Fig 1: Skin abnormalities following galectin-7 overexpression in basal epidermal keratinocytes.(A) Galectin-7 expression vector. The rat galectin-7 cDNA was inserted into the K14 expression plasmid comprising the human K14 promoter, the rabbit ß-globin intron and the K14 polyA sequence [9]. Two independent transgenic lines were obtained. (B) Western blot analysis. Skin protein extracts from wild-type (wt), tg 34 and tg 46 transgenic mice were used. Galectin-7 (14 kDa) and control β-tubulin (55 kDa) proteins were detected. (C) Representative semi-thin sections of wt, tg 34 and tg 46 tail skin are shown. Epidermal thickening, cell disorganization and loose basal lamina are observed in tg 34 and tg 46 skin compared to wt tissue. Scale bar: 20μm. (D) Quantification of cell proliferation. A significantly higher number of EdU-positive keratinocytes per mm of linear epidermis was detected in tg 34 and tg 46 samples compared to wt. A total of 34 mice were used for these measurements (Nwt = 12; Ntg 34 = 10; Ntg 46 = 12). Each bar represents the mean value ± sem. Statistical differences between wt and transgenic animals are noted *p<0.05, ***p<0.001.
Fig 2: Galectin-7 stabilizes E-cadherin at the plasma membrane in mature adherens junctions. (a) Mean surface fluorescence of E-cadherin measured by FACS in non-permeabilized cells. Cells were detached from the plate using trypsin and incubated for the indicated time period at 37 °C to let E-cadherin be re-addressed to the plasma membrane. Mean values ± s.d. are represented (n = 3). (b) Representative images of E-cadherin-GFP signal acquired during FRAP experiments in HaCaT cells (upper panel) and in shGal7 #2 clone (lower panel). Arrows indicate the bleach area. Scale bar = 10 µm. (c) Normalized fitted FRAP curves (dark) and mean of experimental data (red and blue) showing E-cadherin-GFP recovery after photobleaching. Mean values ± s.e.m are represented (n = 39). (d,e) Graphs showing halftime recovery (t1/2) (d) and mobile fraction (e) extracted from one phase association fitting curves. Mean values ± s.e.m are represented.
Fig 3: Galectin-1 and -7 play critical roles in the defense against GAS infection. (A) Galectin-1-knockout HeLa cells were generated using the CRISPR/Cas9 gene-editing system. Immunoblotting was used to detect galectin-1 protein expression in wild-type and galectin-1-knockout cells. (B) Wild-type and galectin-1-knockout HeLa cells were transfected with EmGFP-LC3; after infecting with GAS for 2 or 4 h, immunofluorescence analysis was used to examine the formation of bacterium-containing LC3-vacuoles. (C) Cells harboring GAS-containing LC3-vacuoles were manually counted using immunofluorescence confocal microscopy. In each experiment, >100 autophagosomes were evaluated per sample; bars: means ± SD of 3 independent experiments. (D) Adhesion rate of GAS in wild-type and galectin-1-knockout HeLa cells. Bars: means ± SD of 3 independent experiments. (E) Invasion rate and survival rate of GAS in wild-type and galectin-1-knockout HeLa cells. (F) Galectin-7- or Atg5-knockdown HaCaT cells were generated using siRNAs. Immunoblotting was used to detect galectin-7 and Atg5 protein expression. (G) Adhesion rate of GAS in wild-type and galectin-7- or Atg5-knockdown HaCaT cells. Bars: means ± SD of 3 independent experiments. (H) Invasion rate and survival rate of GAS in wild-type and galectin-7- or Atg5-knockdown HaCaT cells. Bars: means ± SD of 3 independent experiments. *P < 0.02, **P < 0.01.
Fig 4: Tollip is involved in the recruitment of galectin-1 and -7 to GAS-containing LC3-vacuoles. (A) Wild-type and Tollip-knockout HeLa cells transfected with EmGFP-galectin-7 and mCherry-LC3 were infected with GAS; at 4 hpi, immunofluorescence analysis was performed to examine the localization of the expressed proteins. (B) EmGFP-galectin-positive autophagosomes were manually counted using immunofluorescence confocal microscopy. In each experiment, >50 GAS-containing LC3-vacuoles were counted and evaluated per sample; bars: means ± SD of 3 independent experiments. (C) Wild-type and Tollip-knockout HeLa cells transfected with EmGFP-LC3 were infected with GAS; at 4 hpi, immunofluorescence analysis was used to examine the localization of endogenous galectin-1 and EmGFP-LC3; galectin-1 was labeled using a rabbit anti-galectin-1 antibody. (D) Cells containing galectin-1-positive GAS-containing autophagosomes were manually counted using immunofluorescence confocal microscopy. In each experiment, >50 GAS-containing autophagosomes were counted and evaluated per sample; bars: means ± SD of 3 independent experiments. (E) Wild-type and Tollip-knockout HeLa cells were infected with GAS; at 4 hpi, immunofluorescence analysis was performed to examine the SLO protein expression from GAS. Scale bar, 10 µm. (F) Cells containing SLO-positive GAS were manually counted using immunofluorescence confocal microscopy. In each experiment, >200 GAS-infected cells were evaluated per sample; bars: means ± SD of 3 independent experiments. (G) Tollip interacts with galectin-7. HeLa cells were co-transfected with EmGFP-galectin-7 and FLAG-Tollip for 48 h, after which immunoprecipitation was performed using anti-FLAG. The immunoprecipitates were immunoblotted with anti-GFP to detect EmGFP-galectin-7. Experiments were repeated more than three times. *P < 0.05.
Fig 5: Galectin-7 impacts E-cadherin dynamics at the plasma membrane. (a) Total cell surface proteins were purified using labelling with biotin and pull down via avidin affinity. Immunoblots show similar levels of surface E-cadherin in HacaT cells or in shGal7 clones. Transferrin receptor was used as a loading control. (b) Quantification of internalized E-cadherin in HaCaT or shGal7 #2 cells at 15, 30, 60 or 120 min after addition of HECD-1 antibody. (c) Representative images of internalized E-cadherin after 5 min incubation with HECD-1 antibody, directed against E-cadherin ectodomain. Maximum intensity projection of Z-stacks (Z-steps = 0.21 µm) obtained with ImageJ. Scale bar = 10µm (left panels) or 5µm (zoom, right panels). (d,e) Quantification of internalized E-cadherin during 30 min at 37 °C after addition of 0 to 1 µg.ml-1 recombinant galectin-7 (rGal7) in shGal7 #2 clone (d) or control HaCaT (e). (f) Quantification of internalized E-cadherin in HaCaT or shGal7 #2 cells during 30 min at 37 °C after addition of 0 or 0.5 µg.ml-1 recombinant CRD-mutated galectin-7 (R74S). (b,d,e,f) Between 15 and 17 measures per condition were performed in n = 3 independent experiments. Mean values ± s.e.m. are represented. P-values obtained from unpaired t-tests with Welch’s correction are represented. ***p < 0.001; **p < 0.01; *p < 0.05.
Supplier Page from Abcam for Anti-Galectin 7 antibody