Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: (A) The expression of MUC16 protein in HCECs separately transduced with two lentiviral-based shRNA vectors (shMUC16-1 or shMUC16-2) were examined through a Western blot analysis. The a-tubulin was used as a loading control. (B) Representative flow cytometric analyses of fluorescence intensity and MUC16 expressions in transduced HCECs followed by fluorescein staining were shown in contour plots. (C) Representative flow cytometric analyses of fluorescence intensity and MUC16 expression in HCECs cultured in KSFM ( left ) or MPM ( right ) followed by fluorescein staining were presented in contour plots. (D) The frozen tissue sections prepared from fluorescein-stained corneal epithelia were stained with GAL3 ( red , immunofluorescence), and the cell nuclei were counterstained with DAPI ( blue , immunofluorescence).

Article Snippet: To detect the expression of GAL3, cell suspensions were fixed and incubated with anti-galectin-3 (GAL3) mouse antibody (no. A3A12; Novus Biologicals).

Techniques: Expressing, Transduction, shRNA, Western Blot, Control, Fluorescence, Staining, Cell Culture, Immunofluorescence

Journal: International Journal of Stem Cells

Article Title: Mesenchymal Stem Cells Ameliorate Fibrosis by Enhancing Autophagy via Inhibiting Galectin-3/Akt/mTOR Pathway and by Alleviating the EMT via Inhibiting Galectin-3/Akt/GSK3β/Snail Pathway in NRK-52E Fibrosis

doi: 10.15283/ijsc22014

Figure Lengend Snippet: Effects of SF-MSCs-CM on TGF-β-induced NRK-52E fibrosis at different time points. (A) The expression of α-SMA and Gal-3 in NRK-52E cells induced by TGF-β1 was detected by Western Blot after SF-MSCs-CM treatment. (B) Quantification of α-SMA. (C) Quantification of Gal-3. Results were normalized relative to the expression of GAPDH. N=3 (per group). Data are presented as mean±SD and analyzed by two-way ANOVA followed by Tukey post hoc testing. *p<0.05, vs. control group, # p<0.05, vs. TGF-β1＋SF-MSCs-CM (24 h) group, ● p<0.05, vs. TGF-β1＋SF-MSCs-CM (48 h) group, ▲ p<0.05, vs. TGF-β1 ＋SF-DMEM (24 h) group, ◆ p<0.05, vs. TGF-β1＋SF-DMEM (48 h) group.

Article Snippet: The following antibodies are used: GAPDH (1:30,000, Proteintech, USA), α-SMA (1:4,000, Proteintech, USA), Galectin-3 (1:3,000, Cell Signaling, USA), GSK3β (1:1,000, Cell Signaling, USA), Phospho-GSK3β (Ser9) (1:1,000, Cell Signaling, USA), Snail (C15D3) (1:1,000, Cell Signaling, USA), Akt (pan) (1:1,000, Cell Signaling, USA), Phospho-Akt (Ser473) (1:1,000, Cell Signaling, USA), Fibronectin (1:1,000, Abcam, UK), E-Cadherin (1:1,000, Abcam, UK), MMP9 (1:1,000, Abcam, UK), TIMP1 (1:1,000, Cell Signaling, USA), Kim-1 (1:1,000, SAB, USA), PI3K (1:1,000, Abmart, China), Phospho-PI3K (1:1,000, Abmart, China), mTOR (1:1,000, Abmart, China), Phospho-mTOR (1:1,000, Abmart, China), LC3B (1:1,000, Abmart, China), and P62 (1:1,000, Abmart, China) antibodies.

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Stem Cells

Article Title: Mesenchymal Stem Cells Ameliorate Fibrosis by Enhancing Autophagy via Inhibiting Galectin-3/Akt/mTOR Pathway and by Alleviating the EMT via Inhibiting Galectin-3/Akt/GSK3β/Snail Pathway in NRK-52E Fibrosis

doi: 10.15283/ijsc22014

Figure Lengend Snippet: Immunocytochemical staining of E-Cadherin and α-SMA in Gal-3 KD and Gal-3 OE NRK-52E cells. Gal-3 KD reduced the expre-ssion of α-SMA and increased the expression of E-Cadherin in NRK-52E cells, but an opposite trend appea-red in Gal-3 OE. TGF-β1 indeed increased the expression of α-SMA and decreased the expression of E-Cadherin in Gal-3 OE cell, which was more than in Gal-3 KD cells. The treatment of SF-MSCs-CM after TGF-β1 treatment reduced the expression of α-SMA and raised the expression of E-Cadherin in both Gal-3 KD cells and Gal-3 OE cells, but more obvious in Gal-3 KD cells than in Gal-3 OE cells. SF-DMEM treatment also downregulated the expression of α-SMA, worse than SF-MSCs-CM group. Bar=100 μm.

Article Snippet: The following antibodies are used: GAPDH (1:30,000, Proteintech, USA), α-SMA (1:4,000, Proteintech, USA), Galectin-3 (1:3,000, Cell Signaling, USA), GSK3β (1:1,000, Cell Signaling, USA), Phospho-GSK3β (Ser9) (1:1,000, Cell Signaling, USA), Snail (C15D3) (1:1,000, Cell Signaling, USA), Akt (pan) (1:1,000, Cell Signaling, USA), Phospho-Akt (Ser473) (1:1,000, Cell Signaling, USA), Fibronectin (1:1,000, Abcam, UK), E-Cadherin (1:1,000, Abcam, UK), MMP9 (1:1,000, Abcam, UK), TIMP1 (1:1,000, Cell Signaling, USA), Kim-1 (1:1,000, SAB, USA), PI3K (1:1,000, Abmart, China), Phospho-PI3K (1:1,000, Abmart, China), mTOR (1:1,000, Abmart, China), Phospho-mTOR (1:1,000, Abmart, China), LC3B (1:1,000, Abmart, China), and P62 (1:1,000, Abmart, China) antibodies.

Techniques: Staining, Expressing

Journal: The EMBO Journal

Article Title: Microglial colonization of the developing mouse brain is controlled by both microglial and neural CSF-1

doi: 10.1038/s44318-025-00625-8

Figure Lengend Snippet: ( A ) Immunolabeling of coronal brain sections from E14.5 Cx3cr1 gfp/+ embryos showing co-expression of GFP, MAC2 and OPN at the CSA ( n = 3). Close-ups are 75 µm wide. ( B ) Immunolabeling of coronal brain sections from E14.5 Cx3cr1 gfp/+ embryos showing co-expression of GFP and GPNMB at the CSA ( n = 4). ( C ) Immunolabeling of coronal brain sections from P7 Cx3cr1 gfp/+ pups showing co-expression of GFP, MAC2 and OPN at the EDWM ( n = 3). ( D ) Immunolabeling of coronal brain sections from P7 Cx3cr1 gfp/+ pups showing co-expression of GFP and GPNMB at the EDWM ( n = 3). ( E ) Immunolabeling of coronal brain sections from E14.5 Cx3cr1 gfp/+ embryos showing co-expression of GFP and EdU (open arrowheads) at the CSA. ( F ) Percentage of OPN-positive ameboid microglia at the CSA from E14.5 embryos ( n = 13; from at least two distinct litters). ( G ) Percentage of EdU-positive microglia at the CSA at E14.5 amongst OPN-positive or OPN-negative ameboid microglia ( n = 9; from one litter) ( P = 0.0197). ( H ) Immunolabeling of coronal brain sections from P7 Cx3cr1 gfp/+ embryos showing co-expression of GFP and EdU (open arrowheads) at the EDWM. ( I ) Percentage of OPN-positive ameboid microglia at the EDWM from P7 pups ( n = 13; from at least two distinct litters). ( J ) Percentage of EdU-positive ameboid microglia at the EDWM at P7 amongst OPN-positive or OPN-negative ameboid microglia ( n = 8; from two distinct litters) ( P = 0.0272). Data were presented as mean ± SEM. Two-sided unpaired Mann–Whitney test were performed to assess differences ( G , J ). * P < 0.05. Scale bars: 100 µm ( A – D , H ); 50 µm ( E ). Close-ups are 100 µm ( A – D ) and 75 µm ( E , H ) wide. CSA cortico-striato-amygdalar boundary, EDWM early-dorsal white matter, H Hoechst. See Figs. and . .

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated in the same blocking solution with the following primary antibodies overnight at 4 °C: rat anti-CD206 (1/200; Bio-Rad Cat# MCA2235, RRID:AB_324622), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID:AB_10000240), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID:AB_839504), chicken anti-IBA1 (1/400; Synaptic Systems Cat# 234 009, RRID:AB_2891282), rabbit anti-KI67 (1/200; Abcam Cat# ab15580, RRID:AB_443209), rat anti-Lyve1 (ALY7) (1/200; Thermo Fisher Scientific Cat# 14-0443-82, RRID:AB_1633414), rat anti-GAL3 (MAC2) (1/1000; Cedarlane Cat# CL8942AP; RRID:AB_10060357), goat anti-OPN (1/400; R and D Systems Cat# AF808, RRID:AB_2194992), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330; RRID:AB_2112934), rabbit anti-P2RY12 (1/500; AnaSpec; EGT Group Cat# 55043 A, RRID:AB_2298886), and anti-VGlut2 (1/2000; Millipore Cat# AB2251-I, RRID:AB_2665454).

Techniques: Immunolabeling, Expressing, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Microglial colonization of the developing mouse brain is controlled by both microglial and neural CSF-1

doi: 10.1038/s44318-025-00625-8

Figure Lengend Snippet: ( A ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP, MAC2, and OPN at the CSA in E14.5 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at E12.5) ( n control = 5; n mutant = 4). ( B ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP and GPNMB at the CSA in E14.5 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at E12.5). ( C ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP, MAC2 and OPN at the CSA in P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at P3) ( n control = 3; n mutant = 3). ( D ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP and GPNMB at the CSA in P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at P3). ( E ) Immunolabeling of coronal brain sections from control and Cx3cr1 creER/+ ;Csf1 fl/fl mice showing absence of expression of OPN in cortical microglia (IBA1-positive) at P7 (tamoxifen administered at P3) while some white matter microglia do express OPN (solid arrowheads). Close-ups are 200 µm wide. ( F ) Proportion of proliferative microglia in control and Cx3cr1 creER/+ ;Csf1 fl/fl neocortex of P7 mice (tamoxifen administered at P3)( n control = 3 (2 F;1 M) ; n mutant = 4 (2 F;2 M); from one litter). ( G ) Microglial density (IBA1-positive cells/mm²) in control and Cx3cr1 creER/+ ;Csf1 fl/fl neocortex of P7 mice (tamoxifen administered at P3)( n control = 3 (2 F;1 M) ; n mutant = 4 (2 F;2 M); from one litter). ( H ) RNAscope experiments on coronal brain sections from P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl pups showing Csf1 expression in IBA1-positive ameboid microglia at the CSA (solid arrowheads) after tamoxifen administration at P1 and P3. ( I ) Normalized quantification of RNAscope experiments on coronal brain sections from P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos showing decreased Csf1 expression in mutant mice. Left column shows all quantified cells and right column the mean expression per animal ( n control = 3 (1 F,2 M); n mutant = 3 (2 M,1 F); from two distinct litters). ( J ) Immunolabeling of coronal brain sections from control and Cx3cr1 creER/+ ;Csf1 fl/fl mice showing co-expression of IBA1 and OPN in ameboid microglia within the EDWM accumulation at P7 (tamoxifen administered at P1 and P3) ( n control = 3 (1 F,1 M) ; n mutant = 3 (2 F;1 M); from one litter)(all cells, left: P < 0.0001; mean per animal, right: P = 0.10). ( K ) Microglial density (IBA1-positive cells/mm²) in control and Cx3cr1 creER/+ ;Csf1 fl/fl EDWM of P7 mice (tamoxifen administered at P1 and P3) ( n control = 9 (3 F;6 M); n mutant = 6 (3 F;3 M); from three distinct litters) ( P = 0.0028). ( L ) Number of OPN-positive microglia at the EDWM at P7 ( n control = 9 (3 F;6 M); n mutant = 6 (3 F;3 M); from three distinct litters) ( P = 0.0016). Data were presented as mean ± SEM. Two-sided unpaired Mann–Whitney test were performed to assess differences ( F , G , J – L ). ns not significant, ** P < 0.01, *** P < 0.001. Scale bars: 100 µm ( A – D , H , I ); 200 µm ( E ). CSA cortico-striatal-amygdalar boundary, EDWM early-dorsal white matter, H Hoechst, Ncx neocortex.

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated in the same blocking solution with the following primary antibodies overnight at 4 °C: rat anti-CD206 (1/200; Bio-Rad Cat# MCA2235, RRID:AB_324622), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID:AB_10000240), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID:AB_839504), chicken anti-IBA1 (1/400; Synaptic Systems Cat# 234 009, RRID:AB_2891282), rabbit anti-KI67 (1/200; Abcam Cat# ab15580, RRID:AB_443209), rat anti-Lyve1 (ALY7) (1/200; Thermo Fisher Scientific Cat# 14-0443-82, RRID:AB_1633414), rat anti-GAL3 (MAC2) (1/1000; Cedarlane Cat# CL8942AP; RRID:AB_10060357), goat anti-OPN (1/400; R and D Systems Cat# AF808, RRID:AB_2194992), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330; RRID:AB_2112934), rabbit anti-P2RY12 (1/500; AnaSpec; EGT Group Cat# 55043 A, RRID:AB_2298886), and anti-VGlut2 (1/2000; Millipore Cat# AB2251-I, RRID:AB_2665454).

Techniques: Immunolabeling, Expressing, Control, Mutagenesis, RNAscope, MANN-WHITNEY

Journal: Inflammation

Article Title: Nicotinamide N-Methyltransferase Inhibition Mitigates Cerulein-Induced Pancreatic Fibrosis via Galectin-3-Mediated Regulation of Stellate Cell Activation and Macrophage M2 Polarization in Mice

doi: 10.1007/s10753-026-02491-w

Figure Lengend Snippet: Inhibition of NNMT in mPSCs of CP mice limits M2 polarization of mouse macrophages. ( A ) Isolation and culture of bone marrow-derived macrophages (BMDMs) in lentiviral-infected conditional medium. ( B ) The proportion of CD206 + cells was detected by flow cytometry ( n = 3). ( C ) The mRNA levels of Arg1, MRC1, and TGFB1 in the BMDMs ( n = 3). ( D ) Isolation and cultivation of BMDMs in conditional medium with NNMTi. ( E ) The proportion of CD206 + cells was detected by flow cytometry ( n = 3). ( F ) The mRNA levels of Arg1, MRC1, and TGFB1 in the BMDMs ( n = 3).

Article Snippet: The primary antibodies are as follow: NNMT antibody (1:3000, 15123-1-AP, Proteintech), α-SMA antibody (1:20000, 67735-1-Ig, Proteintech), H3K27me3 antibody (1:500, DF6941, Affinity, Liyang, Jiangsu, China), Histone H3 antibody (1:1000, AF0863, Affinity), LGALS3 antibody (1:2000, A11198, Abclonal), Arg1 antibody (1:1000, A1847, Abclonal), MRC1 antibody (1:5000, A21014, Abclonal), GAPDH antibody (1:10000, AF7021, Affinity).

Techniques: Inhibition, Isolation, Derivative Assay, Infection, Flow Cytometry

Journal: Inflammation

Article Title: Nicotinamide N-Methyltransferase Inhibition Mitigates Cerulein-Induced Pancreatic Fibrosis via Galectin-3-Mediated Regulation of Stellate Cell Activation and Macrophage M2 Polarization in Mice

doi: 10.1007/s10753-026-02491-w

Figure Lengend Snippet: Suppression of NNMT impedes macrophage M2 polarization in mice suffering from CP. ( A ) The mRNA levels of Arg1, MRC1, and TGFB1 in mouse pancreatic tissues ( n = 6). ( B ) Immunofluorescence double staining of CD206 and F4/80 in mouse pancreatic tissues ( n = 6). Zoom scale bar = 20 μm, other scale bar = 50 μm. ( C ) Flow cytometry was used to detect the F4/80 + CD206 + cells in mouse pancreatic tissues ( n = 6).

Article Snippet: The primary antibodies are as follow: NNMT antibody (1:3000, 15123-1-AP, Proteintech), α-SMA antibody (1:20000, 67735-1-Ig, Proteintech), H3K27me3 antibody (1:500, DF6941, Affinity, Liyang, Jiangsu, China), Histone H3 antibody (1:1000, AF0863, Affinity), LGALS3 antibody (1:2000, A11198, Abclonal), Arg1 antibody (1:1000, A1847, Abclonal), MRC1 antibody (1:5000, A21014, Abclonal), GAPDH antibody (1:10000, AF7021, Affinity).

Techniques: Immunofluorescence, Double Staining, Flow Cytometry

Journal: Inflammation

Article Title: Nicotinamide N-Methyltransferase Inhibition Mitigates Cerulein-Induced Pancreatic Fibrosis via Galectin-3-Mediated Regulation of Stellate Cell Activation and Macrophage M2 Polarization in Mice

doi: 10.1007/s10753-026-02491-w

Figure Lengend Snippet: NNMT regulates M2 polarization of mouse macrophages via LGALS3. ( A ) Isolation and lentivirus infection in mPSCs of CP mice. ( B ) ELISA assay for the expression of LGALS3 in the supernatant of CP-mPSCs ( n = 3). C - D . The mRNA and protein levels of LGALS3 in the mPSCs with LGALS3 overexpression ( n = 3). E . Isolation and culture of BMDMs in lentiviral-infected conditional medium. F - G . The mRNA and protein levels of Arg1 and MRC1 in the BMDMs ( n = 3). H . The proportion of CD206 + cells was detected by flow cytometry ( n = 3).

Article Snippet: The primary antibodies are as follow: NNMT antibody (1:3000, 15123-1-AP, Proteintech), α-SMA antibody (1:20000, 67735-1-Ig, Proteintech), H3K27me3 antibody (1:500, DF6941, Affinity, Liyang, Jiangsu, China), Histone H3 antibody (1:1000, AF0863, Affinity), LGALS3 antibody (1:2000, A11198, Abclonal), Arg1 antibody (1:1000, A1847, Abclonal), MRC1 antibody (1:5000, A21014, Abclonal), GAPDH antibody (1:10000, AF7021, Affinity).

Techniques: Isolation, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Over Expression, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response

doi: 10.1084/jem.20090121

Figure Lengend Snippet: Galectin-3 promotes angiogenesis in vivo in a dose-dependent manner. (A) Angiogenesis in vivo was evaluated using the mouse corneal micropocket assay. Sustained-release polymer pellets containing various doses of galectin-3 (20–160 ng/pellet) were implanted in the corneas of Gal3 +/+ animals. (i) 5 d after surgery, the vessel area was calculated as described in the text. Data are expressed as mean ± SEM ( n = 4 or more). * , P < 0.01 compared with control pellets (Con) without any protein. (ii) Representative fluorescence images of corneas are shown in A (i). White asterisks indicate pellets. (B–D) Full-length galectin-3 but not truncated galectin-3 (Gal3C) promotes angiogenesis in vitro and in vivo. (B) Modified Boyden Chamber Assay. The cells that migrated from the upper chamber to the lower chamber during the period of 6 h in response to the protein gradients created by full-length galectin-3 and/or Gal3C were counted in three nonoverlapping fields under a microscope (320×). Data are expressed as mean ± SEM ( n = 3/group). * , P < 0.01 compared with cells incubated with media alone (Con). (C) Capillary tubule formation assay. HUVECs premixed with galectin-3 and/or Gal3C were plated on solidified matrigel (4.5 mg/ml) and after 6-h incubation, the extent of capillary tubule formation was evaluated by counting the number of branch points. Data are expressed as mean ± SEM ( n = 3 or more/group). * , P < 0.01 compared with cells incubated with media alone (Con). (D) Angiogenesis in vivo was evaluated using the mouse corneal micropocket assay using pellets containing full-length (Gal3) and truncated galectin-3 (Gal3C; 80 ng/pellet). Data are expressed as mean ± SEM ( n = 4 or more). * , P < 0.01 compared with control pellets (Con). Representative fluorescence images of corneas are shown in D (ii). White asterisks indicate pellets. Bars, 200 µm. Data are representative of three or more independent experiments.

Article Snippet: PCR was performed in triplicates according to the manufacturer’s instructions using cDNA, inventory gene-specific primers (Gal3, Hs00173587_m1; GAPDH, Hs99999905_m; Applied Biosystems Inc.), and master mix containing ROX as a passive reference.

Techniques: In Vivo, Polymer, Control, Fluorescence, In Vitro, Modification, Boyden Chamber Assay, Microscopy, Incubation, Tube Formation Assay

Journal: The Journal of Experimental Medicine

Article Title: Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response

doi: 10.1084/jem.20090121

Figure Lengend Snippet: Galectin-3 inhibitors block VEGF- and bFGF-mediated angiogenesis in vitro. (A) Modified Boyden chamber assay. The effect of galectin-3 inhibitors on VEGF- and bFGF-induced cell migration was evaluated. The cells that migrated from the upper chamber to the lower chamber were counted as described in . (B) Capillary tubule formation assay. HUVEC suspensions premixed with cytokines were plated on solidified matrigel (4.5 mg/ml), and, after 6-h incubation, the extent of capillary tubule formation was evaluated by counting the number of branch points. Representative images from each group are shown in B (ii). Images of cells incubated in media alone (not depicted) were indistinguishable from those incubated in media containing VEGF or bFGF in the presence of β-lactose (Lac). The assays were performed in the absence (Control) and presence of truncated galectin-3 (Gal3C, a dominant-negative inhibitor of galectin-3), full-length galectin-3 (Gal3), 0.1 M β-lactose (Lac, a competing saccharide), and 0.1 M sucrose (Suc, a noncompeting control saccharide). Data are expressed as mean ± SEM ( n = 3 or more/group), * , P < 0.01; **, P < 0.05 compared with respective controls incubated without galectin-3 inhibitors (Control) or with a noncompeting saccharide (Suc). Bar, 100 µm. Data are representative of three or more independent experiments.

Article Snippet: PCR was performed in triplicates according to the manufacturer’s instructions using cDNA, inventory gene-specific primers (Gal3, Hs00173587_m1; GAPDH, Hs99999905_m; Applied Biosystems Inc.), and master mix containing ROX as a passive reference.

Techniques: Blocking Assay, In Vitro, Modification, Boyden Chamber Assay, Migration, Tube Formation Assay, Incubation, Control, Dominant Negative Mutation

Journal: The Journal of Experimental Medicine

Article Title: Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response

doi: 10.1084/jem.20090121

Figure Lengend Snippet: Galectin-3 knockdown reduces VEGF- and bFGF-mediated angiogenesis in vitro. (A and B) HUVECs were transfected with siRNA duplexes directed against galectin-3 (Gal3 siRNA) or control nontargeting siRNA duplexes (Control siRNA). The knockdown of galectin-3 was evaluated by RT-qPCR (A) and Western blot analysis (Bi: a representative image, Bii: quantification of protein knockdown by ImageJ). (C and D) VEGF and bFGF induced cell migration and capillary tubule formation is reduced in galectin-3–knockdown cells. The ability of 25 ng/ml VEGF (open bars) and 10 ng/ml bFGF (solid bars) to promote chemotaxis (C) and capillary tubule formation (D) in galectin-3 siRNA (Gal3 siRNA) transfected and nontargeting siRNA-transfected (control siRNA) HUVECs, as well as galectin-3 siRNA transfected HUVECs in the presence of 10 µg/ml exogenous galectin-3 (Rescue) was compared. A value of 1.0 was assigned to cell migration and capillary tubule formation values of control cells. The values of all other cells were calculated as a fold change with respect to control cells. Data are expressed as mean ± SEM ( n = 3/group). * , P < 0.01 and **, P < 0.05 as compared with nontargeting siRNA transfected HUVECs). Representative images of the cells that migrated from the upper chamber to the lower chamber and capillary tubule formation in response to VEGF and bFGF stimulation are shown in C (ii) and D (ii), respectively. Bars: (C, ii) 100 µm; (D; ii) 200 µm. Data are representative of two or more independent experiments.

Article Snippet: PCR was performed in triplicates according to the manufacturer’s instructions using cDNA, inventory gene-specific primers (Gal3, Hs00173587_m1; GAPDH, Hs99999905_m; Applied Biosystems Inc.), and master mix containing ROX as a passive reference.

Techniques: Knockdown, In Vitro, Transfection, Control, Quantitative RT-PCR, Western Blot, Migration, Chemotaxis Assay

Journal: The Journal of Experimental Medicine

Article Title: Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response

doi: 10.1084/jem.20090121

Figure Lengend Snippet: Integrin αvβ3 serves as a galectin-3 counter-receptor. (A) HUVEC lysates were applied to a galectin-3 affinity chromatography column and bound proteins were sequentially eluted with a noncompeting saccharide, 0.1 M sucrose (Suc), and a competing saccharide, 0.1 β-lactose (Lac). Proteins eluted from the affinity column were identified by Western blot analysis. Both αv and β3 integrins were detected in the lactose eluate, but not in the sucrose eluate. (B and C) Integrin-blocking antibodies directed against integrins αv, β3, and αvβ3 inhibit galectin-3–induced cell migration (B) and capillary tubule formation (C). The ability of 10 µg/ml galectin-3 to promote chemotaxis (Boyden chamber assay) and capillary tubule formation in the presence and absence of 10 µg/ml αv, β3, and αvβ3 integrin-blocking antibodies or control mouse IgG was compared. Data are expressed as mean fold change over control cells incubated in media alone ± SEM ( n = 3/group). **, P < 0.05; * , P < 0.01 as compared with cell migration and capillary tubule formation induced by galectin-3. (D) Galectin-3 promotes clustering of integrin αvβ3. HUVECs were serum starved overnight and treated with 10 µg/ml galectin-3 for 10 min. At the end of the incubation period, the distribution of integrin αvβ3 was evaluated by confocal microscopy after immunostaining with an anti–human integrin αvβ3 monoclonal antibody. Treatment with galectin-3 resulted in a marked redistribution of integrins into a punctate pattern, indicative of integrin clustering. Overlapping Normanski images are shown on the bottom (NM). Bar, 50 µm. (E) Galectin-3 mediates FAK activation. HUVECs were serum starved for 2 h and treated with 10 µg/ml of galectin-3 for various time periods (0, 0.5, 1, 2, 5, and 10 min). HUVEC lysates were then subjected to immunoblot analysis for pFAK (Y397; top) and FAK (bottom). Galectin-3 treatment promoted a time-dependent activation of FAK that started at 1 min and peaked at 5 min. Total FAK levels remained unchanged at all time periods. Data are representative of three or more independent experiments.

Article Snippet: PCR was performed in triplicates according to the manufacturer’s instructions using cDNA, inventory gene-specific primers (Gal3, Hs00173587_m1; GAPDH, Hs99999905_m; Applied Biosystems Inc.), and master mix containing ROX as a passive reference.

Techniques: Affinity Column, Western Blot, Blocking Assay, Migration, Chemotaxis Assay, Boyden Chamber Assay, Control, Incubation, Confocal Microscopy, Immunostaining, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response

doi: 10.1084/jem.20090121

Figure Lengend Snippet: Disruption of GnTV reduces binding of galectin-3 to cell surface glycoprotein receptors with a concomitant reduction in galectin-3 and growth factor-mediated angiogenesis in vitro and in vivo. (A) HUVECs were transfected with lentiviral particles expressing shRNA directed against GnTV (shGnTV) or nontargeting shRNA control (shControl). The knockdown of endogenous GnTV was evaluated at the mRNA level by RT-qPCR. The value for HUVECs transfected with GnTV and control shRNA is expressed as the change in the expression level with respect to untransfected cells which served as a calibrator. * , P < 0.01 compared with control cells. (B) Expression of GnTV product, core β 1,6-branched saccharides, in HUVECs transfected with lentiviral particles was evaluated by staining with rhodamine-conjugated l -PHA, which specifically reacts with core β 1,6-branched saccharides. Bar, 100 µm. (C) The cell surface proteins of HUVECs transfected with lentiviral particles expressing shRNA directed against GnTV (shGnTV) or nontargeting shRNA control (shControl) were biotinylated and cross-linked in a single step, the biotinylated proteins were isolated by streptavidin pull-down, electrophoresed in reducing conditions and then probed with anti–galectin-3 mAb. Regardless of whether the Western blot analysis was performed using the streptavidin pull-down proteins (PD:Strep) or whole-cell lysates (WCL) of the labeled cells, reduced level of galecin-3 was detected in the GnTV knockdown cells compared with the cells transfected with control, nontargeting lentiviral particles. (D) GnTV modifies integrins αv and β3. RIPA buffer cell lysates (WCL) of untransfected HUVECs (H) and HUVECs transfected with control (C) and GnTV (M) targeting lentiviral particles were incubated with either l -PHA-agarose or ConA-agarose (1 h at 4°C). Bound proteins were eluted from the agarose beads by boiling in SDS-PAGE sample buffer, separated on SDS-PAGE, and immunostained to detect integrins αv and β3. Note that GnTV knockdown cell lysates show markedly reduced binding of integrins αv and β3 to l -PHA indicating that integrins αv and β3 are modified by GnTV. (E and F) The ability of galectin-3 (10 µg/ml), VEGF (25 ng/ml), and bFGF (10 ng/ml) to promote cell migration (E, Boyden chamber assay) and capillary tubule formation (F) is substantially reduced in GnTV knockdown cells. Data are expressed as mean fold change over control cells incubated in media alone ± SEM ( n = 3/group), *, P < 0.01; **, P < 0.05 compared with untransfected controls. (G) VEGF- and bFGF-induced angiogenesis is reduced in GnTV −/− mice. Angiogenesis in vivo was evaluated by the mouse corneal micropocket assay using VEGF and bFGF pellets. On day 5 after surgery, the animals were perfused with FITC-BS1, and the extent of angiogenesis was evaluated by examining the flat mounts of corneas by fluorescence microscopy. Blood vessel area was calculated using ImageJ. (i) Vessel area of neovascularization expressed in pixel 2 x 10 4 . Data are expressed as mean ± SEM (n = at least 4/group). *, P < 0.05 compared with GnTV +/+ control animals. (ii) Representative fluorescence images of corneas. White asterisks indicate pellets. Bar, 100 µm. Control pellets, which did not contain any protein, did not promote angiogenesis [not depicted; images identical to that shown in (ii)]. Data are representative of two independent experiments each repeated in triplicate.

Article Snippet: PCR was performed in triplicates according to the manufacturer’s instructions using cDNA, inventory gene-specific primers (Gal3, Hs00173587_m1; GAPDH, Hs99999905_m; Applied Biosystems Inc.), and master mix containing ROX as a passive reference.

Techniques: Disruption, Binding Assay, In Vitro, In Vivo, Transfection, Expressing, shRNA, Control, Knockdown, Quantitative RT-PCR, Staining, Isolation, Western Blot, Labeling, Incubation, SDS Page, Modification, Migration, Boyden Chamber Assay, Fluorescence, Microscopy

Journal: Molecular immunology

Article Title: Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

doi: 10.1016/j.molimm.2014.12.010

Figure Lengend Snippet: The mice were infected with influenza strain PR8 followed by a secondary bacterial infection with Streptococcus pneumoniae (Sp3) after 14 days. Each group of mice, ranging from two to four animals, was euthanized according to the following schedule: 3 days post-PR8 (PR8 3d), 7 days post-PR8 (PR8 7d), 10 days post PR8 (PR8 10d), 14 days post PR8 without Sp3 challenge (PR8 14d), 14 days post PR8 and 1 hour post Sp3 challenge (PR8 14d + Sp3 1h), 14 days post PR8 and 18 hours post Sp3 (PR8 14d + Sp3 18h), ctrl: non-challenged mice. Transcript levels of galectin-1 (Gal1) and galectin-3 (Gal3) were assessed by RT-PCR in lung tissues (A) or bronchoalveolar lavage (BAL) cells (B). Protein levels of selected galectins (Gal1 and Gal3) were assessed by Western blot in lungs (C) or BAL fluid (D) collected from control (non-challenged) or challenged groups of mice. (E) Sialylation status of lung tissues was assessed by immunofluorescence with FITC-PNA, which revealed the exposed galactosyl moieties. Bar graphs represent fold change in galectin transcript or protein levels from challenged mice in comparison with unchallenged mice, normalized to β-actin Images and the bar graphs are representative data from at least two independent experiments. *p <0.05; **p<0.001, non-paired Student’s t test.

Article Snippet: Antibodies against galectin-1 (Gal1) and galectin-3 (Gal3) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Molecular immunology

Article Title: Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

doi: 10.1016/j.molimm.2014.12.010

Figure Lengend Snippet: Transcript (A) or protein (B) levels of selected galectins (Gal1 and Gal3) were assessed with RT-PCR or Western blot, respectively, in lung carcinoma epithelial cells (A549), small airway epithelial cells (SAEC), or mouse lung tissues (lg). RT-PCR was performed with the total RNA extracted from A549, SAEC, or mouse lungs. Western blot was performed from cell lysate (c), culture supernatant (sn), lung tissue lysates (lg), or bronchoalveolar lavage (bal).

Article Snippet: Antibodies against galectin-1 (Gal1) and galectin-3 (Gal3) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Molecular immunology

Article Title: Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

doi: 10.1016/j.molimm.2014.12.010

Figure Lengend Snippet: Exposure of A549 cells to PR8 (A) or neuraminidase (B) modulates galectin expression (A1 and B1) and secretion (A2 and B2). A549 cells were infected with PR8 (MOI 5) for 72h (A), or treated with a bacterial neuraminidase cocktail (Arthrobacter ureafaciens and Clostridium perfringens; Neu K) (B). Galectin (Gal1 and Gal3) expression or secretion was assessed from cell lysates (A1 and B1) or culture supernatant (A2 and B2) with Western blot. Bar graphs show fold changes of galectin expression from PR8-infected or neuraminidase-treated cells (Neu K) in comparison with untreated cells (ctrl) normalized to tubulin. (C) Neuraminidase-treated (Neu K) or untreated (ctrl) cells were incubated with 15 µg/ml of biotinylated rhGal1, rhGal3, or galectins in the presence of lactose (0.1M) (Gal1+lac, Gal3+lac), followed by streptavidin-APC for flow cytometry analysis. The exogenous galectin binding to A549 cells was normalized to A549 cells without exogenous galectin (ctrl). (D) Effects of influenza viral neuraminidase (Neu N2) and bacterial neuraminidases (Arthrobacter ureafaciens and Clostridium perfringens; Neu K) on galectin binding to A549 cells. A549 cells grown in ELISA plates were subject to neuraminidase treatment (Neu K or Neu N2), followed by incubation with 15 µg/ml of exogenous rhGal1 or rhGal3. Galectin binding to the cells was assessed using primary antibodies against Gal1 or Gal3 followed by an HRP-conjugated secondary antibody. The galectin binding levels to neuraminidase-treated cells were normalized to the binding to the untreated cells (ctrl). Data shown and the bar graphs are representative data from at least three independent experiments. *p <0.05; **p<0.001, non-paired Student’s t test.

Article Snippet: Antibodies against galectin-1 (Gal1) and galectin-3 (Gal3) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Infection, Western Blot, Incubation, Flow Cytometry, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular immunology

Article Title: Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

doi: 10.1016/j.molimm.2014.12.010

Figure Lengend Snippet: (A) Binding of rhGal1 and rhGal3 to influenza hemagglutinin (HA0, precursor HA containing a hydrophobic signal sequence; HA1 and HA2, subunits of HA). Lysates from PR8 virus were prepared and subjected to Western blot. The total viral proteins were revealed by an α-PR8 antibody, and the viral HA by an α-HA antibody. Galectin binding was performed by overlaying membrane with rhGal1, rhGal3, and the specificity of the binding assessed by preincubation of the galectins with lactose (0.2 M) (rhGal1+lac, rhGal3+lac), followed by anti-Gal1 or anti-Gal3 antibodies, followed by HRP-conjugated secondary antibodies. To confirm the carbohydrate specificity of the galectin-HA interactions, HA was treated with PNGase F (HA* in a) or β-galactosidase (HA** in b or c) and subjected to lectin blot (a and c) as described above, or ELISA (b). (B) SPR sensogram of binding rhGal1 to HA. SPR was measured with immobilized rhGal1 and using HA (left panel) or desialylated HA (right panel) as analytes. HA starts at 12.5µg/ml, with 2-fold serial dilution; asialo-HA starts at 6.25 µg/ml. (C) SPR sensogram of binding of rhGal3 to HA. SPR was measured with immobilized rhGal3 and using HA (left panel) or desialylated HA (right panel) as analytes. HA starts at 50 µg/ml, with 2-fold serial dilution; asialo-HA starts at 6.25 µg/ml.

Article Snippet: Antibodies against galectin-1 (Gal1) and galectin-3 (Gal3) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Sequencing, Western Blot, Enzyme-linked Immunosorbent Assay, Serial Dilution

Journal: Molecular immunology

Article Title: Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

doi: 10.1016/j.molimm.2014.12.010

Figure Lengend Snippet: (A) A549 cells grown in ELISA plates were treated with S. pneumoniae neuraminidase (Neu Sp) or untreated (ctrl) followed by incubation with 15 µg/ml of exogenous rhGal1 or rhGal3. Galectin binding to the cells was assessed using primary antibodies against Gal1 or Gal3 followed by and HRP-conjugated secondary antibody. The galectin binding levels to the neuraminidase-treated cells were normalized to the untreated cells (ctrl). (B) A549 cells grown on the 10 cm plates were treated with bacterial neuraminidases (Arthrobacter ureafaciens and Clostridium perfringens; Neu K) or S. pneumoniae neuraminidase (Neu Sp), and subsequently incubated with S. pneumoniae (MOI 10) in the absence (Sp) or presence of 15 µg/ml of exogenous rhGal1 (Sp + rhGal1) or rhGal3 (Sp + rhGal3). The bound bacteria were released in water then quantified after 24h incubation on 5% sheep’s blood agar plates by counting colony forming units (CFU). The bar graphs represent the fold change of CFU from galectin-mediated Sp3 adhesion compared to the Sp3 adhesion in the absence of exogenous galectin. (C) A549 cells grown on the 10 cm plates were exposed to PR8 (MOI 5) for 72 h, then incubated with Sp3 (MOI 10) without (Sp) or with 15 ug/ml of exogenous rhGal1 (Sp + rhGal1) or rhGal3 (Sp + rhGal3) for bacterial adhesion. The colony forming units (CFU) released from bound bacteria were counted as described above. The bar graphs represent the fold change of CFU from galectin-mediated Sp adhesion compared to the Sp adhesion in the absence of exogenous galectin. Data shown and the bar graphs are representative data from at least three independent experiments. *p <0.05; **p<0.001.

Article Snippet: Antibodies against galectin-1 (Gal1) and galectin-3 (Gal3) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

Journal: bioRxiv

Article Title: Spatial regulation of the glycocalyx component Podocalyxin is a switch for pro-metastatic function

doi: 10.1101/2022.11.04.515043

Figure Lengend Snippet: (A) Schema, multi-omic (interactome, proteome, transcriptome) profiling to identify high-surface PODXL interactors. (B) Network diagram of subgroup interactions among identified PODXL binding partners. Networks, STRING analysis; layout, Cytoscape analysis. (C) Bubble-plot, PODXL binding partners sorted by subgroup then interaction score with high-surface PODXL. For proteomics, colour represents fold change (Log 2 ) interaction with PODXL compared to GFP alone; Interactome, blue-to-red; Proteome, greyscale. For RNA-seq, colour represents fold change (Log 2 ) endogenous high-surface compared to low-surface PODXL expression. Bubble size, significance (-log 10 ). (D) Cartoon, GFP-PODXL domains and known PODXL interactors. SP, signal peptide; TM, transmembrane domain; FBM, FERM-binding-motif; PBM, PDZ-binding-motif. (E) GFP-Trap immunoprecipitation of GFP or GFP-PODXL WT from PC3 cells. Western blotting of input and immunoprecipitates for GAL3, PODXL, GFP and GAPDH (loading control for GFP). (F) Quantitation of relative GAL3 to precipitated GFP vs GFP-PODXL WT in PC3 cells. Floating bar graph: dots, experiments; midline, mean; n=3 independent experimental culturing, lysate preparation and protein isolation. p-values (unpaired Student’s 2-tailed t-test), ns, not significant,*P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001.

Article Snippet: FFPE sections for Galectin-3 (Gal3) (87985, Cell Signaling) IHC staining were dewaxed using an Agilent pre-treatment module.

Techniques: Binding Assay, RNA Sequencing, Expressing, Immunoprecipitation, Western Blot, Control, Quantitation Assay, Isolation

Journal: bioRxiv

Article Title: Spatial regulation of the glycocalyx component Podocalyxin is a switch for pro-metastatic function

doi: 10.1101/2022.11.04.515043

Figure Lengend Snippet: (A-B) Western blot and quantitation, PC3-high expressing Scramble or GAL3 shRNAs for GAL3 or GAPDH (loading control). Lines, not adjacent in gel. Normalised to Scramble, mean ± SD, n=3 independent experiments. (C-D) Phase images and quantitation of (A). Cysts, yellow outlines. Outlines coloured by time in single frames and overlayed. Heatmap, proportions of Round, Spread, Tunnel-forming, Z-score normalised. n=3 independent experiments, 4 technical replicates/condition, 1063-1789 cysts/condition/experiment. (E-F) Phase images and quantitation of PC3-high cysts (control) with recombinant GAL3 (Re:GAL3). Cysts, yellow outlines. Outlines coloured by time in single frames and overlayed. Heatmap, proportions of Round, Spread, Tunnel-forming, Z-score normalised. n=3 independent experiments, 3 technical replicates/condition, 297-577 cysts/condition/experiment. (G-H) Western blot and quantitation, PC3 overexpressing GFP-PODXL WT, co-expressing either TagRFP-T, TagRFP-T-GAL3 (WT or R186S mutant), and Scramble or GAL3 shRNA for RFP, GAL3, GFP or GAPDH (loading control for RFP). Combined band intensity of GAL3 relative to control. Mean ± SD, n=4 independent experiments. (I-J) Phase images and quantitation of cysts from (G). Cysts, yellow outlines. Outlines coloured by time in single frames and overlayed. Heatmap, proportions of Round, Spread, Tunnel-forming, Z-score normalised. n=2 independent experiments, 3 technical replicates/condition, 287-551 cysts/condition/experiment. (K-L) Representative plot and geometrical mean of surface PODXL levels in Scramble, PODXL or GAL3 shRNA cells. Bar chart: dots, experiments; midline, mean; boundaries, min and max values; n=3 independent experiments. Heatmap p-values, Cochran-Mantel-Haenszel test, Bonferroni adjusted, comparing proportion to control, greyscale as indicated. Scale bars, 300 μm. p-values, unpaired Student’s 2-tailed t-test; ns, not significant, *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001.

Article Snippet: FFPE sections for Galectin-3 (Gal3) (87985, Cell Signaling) IHC staining were dewaxed using an Agilent pre-treatment module.

Techniques: Western Blot, Quantitation Assay, Expressing, Control, Recombinant, Mutagenesis, shRNA

Journal: bioRxiv

Article Title: Spatial regulation of the glycocalyx component Podocalyxin is a switch for pro-metastatic function

doi: 10.1101/2022.11.04.515043

Figure Lengend Snippet: (A-H) Receptor internalisation by capture-ELISA from GFP-PODXL WT-expressing PC3 cells co-expressing Scramble or GAL3 shRNA (A-D) or in cells with GFP-PODXL WT versus 4K>R mutant expression (E-H). Representative graphs, percentage of GFP-PODXL (A), a5 1-integrin (C), GFP-PODXL WT versus 4K>R (E, G) internalisation at indicated timepoints. Values, mean ± SEM, n=3 independent experiments. Graphs showing relative endocytosis at timepoint 2 minutes (B, D) or 4 minutes (F, H) normalised to control; 3 independent experiments combined. (I-L) Western blotting and quantitation, PC3 expressing Scramble or ITGB1 shRNA for ITGB1, PODXL, GAL3 and GAPDH (loading control). Quantitation across replicates. Mean ± SD, n=5 independent experiments. (M-N) Co-immunoprecipitation, western blotting and quantitation of endogenous PODXL and GAL3 from PC3 in (I) for ITGB1, GAL3, PODXL and GAPDH (loading control). Quantitation across replicates. Mean ± SD, n=5 independent experiments. (O-R) Phase images and quantitation from cells expressing (O, P) Scramble or GAL3 shRNAs or (Q, R) GFP-PODXL 4K>R mutant (IgG control) with 1-integrin blockade (AIIB2, blocking antibody). Cysts, yellow outlines. Outlines coloured by time in single frames and overlayed. Heatmap, proportions of Round, Spread, Tunnel-forming, Z-score normalised. (P) n=4 independent experiments, 3 technical replicates/condition, 444-702 cysts/condition/experiment. (R) n=3 independent experiments, 2 technical replicates/condition, 137-354 cysts/condition/experiment. Bar graphs, p-values, unpaired Student’s 2-tailed t-test; ns, not significant, *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001. Heatmap, p-values, Cochran-Mantel-Haenszel test Bonferroni adjusted, to compare area and proportion of each classification to control, greyscale as indicated. Scale bar 100 μm.

Article Snippet: FFPE sections for Galectin-3 (Gal3) (87985, Cell Signaling) IHC staining were dewaxed using an Agilent pre-treatment module.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, shRNA, Mutagenesis, Control, Western Blot, Quantitation Assay, Immunoprecipitation, Blocking Assay

Journal: bioRxiv

Article Title: Spatial regulation of the glycocalyx component Podocalyxin is a switch for pro-metastatic function

doi: 10.1101/2022.11.04.515043

Figure Lengend Snippet: (A-R) Primary tumour (PT) and metastasis in mice injected with PC3 (A-K) expressing Scramble or PODXL shRNA, n=12 mice/condition or (L-R) sorted for low or high-surface PODXL, n=10 mice/condition. (A, L) PT incidence. In mice with PT, (B, M) prostate volume by ultrasound, (C, N) prostate weight, (D, P) number of mice with metastases, (E, Q) number of metastasis-containing organs/mouse, in mice with metastasis, (F, R) metastasis site tropism. (G-H) Heatmap, weighted histoscore for PODXL or GAL3 across six control mice (Scramble), for PT (Prostate) or metastatic sites. (I-J) Pearson Correlation of Histoscore from PT of six control mice (Scramble) with number of organs in bearing macrometastasis. (K) Serial sections from Mouse ID=3 of the PT (prostate) and metastasis (Abdominal Wall), stained for H&E, PODXL or GAL3. Scale bar, 100μm. Arrowhead, cortical PODXL. (O) Images at intermediate magnification (H&E, scale bar 200μm) with high magnification inserts (H&E, scale bar 100μm). Low-surface PODXL tumours (i-ii), black arrows, solid growth with rounded contours, black arrowheads, geographic necrosis with sparse islands of viable tumour around capillaries. High surface PODXL tumours (iii-iv), yellow arrowheads, looser architecture, infiltrative growth with frequent lymphovascular space invasion, yellow arrow, infiltrative focus. Box-and-whiskers plots, dots, values from individual mice; +, mean; midline, median; boundaries, quartiles; error bars, min to max values; n=3-10 mice that developed PT. p-values (chi-square test on raw counts for contingency tables or unpaired Student’s 2-tailed t-test for box-and-whiskers plots, ns, not significant,*P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001.

Article Snippet: FFPE sections for Galectin-3 (Gal3) (87985, Cell Signaling) IHC staining were dewaxed using an Agilent pre-treatment module.

Techniques: Injection, Expressing, shRNA, Control, Staining

Journal: bioRxiv

Article Title: Spatial regulation of the glycocalyx component Podocalyxin is a switch for pro-metastatic function

doi: 10.1101/2022.11.04.515043

Figure Lengend Snippet: (A-B) PODXL mRNA expression in (A) normal prostate, primary tumour and metastasis or (B) primary tumours in Disease-free or recurred patients. (C) Progression-free survival (% patients, over months) in patient cohorts of high (quartile 4, Q4) versus not high (quartiles 1-3, Q1-3) PODXL mRNA expression. (D-E) LGALS3 mRNA expression in (D) normal prostate, primary tumour and metastasis or (E) primary tumours in disease-free or recurred patients. (F) Progression-free survival (% patients, over months) in patient cohorts of low (quartile 1, Q1) versus not low (quartiles 2-4, Q2-4) LGALS3 mRNA expression. (G-L) Clinical parameters in patient cohorts for combinations of high PODXL (Q4) and low LGALS3 (Q1). (G) Sample type (Normal, Primary Tumour, Metastasis; % patients). (H) Disease-free versus recurred status (% patients). (I) Tumour stage (% samples). (J). Radiation Therapy status (% samples). (K) New neoplasm post initial therapy status (% samples). (L) Progression-free survival (% patients, over months). (M) Model, PODXL-GAL3 interplay controlling invasion. Box-and-whisker graphs (A, B, D, E): 10-90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. Patient numbers presented in figure. Statistical analyses, Kruskal–Wallis Test (A, D), Welch’s t-test (B, E), Log-rank t-test (C, F, L) or chi-square test (G, H, J, K, L). p values: ns, not significant,*P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001. Data, Taylor (GSE21032) or TCGA (PRAD cohort).

Article Snippet: FFPE sections for Galectin-3 (Gal3) (87985, Cell Signaling) IHC staining were dewaxed using an Agilent pre-treatment module.

Techniques: Expressing, Whisker Assay