Article Figures & Data
Figures
- Figure 1. LecB localisation in chronically infected human wounds.
(A, B) Tissue sections of human infected wounds embedded in paraffin and stained for (A) P. aeruginosa (green) and for (B) LecB (green). Normal skin is used as negative control. Note: green signal in the upper left panel (A) is due to unspecific staining in the stratum corneum, not present in wounds. Rectangular squares refer to the zoomed area. Arrows point at LecB localised in the epidermal layers; arrowheads indicate LecB distributed in the dermis.
- Figure 2. LecB depletes IGF-1R from the plasma membrane without inducing its activation.
(A) Western blot of eluted samples from pull-down assay. NHKs were stimulated in the presence of 5 μg/ml biotinylated LecB (106 nM). The lysates were incubated with streptavidin beads and further eluted. Western blot was performed and IGF-1R and LecB were detected in the precipitated fractions. (B, C) Surface staining of keratinocytes treated with LecB (5 μg/ml) for the indicated time points. (B) Images show maximum intensity projections. After stimulation, the cells were stained for IGF-1R (red) and DAPI (blue). Scale bar: 10 μm. (C) Quantification of IGF-1R surface intensity from n = 4 independent experiments. Bars display the mean value ± SEM. ** denotes P < 0.01; **** denotes P < 0.0001; one-way ANOVA was used for statistical analysis. (D) Representative blots from lysates after LecB (5 μg/ml) and IGF-1 (100 ng/ml) stimulation for the indicated times. Antibodies against different phosphorylation sites of IGF-1R (Tyr1131 and Tyr1135/1136) and against total IGF-1R were used. Tubulin was used as loading control. (E, F, G) Blot quantification. Phosphorylated IGF-1R (E, F) and total IGF-1R levels (G) are represented as fold change compared with the loading control from n = 3 independent experiments. * denotes P < 0.05; *** denotes P < 0.001; two-way ANOVA was used for statistical analysis.
- Figure S1. LecB leads to IGF-1R degradation.
(A, B) Western blot of eluted samples from pull-down assay using biotinylated LecB (5 μg/ml) with or without 30 mM L-fucose. Membranes were probed for IGF-1R, EGFR, LecB, and GAPDH. M indicates marker lanes. (C) Whole-cell staining of IGF-1R (red) and LAMP1 (green) upon LecB treatment from 30 min to 6 h. Arrows point at IGF-1R accumulations, whereas arrowheads indicate colocalisation between LAMP1 and IGF-1R. Scale bar: 10 μm. (D) Quantification of LAMP1 intensity. Bars show the mean value ± SEM of N = 3 independent experiments. **** denotes P < 0.0001; one-way ANOVA was used for statistical analysis. (E) Western blot of IGF-1R after 3 h treatment with LecB with or without 100 nM bafilomycin A1. (F) Quantification of the fold change of the receptor levels normalised to actin. Bars show the mean value ± SEM of N = 8 independent experiments. * denotes P < 0.05, ** denotes P < 0.01, ns denotes not significant; one-way ANOVA was used for statistical analysis.
- Figure S2. IGF-1R surface expression upon IGF-1 stimulation.
(A) Surface staining of IGF-1R (red) upon stimulation of keratinocytes with 100 ng/ml IGF-1 for the indicated times. Maximum intensity projections are depicted. Nuclei are shown by DAPI (blue). (B) Quantification of IGF-1R surface intensity. Bars show the mean value ± SEM of N = 3 independent experiments. **** denotes P < 0.0001; one-way ANOVA was used for statistical analysis.
- Figure 3. LecB impairs cell survival signalling pathways and leads to cell cycle arrest.
(A, B, C, D, E) Representative blots and relative quantifications of N = 3 independent experiments. NHKs were treated with 5 μg/ml LecB for the indicated time and lysates were subjected to SDS–PAGE and Western blot analysis using the designated anti-phospho (A) and anti-pan antibodies (B). GAPDH was added as loading control. Graphs (C, D, E) depict the fold change of the phosphorylated protein compared with pan levels and represent the mean value ± SEM. ** denotes P < 0.01, **** denotes P < 0.0001, ns denotes not significant; multiple t tests were used for statistical analysis. (F) Western blot showing cyclin D1 levels after 12 and 24 h of LecB stimulation (5 or 10 μg/ml). (G) Quantification of cyclin D1 relative to tubulin. The mean value ± SEM of N = 3 independent experiments is reported. * denotes P < 0.05, ** denotes P < 0.01; one-way ANOVA was used for statistical analysis. (H) MTT assay assessing the cytotoxic effect of LecB. NHKs were treated with the indicated LecB concentration with or without 30 mM L-fucose for 24 h. Staurosporine (1 μM) was used as positive control. MTT was added to the medium and left for 4 h. The absorbance at 570 nm was measured and plotted as fold change compared with the untreated or L-fucose–treated sample. The mean value ± SEM of N = 5 is plotted. ** denotes P < 0.01; one-way ANOVA was used for statistical analysis. (I) Representative images of keratinocyte monolayers after 24-h exposure to 5 μg/ml LecB with or without 30 mM L-fucose. White arrows in the zoomed image point at vacuolar structures induced by lectin treatment. Scale bar: 200 μm.
- Figure S3. AMPK activation is blocked by L-fucose supplementation.
(A, B) Whole-cell lysates were analysed by Western blot to detect levels of pAMPK upon 4-h incubation with LecB (5 μg/ml) ± 30 mM L-fucose. The phosphorylated protein levels were normalised to actin. The graph reports the mean value ± SEM of N = 3 independent experiments. ** denotes P < 0.01, ns denotes not significant; one-way ANOVA was used for statistical analysis.
- Figure 4. The cytotoxic effect of LecB is preceded by the formation of intraluminal vesicle-containing vacuoles.
(A, B) NHKs were incubated with 5 μg/ml LecB and processed for conventional electron microscopy at 12 h post-incubation. (A, B) Representative electrographs of the untreated (A) and LecB-treated (B) cells. Black hashtags point at Category 1 vacuoles, whereas black and red asterisks indicate Category 2 and Category 3 vacuoles, respectively. Zoomed image of panel (B) shows a higher magnification of the Category 3 vacuoles containing intraluminal vesicles. Scale bars: 1 μm. (C, D, E, F) Cells treated with 5 μg/ml biotinylated LecB or un-tagged LecB for 12 h were subjected to immuno-EM. (C, E) Control staining showing no aspecific antibody binding. (D) LecB localises in internal vesicles, either in the lumen (panel [D] zoom, black arrowheads) or at the limiting membrane (white arrowheads). (F) LecB localisation at the plasma membrane, in the ruffle-like region. Scale bars: 500 nm. Scale bar zoom panel (D): 200 nm. E, endosome; ER, endoplasmic reticulum; G, Golgi apparatus; k, keratin; M, mitochondrion; N, nucleus; PM, plasma membrane.
- Figure S4. LecB containing vesicles originate from ruffle-like regions at the plasma membrane.
(A, B, C, D, E) Representative electrographs of keratinocytes treated with 5 μg/ml LecB for the indicated time points and processed for conventional EM. (A′, B′, C′, D′, E′) Additional area for each indicated condition with higher magnification. Black hashtags point at Category 1 vacuoles, whereas black and red asterisks indicate Category 2 and Category 3 vacuoles, respectively. Rectangular box highlights ruffle-like regions. Scale bars: 1 μm. E, endosome; ER, endoplasmic reticulum; G, Golgi apparatus; k, keratin; M, mitochondrion; N, nucleus; PM, plasma membrane.
- Figure 5. LecB localises in LC3- and RAB9-positive compartments.
(A, B) Confocal micrographs with respective colocalisation quantification of keratinocytes stimulated with 5 μg/ml of fluorescently labelled LecB (green). Panels indicated as “+LecB” refer to 12 h incubation. (A, B) After fixation and permeabilisation, the cells were stained for LC3 (A) and RAB9 (B) (both in red). Graphs report the mean value ± SEM of Mander’s overlap coefficients calculated from at least three independent experiments. **** denotes P < 0.0001; one-way ANOVA was used for statistical analysis. Scale bar: 10 μm. (C, D) Western blot of eluted samples from time series pull-down assays using biotinylated LecB (5 μg/ml). (C, D) Membranes were probed for LecB, IGF-1R (C) and ubiquitin (D).
- Figure S5. LecB colocalises with LAMP1 positive compartments.
(A, B) Confocal micrographs of NHKs stimulated with 5 μg/ml of fluorescently labelled LecB (green) permeabilised and stained for (A) LAMP1 and (B) RAB11 (both in red). Panels indicated as “+LecB” refer to 12-h incubation. Note that LAMP1 signal in the untreated panel is not visible due to the high difference in intensity between untreated and treated conditions. Scale bar: 10 μm. Graphs right to the panels show the quantification of Mander’s overlap coefficients between LecB and LAMP1 and LecB and RAB11. Error bars indicate means ± SEM of N = 3 independent experiments. ** denotes P < 0.01, **** denotes P < 0.0001; one-way ANOVA was used for statistical analysis.
- Figure 6. IGF-1R is sorted to degradation by LecB.
(A, B) NHKs were incubated with LecB (5 μg/ml) for the indicated time points and whole cell lysates were immunoblotted for LC3 and β-actin (A). (B) The levels of LC3-II were normalised to β-actin and depicted as fold change increase to the untreated condition (B). Error bars indicate means ± SEM of N = 5 independent experiments. * denotes P < 0.05, ** denotes P < 0.01; one-way ANOVA was used for statistical analysis. (C) Confocal micrographs of keratinocytes treated with 5 μg/ml of fluorescently labelled LecB (pink) and stained for LC3 (red) and IGF-1R (green). Panel indicated as “+LecB” refers to 12 h incubation. White arrows point at colocalisation among LecB, LC3, and IGF-1R. Scale bar: 10 μm. N = 3. (D, E) Representative images of NHKs treated with LecB and stained for (D) IGF-1R (green), LC3 (red), and (E) TfR (green). Intensity profiles of single cells, along the yellow line are shown. Scale bar: 10 μm.
- Figure S6. LecB-mediated increase of LC3-II is ATG13 independent.
(A, B, C, D) Representative blots and relative quantifications. The cells were treated with 5 μg/ml of LecB for 12 h ± 200 nM bafilomycin A1 (Bafi) or ±20 μg/ml cycloheximide (Cyclo). Whole-cell lysates were probed for LC3 and β-actin was used as loading control. (A, B, C, D) Graphs indicate the fold change of LC3-II levels, expressed as means ± SEM of N = 4 (A, B) and N = 6 (C, D) independent experiments. *** denotes P < 0.001, **** denotes P < 0.0001, ns denotes not significant; one-way ANOVA was used for statistical analysis. (E, F, G) ATG13 was silenced via siRNA and its expression quantified by Western blot (F). (G) Silenced cells were treated with LecB for 3 h and LC3-II levels were quantified (G). Graphs report the mean value ± SEM of N = 4 independent experiments. ** denotes P < 0.01, **** denotes P < 0.0001, ns denotes not significant; one-way ANOVA was used for statistical analysis.
- Figure S7. Cytochalasin D reduces LecB uptake.
(A) Confocal micrographs after 45-min incubation with 5 μg/ml of fluorescently labelled LecB (green) with or without cytochalasin D (0.5 μg/ml). Cells were stained for the early endosome marker EEA1 (red) and for the nuclei (blue). Scale bar: 10 μm. (B) Quantification of LecB intensity per cell. Graphs report the mean value ± SEM of N = 4 independent experiments. * denotes P < 0.05, unpaired t test was used for statistical analysis.
- Figure S8. IGF-1 does not affect LC3 distribution.
Representative confocal images depicting NHKs treated with 100 ng/ml IGF-1 or 100 nM bafilomycin A1 for 12 h and stained for LC3 (green) and IGF-1R (red). Scale bar: 10 μm. Figure S9. Western blot of eluted samples from pull-down assays with biotinylated LecB (5 μg/ml) with or without 30 mM L-fucose. Membranes were probed for TrR and LecB. M indicates the marker lane.
- Figure S9. Transferrin receptor is not coprecipitated upon LecB treatment.
Supplementary Materials
Table S1 List of coprecipitated growth factor receptors identified by mass spectrometry analysis.
Source data are available for this table.
Source Data for Table S1[LSA-2019-00422_Sdata_TS1.xlsx][LSA-2019-00422_Sdata_TS2.txt]
Table S2 List of primary antibodies used.
Table S3 List of secondary antibodies used.
