Journal: bioRxiv

Article Title: A novel subtype of reactive astrocytes critical for HIV associated pain pathogenesis

doi: 10.1101/2022.08.03.502665

Figure Lengend Snippet: a . An overview of cell clusters. Major spinal cell types revealed by integrated clustering analysis (by UMAP) of the snRNA-seq datasets from Wt and gp120Tg lumbar spinal cords. b and c . Identification of astrocytes. The astrocytic cluster (red circle) identified by the feature plots of astrocyte marker genes Aqp4 (b) and Gja1 (c). d . Astrocytic subtypes. The snRNA-seq data of astrocytes from from Wt and gp120Tg mice were pooled for re-clustering. Eight astrocytic subtypes were identified. e and f . Differential astrocytic subtypes in Wt and gp120Tg spinal cords. Splitting the merged astrocyte clusters (d) based on their genotypes revealed marked differences in specific subtypes from Wt (e) and gp120Tg (f) mice; note the marked decrease of clusters 1 and 2 and increase of cluster 5. Cluster 7 was a subtype specifically in gp120Tg mice. g . Quantitation of individual subtypes in Wt and gp120Tg mice. Shown here were fractions of nuclei of individual clusters in the total Wt or total gp120Tg lumbar spinal astrocytes, respectively. Note that clusters 1 and 2, the two major clusters in the Wt were dramatically decreased whereas cluster 5 was increased in gp120Tg mice. Cluster 7 was present in gp120Tg but not Wt spinal cords. h . Cluster-specific markers in the Wt astrocytes. Heatmap shows the expression of top 10 cluster-specific markers in astrocytes from the Wt mice. Cluster-specific markers are the upregulated genes with adjusted p-value<0.05 of the single astrocytic cluster compared to all other astrocytic clusters. Each column represented a nucleus, each row represented a gene. The markers in clusters 1 and 2 (green rectangles) were exemplified in the list on left. Note cluster 1 and 2 express high level of astrocyte transporter genes Slc6a11 (red arrow) and Slc7a10 (blue arrow) and very low level of Gfap (black arrow). On the contrary, Cluster 3 and 4 express very low level of Slc6a11 and Slc7a10 but high level of Gfap. i . Expression of the Wt astrocytic cluster markers (h) in the gp120Tg astrocytes. Note cluster 5 in the gp120Tg mice expressed the marker genes of both cluster 1 and cluster 2 from the Wt mice. Cluster 3 and 4 from the Wt and gp120Tg mice express the same set of markers. j . Cluster-specific markers in gp120Tg astrocytes. Heatmap showed the expression of the top10 cluster-specific markers in astrocytes from the gp120Tg mice. Note cluster 7 expressed a unique set of gene markers (red rectangle). k . Expression profiles of selected genes in different astrocyte subtypes shown by a dot plot. Cluster 7 has low levels of expression of genes related to astrocytic homeostatic function (orange rectangle), but high levels of expression of genes related to inflammation (red rectangle) and phagocytosis (blue rectangle). scale bar: Log value. Red arrow points to the expression profiles of Lgals3 (gene encoding Gal3). Note the selective expression of Lgal3 in cluster 7.

Article Snippet: The primary antibodies used included anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938), anti-GFAP antibody (1:500, Millipore, cat. No. AB5541), anti-Iba1 antibody (1:500, 1:1000; Wako, cat. No. 016–20001), ant-Vimentin antibody (1:500, abcam, cat. no. ab137321), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, catalog #9661), anti-NeuN antibody (1:500, Millipore, cat. no. ABN90), and anti-vGlut1 antibody (Synaptic systems, cat. no. 35 303).

Techniques: Marker, Quantitation Assay, Expressing

Journal: bioRxiv

Article Title: A novel subtype of reactive astrocytes critical for HIV associated pain pathogenesis

doi: 10.1101/2022.08.03.502665

Figure Lengend Snippet: a and b Double immunofluorescent staining (IF) of Gal3 (red) with astrocytic marker (Gfap, white) and microglial marker Iba1 (green) respectively showed most Gal3+ cells were also GFAP+ (white open arrows) (a) in the gp120Tg lumbar spinal cord, although Gal3+ microglia were occasionally observed (white filled arrowhead) (b). SDH: spinal dorsal horn. c , Quantitative summary of Gal3+ microglia and astrocytes. d , A 3D confocal image of a cell clump of microglia (filled arrow) and an astrocyte (open arrow) with Gal3 signals (red). e-g : Different optical sections of the clump showed that Gal3 was exclusively in the astrocyte (open arrow) but not in the nearby microglia (filled arrow).

Article Snippet: The primary antibodies used included anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938), anti-GFAP antibody (1:500, Millipore, cat. No. AB5541), anti-Iba1 antibody (1:500, 1:1000; Wako, cat. No. 016–20001), ant-Vimentin antibody (1:500, abcam, cat. no. ab137321), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, catalog #9661), anti-NeuN antibody (1:500, Millipore, cat. no. ABN90), and anti-vGlut1 antibody (Synaptic systems, cat. no. 35 303).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: A novel subtype of reactive astrocytes critical for HIV associated pain pathogenesis

doi: 10.1101/2022.08.03.502665

Figure Lengend Snippet: a . HIPA marker Gal3 was significantly upregulated in the spinal cords of gp120Tg mice. b . IF revealed Gal3+ cells were predominantly at the central canal (CC) region (arrow) in Wt mice. c . The spatial distribution of HIPAs in the gp120Tg spinal cord shown in a low power image. HIPAs were scattered in the grey matter. Compared with Wt , Gal3 signals around the central canal (arrow) was diminished. Dash lines encircled the gray matters; scale bar (b∼c): 200µm. d . An IF image of HIPAs (white arrows) that form a trajectory from the CC region to spinal dorsal horn (SDH) of the gp120Tg mice. Dash lines encircled the gray matters; scale bar: 100µm. e∼l , High power images showed the distribution of Gal3 (red) protein in spinal CCs (encircled by dash lines) of Wt and gp120Tg mice. e, In the Wt mice, Gal3 protein was strictly and strongly distributed in Gal3+ cells lined in the CC (wide filled arrow); f∼h, Gal3+ cells in the Wt CC did not expressed astrocytic marker Gfap; h, no Gal3+Gfap+ astrocytes (HIPAs) were detected outside the Wt CC. i, In the gp120Tg mice, the expression of Gal3 in CC was severely diminished (wide open arrow), instead strong Gal3 protein were expressed outside CC (wide filled arrows); j and k, Compared to Wt mice (f, g), cells in the gp120Tg CC expressed Gfap (thin arrows); l, HIPAS were induced outside the gp120Tg CC (thin arrows). m∼o , Confocal images showing the heterogenous morphology of HIPAs in different locations in the gp120Tg spinal cord. m, Most HIPAs (wide filled arrows) near the CC region (encircled by dash line) were small and had Gal3 protein concentrated in the nucleus area. n, In the SDH, HIPAs were larger in size with various shapes and Gal3 was distributed throughout the whole astrocyte including the processes (open arrows); m and n, Gal3-negative astrocytes (thin arrows) were smaller in size with fewer short processes. o . A stacked confocal image to show the various morphologies of large HIPAs in SDH. Filled arrow pointed to a HIPA with fewer and long processes. Open arrow pointed to a HIPA with swollen cellular body and many short processes. Note the expression of Gal3 (red) across the whole cells and the scattered Gfap fragments (white) in these HIPAs. Scale bar (e∼o): 20µm.

Article Snippet: The primary antibodies used included anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938), anti-GFAP antibody (1:500, Millipore, cat. No. AB5541), anti-Iba1 antibody (1:500, 1:1000; Wako, cat. No. 016–20001), ant-Vimentin antibody (1:500, abcam, cat. no. ab137321), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, catalog #9661), anti-NeuN antibody (1:500, Millipore, cat. no. ABN90), and anti-vGlut1 antibody (Synaptic systems, cat. no. 35 303).

Techniques: Marker, Expressing

Journal: bioRxiv

Article Title: A novel subtype of reactive astrocytes critical for HIV associated pain pathogenesis

doi: 10.1101/2022.08.03.502665

Figure Lengend Snippet: a . Confocal images of HIPAs (Gal3+) and neurons (NeuN+) revealed by double immunofluorescent staining. Shown were two representative HIPAs (red) (open wide arrows) in close interactions with neurons (green) (thin arrows). b-g , Imaris 3D image segmentation of the selected area in a. This image analysis removed all the signals that were not interact with HIPAs to visualize NeuN signals engulfed by HIPAs. b-d, Imaris image segmentations of one region in a to show intact neuronal somas (thin white arrows) engulfed by a HIPA. e-g. Imaris image segmentations of another regions in a to show neuronal debris (thin red arrows) inside a HIPA. The engulfed neuronal somas revealed by creating the solid opaque surfaces (open arrow)(b and e), transparent surfaces (open arrow) (c and f), and by removing HIPA components (d and g). h , Confocal image of HIPAs (wide filled arrows) and synapses (green) in the SDH of gp120Tg mice. i and j . Imaris segmentation of the image in h to reveal HIPA-synapse interaction. i, This image analysis removed all the synapse signals (green dots) outside HIPAs. HIPAs were revealed by creating the solid opaque surfaces (wide filled and open arrows). j. The engulfed synapses (thin arrows) were viewed by creating transparent surface of HIPAs in i; k . The imaris clipping plane tool truncated a 3D HIPA to view the synapses (green) (arrows) inside a representative HIPA (open arrow in i).

Article Snippet: The primary antibodies used included anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938), anti-GFAP antibody (1:500, Millipore, cat. No. AB5541), anti-Iba1 antibody (1:500, 1:1000; Wako, cat. No. 016–20001), ant-Vimentin antibody (1:500, abcam, cat. no. ab137321), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, catalog #9661), anti-NeuN antibody (1:500, Millipore, cat. no. ABN90), and anti-vGlut1 antibody (Synaptic systems, cat. no. 35 303).

Techniques: Staining

Journal: bioRxiv

Article Title: A novel subtype of reactive astrocytes critical for HIV associated pain pathogenesis

doi: 10.1101/2022.08.03.502665

Figure Lengend Snippet: Western blot (a) and quantification (b and c) showed that Gal3 knockout significantly decreased gp120-induced Vim (b) and Gfap (c) protein expression in the spinal cord lumbar of gp120Tg mice.

Article Snippet: The primary antibodies used included anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938), anti-GFAP antibody (1:500, Millipore, cat. No. AB5541), anti-Iba1 antibody (1:500, 1:1000; Wako, cat. No. 016–20001), ant-Vimentin antibody (1:500, abcam, cat. no. ab137321), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, catalog #9661), anti-NeuN antibody (1:500, Millipore, cat. no. ABN90), and anti-vGlut1 antibody (Synaptic systems, cat. no. 35 303).

Techniques: Western Blot, Knock-Out, Expressing

Journal: bioRxiv

Article Title: A novel subtype of reactive astrocytes critical for HIV associated pain pathogenesis

doi: 10.1101/2022.08.03.502665

Figure Lengend Snippet: a . A UMAP cluster plot of the integrated snRNA-seq datasets from Wt, gp120Tg, gp120Tg/Gal3 -/- , and Gal3 -/- mice to show the clusters of the main cell types in lumbar spinal cords. b and c . Feature plots of astrocyte marker genes Aqp4 (b) and Gfap (c) to show the astrocytic cluster in the combined UMAP cluster plot. d . A UMAP cluster plot of subset analysis of combined astrocytes from of Wt, gp120Tg, gp120Tg/Gal3 -/- , and Gal3 -/- lumbar spinal cords. e-h . UMAP cluster plots of astrocytes from different genotypes of mice after splitting the overlayed astrocytic clusters (d). Compared with the Wt astrocyte clusters (e), there were evident shifts of several gp120Tg clusters (f), including marked decrease of clusters 1 and 2 and increase of cluster 5. Note that cluster 7 was almost exclusively in the gp120Tg plot (f), with only a small number of homologous cells in the counterpart cluster in the Wt plot (e). Strikingly, these gp120-induced shifts of astrocyte cluster sizes were reversed by Gal3 KO (g). Compared with clusters in the plot of gp120Tg (f), the clustering pattern of gp120Tg/Gal3 KO astrocytes (g) appeared to shift toward the pattern of Wt astrocytes (e), with increased sizes of clusters 1 and 2 and decreased size of cluster 5. Notably, the gp120Tg-specific cluster 7 (i.e., HIPA) was almost completely depleted in gp120Tg/Gal3 KO mice (g). The overall cluster pattern of gp120Tg/Gal3 KO (g) was more similar to that of Wt (e) than gp120Tg (f). h, A UMAP cluster plot of astrocytes from Gal3 KO mice. The cluster pattern of Gal3 KO astrocytes (h) was similar to that of Wt (e). i . Quantitative analysis of the relative population sizes of nuclei of individual clusters from different genotypes. Note that Gal3 KO depleted HIPAs (cluster 7) in gp120Tg mice, and prevented the decrease of the two homeostatic astrocytic subpopulations (cluster 1, 2), and attenuated the increase of cluster 5. j-m . Comparison of the expression of the top 50 upregulated genes in HIPA (cluster 7 in f) in gp120Tg (k) with their counterpart genes in Wt (j), gp120Tg/Gal3 KO (l), or Gal3 KO astrocytes in heatmaps. These genes included those implicated in inflammatory and phagocytotic pathways. Each row represented a gene, and each column represented a cell. The color bars on top of individual heatmaps represented individual clusters, and the color scale bar on the right represented the expression levels (log2 values). The red arrows point to cluster 7 in individual heatmaps. Note there is no cluster 7 in Gal3 KO mice. Compared with Wt (j), these genes were significantly upregulated in cluster 7 of gp120Tg astrocytes (k), but the upregulation was blocked in gp120Tg/Gal3 KO astrocytes (l). Compared with the heatmap of Wt astrocytes (j), Gal3 KO only (m) did not evidently alter the expression of these genes.

Article Snippet: The primary antibodies used included anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938), anti-GFAP antibody (1:500, Millipore, cat. No. AB5541), anti-Iba1 antibody (1:500, 1:1000; Wako, cat. No. 016–20001), ant-Vimentin antibody (1:500, abcam, cat. no. ab137321), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, catalog #9661), anti-NeuN antibody (1:500, Millipore, cat. no. ABN90), and anti-vGlut1 antibody (Synaptic systems, cat. no. 35 303).

Techniques: Marker, Comparison, Expressing

Journal: bioRxiv

Article Title: A novel subtype of reactive astrocytes critical for HIV associated pain pathogenesis

doi: 10.1101/2022.08.03.502665

Figure Lengend Snippet: a . Von Frey test of mechanical hyperalgesia. Gp120Tg mice (5 months) developed mechanical hyperalgesia. Gal3 KO blocked the expression of mechanical hyperalgesia of gp120Tg mice. b . Association of HIPAs with degenerative neurons in gp120Tg spinal cords. Combination of FJ-C staining and Gal3 IF staining revealed that extensive overlap (arrows) of signals of degenerative neurons (FJ-C staining) and HIPAs (Gal3 IF staining). c-e . FJ-C staining of lumbar spinal dorsal horns (SDH) from Wt (c), gp120Tg (d), gp120Tg/Gal3-/-(e) mice. FJ-C staining signals (arrows) were markedly increased in gp120Tg mice (d), which were attenuated by Gal3 KO (e). f . Quantification of FJ-C staining signals. g . Comparison of spinal protein levels of selected molecular markers of neuroinflammation in Wt, gp120Tg, gp120Tg/Gal3 -/- mice. Protein lysates of lumbar spinal cords were analyzed by immunoblotting of western. h-i . Quantification of active Il-1β (h), pro-IL-1β (i), active-caspase1 (j), Wnt5a (k), and Iba1 (l).

Article Snippet: The primary antibodies used included anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938), anti-GFAP antibody (1:500, Millipore, cat. No. AB5541), anti-Iba1 antibody (1:500, 1:1000; Wako, cat. No. 016–20001), ant-Vimentin antibody (1:500, abcam, cat. no. ab137321), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, catalog #9661), anti-NeuN antibody (1:500, Millipore, cat. no. ABN90), and anti-vGlut1 antibody (Synaptic systems, cat. no. 35 303).

Techniques: Expressing, Staining, Comparison, Western Blot

Journal: bioRxiv

Article Title: A novel subtype of reactive astrocytes critical for HIV associated pain pathogenesis

doi: 10.1101/2022.08.03.502665

Figure Lengend Snippet: HIPAs are a novel subpopulation of reactive astrocytes, with the expression of phagocytosis-related genes (Gal3, C3 etc.) and inflammation-related genes (Anxa3, Nox4, Adamts5 etc.), in the lumbar spinal cord of gp120Tg mice. HIPAs originate from the Gal3+ stem cells lined in spinal central canal, migrating along a clear trajectory toward the spinal marginal area with gradual increase of cell volume and complexity. During this process, Gal3 protein is progressively translocated from the nucleus to the cytoplasm and plasma membrane. HIPAs are eventually developed into enormous, big phagocytes. Gal3 (and probably other proteins such as C3) released from HIPAs may opsonize nearby neurons and synapses for phagocytosis. HIPAs also express inflammatory proteins such as Anax3, Nox4 and Adamts5. The upregulation of caspase1 in HIPAs may lead to the activation of inflammasome pathway that activate IL-1β. These inflammatory cytokines released from HIPAs may activate microglia, which further elevate spinal inflammation. Via an unknown mechanism, HIPAs may also regulate the expression of Wnt5a, a key protein that regulates neuroinflammation during the pathogenesis of gp120-induced pain. Both the phagocytotic and inflammatory activities of HIPAs may contribute to the damages of neural circuits in the spinal dorsal horn and the expression of pathological pain induced by HIV-1 gp120.

Article Snippet: The primary antibodies used included anti-Gal3 antibody (1:500, Santa Cruz, cat. no. sc-23938), anti-GFAP antibody (1:500, Millipore, cat. No. AB5541), anti-Iba1 antibody (1:500, 1:1000; Wako, cat. No. 016–20001), ant-Vimentin antibody (1:500, abcam, cat. no. ab137321), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, catalog #9661), anti-NeuN antibody (1:500, Millipore, cat. no. ABN90), and anti-vGlut1 antibody (Synaptic systems, cat. no. 35 303).

Techniques: Expressing, Clinical Proteomics, Membrane, Activation Assay

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A: paraffin sections of anterior chamber angle from a normal human eye were immunostained with anti-Gal8 antibody. (i) anti-Gal8 IgG reacted intensely with cells on the trabecular beams (arrows) and with cells in the juxtacanalicular portion of TM (arrowheads). Staining was also observed in the ECM of both portions of TM (JCT and CS) and in the wall of Schlemm’s canal. (ii) No staining was observed when the sections were not exposed to the primary antibody. SC : Schlemm’s canal, JCT : juxtacanalicular TM; CS beams : corneoscleral beams. Bar: 25 µm. B: (i) RT-PCR . Total RNA (1.0 µg) from confluent cultures of normal human TM cells was subjected to RT-PCR. The expected 191 bp fragment was amplified using Gal8- specific-primers. In each case, no components were amplified when reaction mixtures lacked reverse transcriptase (RT). (ii) qRT-PCR . Total RNA was subjected to Taq-Man RT-PCR using Gal8 specific primers. Original amplification plots of Gal8 and GAPDH mRNAs genes are shown (Ct 37.37 and 33.38 for Gal8 and GAPDH, respectively). N = 3 for each experiment; all experiments were performed twice using TM cells from two different donors with reproducible results. (iii and iv) Western Blot Analysis . Protein extracts from confluent cultures of normal human TM cells were incubated with lactogel beads and eluted first with sucrose, and then with lactose. Eluted proteins were electrophoresed, the protein blot of the gel was stained with Ponceau S (iii) and was then processed for immunostaining with goat anti-Gal8 (iv). Both the total cell extract ( T ) and the lactose eluate ( L ) contained a major 36-kDa anti-Gal8 reactive component. This component was not detected in the unbound fraction ( UB ) and in the sucrose eluate ( S ).

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Reverse Transcription, Quantitative RT-PCR, Western Blot, Incubation, Immunostaining

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: Normal human TM cells were incubated on microtiter wells coated with BSA, fibronectin, or Gal8 in DPBS, in the presence and absence of a function-blocking anti-β 1 integrin antibody (JB1A), or control mouse IgG. Following incubation at 37°C for 30 min, cells were fixed and stained with crystal violet. Attached cells in fibronectin-coated wells are set as 100% (positive control); attached cells in other wells are presented as percent of positive control. Data are expressed as mean±SEM and analyzed with one-way ANOVA. * P <0.05 vs IgG; ** P <0.01 vs media or IgG; *** P <0.001 vs media. B and C: Cell spreading assay. TM cells were fixed with 4% paraformaldehyde after adhesion for 30 min. F-actin was stained with rhodamine-labeled phalloidin and cell nuclei were labeled with DAPI. Random fields of each experimental condition were photographed, and spread areas of individual cells were quantified with ImageJ. Representative micrographs of TM cells incubated in the presence and the absence of anti-β 1 integrin antibody are shown in C. Data are presented as Box–whisker plot (after Tukey) and analyzed with one-way ANOVA. *** P <0.001 vs media or IgG. This experiment was performed three times with reproducible results. Bar: 100 µm.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Incubation, Blocking Assay, Control, Staining, Positive Control, Labeling, Whisker Assay

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A: Normal human TM cells were plated on eight-chamber glass slides coated with 20 µg/ml of recombinant human Gal8 (i–iii), 20 µg/ml of fibronectin (iv–vi), or 100 µg/ml of poly-L-lysine (vii–ix) in serum-free DMEM at 37°C for 30 min (i, iv, vii), 1 hr (ii, v, viii), and 2 hr (iii, vi, ix). Following the incubation period, cells were fixed with 4% paraformaldehyde and stained with rhodamine-labeled phalloidin. Bar: 50 µm. B: Quantification of stress fiber formation. Random fields were photographed, and cells with robust stress fibers were counted. N = 225 to 362. Data are expressed as mean±SEM and analyzed with one-way ANOVA. *** P <0.001 vs poly-L-lysine at different time points. This experiment was performed three times with reproducible results.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Recombinant, Incubation, Staining, Labeling

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A and C: Serum-starved human TM cells were incubated on chamber glass slides coated with recombinant human Gal8 in the presence of the Rho inhibitor, C3 transferase or ROCK inhibitor, Y27632, at different concentrations. After 2 hr, cells were stained with rhodamine-labeled phalloidin, and cells with robust stress fibers were enumerated. Data are expressed as mean±SEM. B and D: Cells were treated C3 transferase at 2 µg/ml (B) or Y27632 at 20 µM (D), stained with rhodamine-labeled phalloidin, and random fields were photographed. Note that cells treated with Y27632 or C3 transferase are not spread and exhibit dendrite-like structures. Bar: 50 µm.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Incubation, Recombinant, Staining, Labeling

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A: Gal8 induces phosphorylation of MLC2 in a time-dependent manner. Normal human TM cells were incubated on 100-mm dishes coated with Gal8 for 0.5, 1, and 2 hr. Following incubation, cells were lysed, and protein extracts were subjected to electrophoresis in 12% SDS-PAGE gels. Blots were probed with anti-phosphorylated myosin light chain 2 (ppMLC2) (Thr18/Ser19) antibody. The blots were subsequently stripped and reprobed with anti-MLC antibody. A representative Western blot is shown in the top panel. Images were acquired by Odyssey Infrared Imaging System, and band intensity was quantified by ImageJ (bottom panel). N = 3. B: Phosphorylation of MLC2 is inhibited by Rho and ROCK inhibitors. Normal human TM cells were serum-starved overnight and treated with C3 transferase (2 µg/ml) and Y27632 (20 µM) for 4 hr. Treated cells were detached and plated on Gal8-coated dishes for 2 hr in the presence or absence of inhibitors and were then examined for the expression levels of ppMLC2 as described in the legend to panel A. Top: A representative Western blot; bottom: quantification of ppMLC2. Data are expressed as mean±SEM and analyzed with one-way ANOVA. *** P <0.001 vs control. N = 3.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Phospho-proteomics, Incubation, Electrophoresis, SDS Page, Western Blot, Imaging, Expressing, Control

Journal: Food Science & Nutrition

Article Title: Pinellia pedatisecta Schott‐Derived Exosome‐Like Nanovesicles Promote Apoptosis in Colorectal Cancer by Regulating the Lysosome‐Mediated Mitophagy Pathway

doi: 10.1002/fsn3.71500

Figure Lengend Snippet: Lysosome inhibitor suppressed mitophagy in PPS‐ELNs‐treated HCT116 cells. (A) The levels of ROS, SOD, and MDA were detected by commercial kits. (B) Autophagosomes in HCT116 cells were observed by TEM. Scale bar = 1 μm. (C) Lysosomes in HCT116 cells were detected by LysoTracker Red staining. Scale bar = 100 μm. (D) The co‐localization of LC3 and mitochondrial protein TOM20 was detected by immunofluorescence. Scale bar = 100 μm. (E) The expression of p62, PINK1, Parkin, Gal3, TOM20, and LC3‐II/I was detected by Western blot. ** p < 0.01 vs. Control group. # p < 0.05, ## p < 0.01 vs. PPS‐ELNs group. CQ, chloroquine.

Article Snippet: Subsequently, sections were incubated with primary antibodies including Ki67 (1:300, Affinity, AF0198) and Gal3 (1:100, CST, 87985) at 4°C overnight.

Techniques: Staining, Immunofluorescence, Expressing, Western Blot, Control

Journal: Food Science & Nutrition

Article Title: Pinellia pedatisecta Schott‐Derived Exosome‐Like Nanovesicles Promote Apoptosis in Colorectal Cancer by Regulating the Lysosome‐Mediated Mitophagy Pathway

doi: 10.1002/fsn3.71500

Figure Lengend Snippet: PPS‐ELNs regulated lysosome‐mediated mitophagy pathway in CRC mice. (A) The expression of Gal3 in tumors was detected by immunohistochemistry. Scale bar = 20 μm. (B) Autophagosomes in tumor tissues were observed by TEM. Scale bar = 1 μm. (C) The expression of p62, TOM20, LC3‐II/I, PINK1, and Parkin was detected by Western blot. * p < 0.05, ** p < 0.01 vs. Control group.

Article Snippet: Subsequently, sections were incubated with primary antibodies including Ki67 (1:300, Affinity, AF0198) and Gal3 (1:100, CST, 87985) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Western Blot, Control

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Knockdown of Gal3 ameliorates HD symptoms in R6/2 mice. Mice of 6 weeks were intrastriatally injected with the sh Lgals3 -expressing or shGFP -expressing lentivirus, and monitored for an additional 7 weeks ( n = 9–11 mice in each group). a The motor functions of R6/2 mice and their littermate controls were assessed by rotarod performance. b Kaplan–Meier plots of R6/2 and WT mice ( P = 0.0089). c Body weight of the indicated animal was measured from 7 to 13 weeks of age. Brain tissues were carefully removed and subjected to immunofluorescence staining to determine the amount of mHTT aggregates (green, EM48; d , e ) or DARPP32 (green, a marker for the medium spiny neurons (MSNs), g , h ), on the neurons (NeuN, red; a neuronal marker; d , f , g ). Nuclei were stained with Hoechst (blue). Four animals were analyzed in each group. The results were analyzed by two-way ANOVA followed by Tukey’s post hoc test. Data are presented as the means ± SEM. *Specific comparison between WT and R6/2 mice infected with the same lentivirus; #Specific comparison between mice of the same genotype. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Same P -value denotation for #. Scale bar: 10 µm. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Knockdown, Injection, Expressing, Immunofluorescence, Staining, Marker, Comparison, Infection