA. (H) RLU levels detected from cultured mDA neurons transduced with FLuc LGALS3 and either untreated or treated with SNCA fibrils at 48 h post-transduction. (I) Fold difference in LGALS3 present in the cultured media from the untreated control or SNCA fibrils treated mDA neurons after 24 h. (J, K) Lysosomal dysfunction assay shows the normalized RFUs from mDA neuronal cultures loaded with Magic Red CTSB dye. Data from J and K are from the same experiments; the data is displayed in 2 graphs to increase clarity. RFU normalization was calculated from the endpoint of the control siRNA plus vehicle (0.1% DMSO) condition. (L) Quantification of the area under the curve for each stated condition. The vehicle minus Baf-A1 was used to determine lysosomal activity. All data are expressed as M ± SE (A, n = 6; B, n = 3; C, n = 11; D and E, n = 6; F, n = 11; G, n = 4; H, n = 5; I, n = 6; J-L; pooled data from n = 4 independent experiments with 1–3 conditions per experiment). Statistical significance was determined by evaluating Pearson’s r (C), paired t -test (A, D, E, F, G, and H), paired one-way ANOVA with Dunnett’s post hoc tests (L), or ratio paired t -test (B and I) to better account for the distribution of values < 1. For all statistical tests *, **, ***, ****, p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Article Snippet: Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions.

Techniques: Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Transfection, Activity Assay, CRISPR, Selection, Transduction, Concentration Assay, Plasmid Preparation

Journal: Autophagy

Article Title: LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons

doi: 10.1080/15548627.2021.1967615

Figure Lengend Snippet: Neuronal Depletion of TRIM16 or ATG16L1 LGALS3 decreases SNCA and LGALS3 secretion. (A) Relative fold difference in SNCA (left) and LGALS3 (middle) in the cultured media of mDA neuronal cultures at 24 to 48 h post-transfection, relative depletion of TRIM16 (right), and representative KD (inset). Values were determined by ELISA and normalized to the mean of the control group (i.e. mean control). The different colors represent matched replicates. (B) A plot of the data shown in (A) illustrating the linear relationship between relative TRIM16 depletion (x-axis) and mean fold SNCA secretion (B, left, y-axis) or LGALS3 secretion (B, right, y-axis). (C) Relative fold difference in SNCA (left) and LGALS3 (middle) in the cultured media of mDA neuronal cultures at 48 to 72 h post-transfection, relative depletion of ATG16L1 (right), and representative KD (inset). (D) A plot of the data shown in (C) illustrating the linear relationship between relative ATG16L1 depletion (x-axis) and mean fold SNCA secretion (D, left, y-axis) or LGALS3 secretion (D, right, y-axis). (E) A representative z-stack from multiple z-stack MIPs of imaged mDA neurons’ cultured media. Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions. Each data point is the mean of 20 images for each independent experiments. There were 4 independent experiments. (I-K) Relative fold difference in SNCA (I, left) and LGALS3 (I, right) in the cultured media of mDA cultures 4 h after vehicle (0.1% DMSO) or LLOME treatment measured by ELISA and normalized to the mean vehicle concentration. (J, K) Relative fold difference in SNCA from cultured media from either control and LGALS3 (J) or control and TRIM16 (K) siRNA transfected mDA neurons followed by 4 h of vehicle or LLOME treatment. Vehicle or LLOME treatment was performed 72 h post control or LGALS3 siRNA transfection, or 48 h post control or TRIM16 siRNA transfection. Representative mDA neuronal lysate western blots demonstrating successful knockdown of LGALS3 (J) or TRIM16 (K). A and C data are expressed as M ± SE , B and D data are expressed as M ± SD (A and B, n = 6 (left), n = 5 (middle), n = 6 (right); C and D, n = 8 (left), n = 7 (middle), control siRNA n = 6, ATG16L1 siRNA n = 8 (right), I, n = 4 (left), n = 6 (right), J and K, n = 5–6. Statistical significance was determined by evaluating Pearson’s r (B and D), ratio paired t -test (A, C, and I), a two-way ANOVA, with Sidak’s post hoc tests (G-H), or a one-way ANOVA with Dunnett’s post hoc tests (J-K) following natural log transformation to better account for the distribution of values < 1. (G-H) * indicates significance relative to control plus vehicle, # indicates significance relative to control plus Baf-A1. For all statistical tests *, **, ***, ****, or #, ##, ###, ####, p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Article Snippet: Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions.

Techniques: Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Concentration Assay, Knockdown, Transformation Assay

Journal: Autophagy

Article Title: LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons

doi: 10.1080/15548627.2021.1967615

Figure Lengend Snippet: SNCA fibril treatment increases the colocalization of TRIM16 and ATG16L1 with mCherry LGALS3 and SNCA fibrils. (A-C) Representative images from mCherry LGALS3 SH-SY5Y cells treated with SNCA fibrils and stained for TRIM16 and LC3B (B), or ATTO 647 labeled SNCA Fibrils and stained for TRIM16 (A) or ATG16L1 (C). The white arrows point to instances of triple colocalization. (D-F) an Imaris masking algorithm built around TRIM16 (A-B, D, and F) or ATG16L1 puncta (C, E). Each data point represents the averaged maximum intensity from the total masked puncta in an individual image. The same algorithm was applied to all experiments and treatment conditions. Intensity data are expressed as M ± SE ( n = 3 independent experiments, 7–10 images per experiment). Statistical significance was determined by unpaired t -tests. For all statistical tests *, **, ***, ****, p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Article Snippet: Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions.

Techniques: Staining, Labeling

Journal: Autophagy

Article Title: LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons

doi: 10.1080/15548627.2021.1967615

Figure Lengend Snippet: Lysosomal rupture increases the formation of amphisomes DSP-SNCA SH-SY5Y cells and the depletion of LGALS3 or TRIM16 reduces the recruitment of SNCA fibrils to autophagosomes in mDA neurons. (A) Representative images from DSP-SNCA SH-SY5Y cells treated vehicle or LLOME for 4 h or SNCA fibrils for 24 h and subsequently stained for LC3B and CD63. The same end point was used for all conditions. The white arrows point to instances of triple labeled events. (B) Quantification of the number of LC3B puncta recognized by a masking algorithm among images. (C, D) Quantification of DSP-SNCA (C) or CD63 (D) intensity in masked LC3B puncta. Each data points represents the averaged maximum intensity from the total masked puncta in an individual image. The same algorithm was applied to all experiments and treatment conditions. (E) Representative images of induced YFP-LC3 (green) transduced mDA neuronal cultures treated with SNCA fibrils (blue) and stained for endogenous LGALS3 (red) shown at the same intensities for each of the channels for each condition. YFP-LC3 induction was initiated 24 h post-transfection. SNCA fibrils treatment was done 48 h post-transfection. Staining was conducted 72 h post-transfection. The white arrows point to triple colocalization of YFP-LC3, LGALS3, and SNCA fibrils. (F) Quantification of the number of YFP-LC3 puncta recognized by a masking algorithm among images for the indicated siRNA pretreated condition. (G, H) Quantification of the degree of masked YFP-LC3 puncta colocalized with SNCA fibrils puncta (G) and LGALS3 puncta (H). Each data point is the mean of 3 randomly selected coverslips ( n = 15–20 images per coverslip). (A-D) Intensity data are expressed as M ± SE ( n = 3 independent experiments, 7–10 images per experiment). Statistical significance was determined by one-way ANOVA with Dunnett’s post hoc tests (A-D) or with Tukey’s post hoc tests (E-H). For all statistical tests *, **, ***, ****, p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Article Snippet: Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions.

Techniques: Staining, Labeling, Transfection

Journal: Autophagy

Article Title: LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons

doi: 10.1080/15548627.2021.1967615

Figure Lengend Snippet: A summary diagram of the hypothesized cellular mechanism. This diagram illustrates a hypothetical mechanism where endocytosed pathological SNCA enters the cell and induces lysosomal rupture. Lysosomal rupture results in MTOR inactivation subsequently increasing ULK1 and RPS6KB1 activity. Concurrently, lysosomal rupture exposures its inner-luminal glycans, which are recognized by LGALS3. Bound LGALS3 recruits TRIM16 and ULK1, and subsequently ATG16L1 to mediate further autophagic machinery assembly, increase cellular autophagy, and promote autophagosome development. This response, ultimately, increases SNCA secretion. (Bottom) LGALS3 recruitment to lysosomes during early membrane stress facilitate membrane repair.

Article Snippet: Cells were first transfected with siRNAs followed by either vehicle (0.1% DMSO) or Baf-A1 treatment 48 h post-transfection for 24 h. (F) A representative western blot that demonstrates successful depletion of LGALS3, TRIM16, or ATG7. (G, H) the Relative number of SNCA puncta (G) or LGALS3 puncta (H) from each image based on the same Imaris masking algorithm as a measure of relative differences between the conditions.

Techniques: Activity Assay, Membrane

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: LAMP3 induces lysosome-dependent cell death by impairing autophagic caspase-8 degradation in Sjögren’s salivary glands

doi: 10.1002/art.42540

Figure Lengend Snippet: ( A ) Representative immunofluorescent images for V-ATPase (green) and galectin-3 (red) staining on labial minor salivary gland sections from SjD patients ( n = 10) or control non-SjD sicca subjects ( n = 5). Original magnification: 40x (inset: 100x). ( B ) Bar chart showing mean (± SD) percentage of galctin-3 puncta-positive cells. ** P < 0.01, t -test.

Article Snippet: Results: Galectin-3 puncta formation was more frequent in SjD patients’ salivary glands compared to control glands.

Techniques: Staining, Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: LAMP3 induces lysosome-dependent cell death by impairing autophagic caspase-8 degradation in Sjögren’s salivary glands

doi: 10.1002/art.42540

Figure Lengend Snippet: ( A , B ) Labial minor salivary gland sections from SjD patients ( n = 80) were stained for ( A ) V-ATPase (green) and galectin-3 (red) (original magnification: 40x) or for ( B ) LAMP3 (green) (original magnification: 20x). Representative immunofluorescent image in each LMP and LAMP3 expression grade are shown. ( C ) Matrix shows the number of patients having indicated combination of LMP and LAMP3 expression grades. ( D ) Bar charts show the number of patients having indicated LMP grade based on positivity of serum anti-SSA/Ro antibodies or salivary gland histology. FLS, focal lymphocytic sialadenitis; NSCS, non-specific chronic sialadenitis; SCS, sclerosing chronic sialadenitis; F/S, combination of FLS and SCS features.

Article Snippet: Results: Galectin-3 puncta formation was more frequent in SjD patients’ salivary glands compared to control glands.

Techniques: Expressing, Staining

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: LAMP3 induces lysosome-dependent cell death by impairing autophagic caspase-8 degradation in Sjögren’s salivary glands

doi: 10.1002/art.42540

Figure Lengend Snippet: ( A ) Western blots showing caspase-8 and α-tubulin (internal control) expressions in control and LAMP3-overexpressing A253 cells. ( B , C ) A253 cells were transfected with CASP8 -GFP and/or empty-GFP expression plasmids. ( B ) Flow cytometry using APC-annexin V and 7-AAD 48 hours after transfection, and bar chart showing the difference in the rate of annexin V-positive cells from that in control ( n = 5). ( C ) Immunofluorescent images for GFP (green) or galectin-3 (magenta) staining, and bar chart showing percentage of galectin-3 puncta-positive cells ( n = 5). ( D-F ) LAMP3-overexpressing A253 cells were treated with negative control or two types of CASP8 siRNA for 72 hours. ( D ) Western blots showing caspase-8 and α-tubulin expressions. ( E ) Flow cytometry using APC-annexin V and 7-AAD, and bar chart showing the difference in the rate of annexin V-positive cells from that in control A253 cells ( n = 3). ( F ) Immunofluorescent images for galectin-3 (magenta) staining, and bar chart showing percentage of galectin-3 puncta-positive cells ( n = 3). Original magnification: 40x. Values shown are mean ± SD. * p < 0.05, ** p < 0.01, t -test with Dunnett’s correction.

Article Snippet: Results: Galectin-3 puncta formation was more frequent in SjD patients’ salivary glands compared to control glands.

Techniques: Expressing, Western Blot, Control, Transfection, Flow Cytometry, Staining, Negative Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: LAMP3 induces lysosome-dependent cell death by impairing autophagic caspase-8 degradation in Sjögren’s salivary glands

doi: 10.1002/art.42540

Figure Lengend Snippet: Control or LAMP3-overexpressing A253 cells were treated with liraglutide, an inducer of lysosomal biogenesis, at indicated concentration or vehicle control for 18 hours. ( A ) Flow cytometry using Lysotracker Deep Red (Black line: without Lysotracker; Red line: with Lysotracker) and bar chart showing mean fluorescent intensity. ( B ) Western blots showing caspase-8 and α-tubulin (internal control) expressions and bar chart showing relative change of caspase-8 expression normalized by α-tubulin expression. ( C , D ) Control or LAMP3-overexpressing A253 cells were treated with liraglutide at 60 nM or vehicle control for 18 hours. ( C ) Immunofluorescent images for galectin-3 (magenta) staining and bar chart showing percentage of galectin-3 puncta-positive cells. Original magnification: 40x. ( D ) Flow cytometry using APC-annexin V and 7-AAD and bar chart showing the difference in the rate of annexin V-positive cells from that in control cells. Values shown are mean ± SD ( n = 4 for all experiments). * p < 0.05, ** p < 0.01, t -test with Dunnett’s correction.

Article Snippet: Results: Galectin-3 puncta formation was more frequent in SjD patients’ salivary glands compared to control glands.

Techniques: Expressing, In Vitro, Control, Concentration Assay, Flow Cytometry, Western Blot, Staining

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: LAMP3 induces lysosome-dependent cell death by impairing autophagic caspase-8 degradation in Sjögren’s salivary glands

doi: 10.1002/art.42540

Figure Lengend Snippet: LAMP3-overexpressing mice received weekly subcutaneous injections of dulaglutide, an inducer of lysosomal biogenesis, at 0.6 mg/kg or placebo for 3 months after developing an SjD-like phenotype. ( A ) Immunofluorescent images of murine submandibular glands for galectin-3 (magenta) staining and bar chart showing the percentage of galectin-3 puncta-positive cells. ( B ) Immunofluorescent images of submandibular glands with terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL, red) and bar chart showing the percentage of TUNEL-positive apoptotic cells. ( C ) Hematoxylin and eosin staining of glands and the percentage of lymphocytic infiltration area in whole glands. ( D ) Pilocarpine-stimulated salivary flow per body weight in 20 minutes before and after treatment. ( E ) Serum anti-Ro/SS-A antibody levels. Original magnification: 40x (inset: 100x) for ( A ) and ( B ), and 4x for ( C ). Values shown are median and quartile ( n = 9, each group). * p < 0.05, ** p < 0.01, Wilcoxon test.

Article Snippet: Results: Galectin-3 puncta formation was more frequent in SjD patients’ salivary glands compared to control glands.

Techniques: Staining, TUNEL Assay