Journal: Journal of Extracellular Vesicles

Article Title: Epidermal Stem Cell‐Derived Extracellular Vesicles Induce Fibroblasts Mesenchymal‐Epidermal Transition to Alleviate Hypertrophic Scar Formation via miR‐200s Inhibition of ZEB1 and 2

doi: 10.1002/jev2.70160

Figure Lengend Snippet: ESC‐EVs were internalized and effectively inducing the MET of HSFs. (a) Confocal imaging showed the internalized PKH67‐labeled ESC‐EVs (green) in ACTIN‐labelled HSFs (red). Scale bar, 25 µm. (b) Western blotting analysis of COLI, α‐SMA, N‐cad, K1, K15 and E‐cad in HSFs incubated with PBS, ESC‐EVs at concentrations of 1.25, 2.5, 5 or 10 µg/mL or FB‐EVs (10 µg/mL). (c) Quantification of the relative band density compared to GAPDH. Data are presented as mean values ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. (d) RT‐qPCR analysis of the mesenchymal markers COLI, α‐SMA and N‐cad and epithelial markers K1, K15 and E‐cad in HSFs treated with ESC‐EVs, FB‐EVs or PBS. Graph represented the expression of the markers relative to that of GAPDH. Data are presented as mean values ± SD ( n = 9). Statistical analysis was performed using one‐way ANOVA. (e) Representative images of COLI, α‐SMA, N‐cadherin (green) immunofluorescence staining in ACTIN‐labelled HSFs (red) incubated with ESC‐EVs or PBS for 24 h. Scale bar, 50 µm. (f) Quantification of the relative fluorescence ratio. Data are presented as mean values ± SD ( n = 4). Statistical analysis was performed using one‐way ANOVA. (g) Representative images of K1, K15 and E‐cadherin (green) immunofluorescence staining in ACTIN‐labelled HSFs (red) incubated with ESC‐EVs or PBS for 24 h. Scale bar, 50 µm. (h) Quantification of the relative fluorescence ratio. Data are presented as mean values ± SD ( n = 4). Statistical analysis was performed using one‐way ANOVA. Significance levels are indicated as * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001.

Article Snippet: Cells were washed and incubated with secondary fluorescence antibodies (1:2000, 4408S, 4409S, 4412S, 4413S, Cell signalling Technology) for 1 h and DAPI for 10 min at room temperature.

Techniques: Imaging, Labeling, Western Blot, Incubation, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Epidermal Stem Cell‐Derived Extracellular Vesicles Induce Fibroblasts Mesenchymal‐Epidermal Transition to Alleviate Hypertrophic Scar Formation via miR‐200s Inhibition of ZEB1 and 2

doi: 10.1002/jev2.70160

Figure Lengend Snippet: miR‐200s were enriched in ESC‐EVs and targeting ZEBs in HSFs. (a) Heatmap of the 20 miRNAs with the largest LogFC in the microarray analysis of the miRNA expression profiles between ESC‐EVs and FB‐EVs. (b) Volcano annotated with miR‐200s probes (ESC‐EVs vs. FB‐EVs). Type, High expression (red), Low expression (blue) and No significance (grey). (c) Chord diagram of GO enrichment terms. (d) Chord diagram of KEGG enrichment terms. (e) RT‐qPCR analysis of the expression of miR‐200s in HSFs treated with ESC‐EVs, FB‐EVs or PBS for 24 h. Graph represented the expression of the markers relative to that of U6. Data are presented as mean values ± SD ( n = 9). Statistical analysis was performed using one‐way ANOVA. (f) RT‐qPCR analysis of the expression of ZEB1 and ZEB2 in HSFs relative to GAPDH. Data are presented as mean values ± SD ( n = 9). Statistical analysis was performed using one‐way ANOVA. (g) Western blotting analysis of ZEB1 and ZEB2 in HSFs incubated with PBS, ESC‐EVs at concentrations of 1.25, 2.5, 5 or 10 µg/mL or FB‐EVs (10 µg/mL). (h) Quantification of the relative band density compared to GAPDH. Data are presented as mean values ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. (i) Representative images of ZEB1 and ZEB2 (green) immunofluorescence staining in ACTIN‐labelled HSFs (red). Scale bar, 50 µm. (j) Quantification of the relative fluorescence ratio. Data are presented as mean values ± SD ( n = 5). Statistical analysis was performed using one‐way ANOVA. Significance levels are indicated as * p < 0.05, ** p < 0.005, *** p <0.0005 and **** p <0.0001.

Article Snippet: Cells were washed and incubated with secondary fluorescence antibodies (1:2000, 4408S, 4409S, 4412S, 4413S, Cell signalling Technology) for 1 h and DAPI for 10 min at room temperature.

Techniques: Microarray, Expressing, Quantitative RT-PCR, Western Blot, Incubation, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Epidermal Stem Cell‐Derived Extracellular Vesicles Induce Fibroblasts Mesenchymal‐Epidermal Transition to Alleviate Hypertrophic Scar Formation via miR‐200s Inhibition of ZEB1 and 2

doi: 10.1002/jev2.70160

Figure Lengend Snippet: HS exhibited miR‐200s deficiency and ZEBs accumulation. (a) FISH stains of miR‐200s in normal skin (NS), hyperplasia stage (HS) and mature stage of hypertrophic scar (MS). Scale bar, 20 µm. (b) Quantification of the relative fluorescence ratio. Data are presented as mean values ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. (c) mIHC stains of α‐SMA, ZEB1, ZEB2, COLI, α‐SMA, N‐Cad and K14, E‐Cad in normal skin, hyperplasia stage or mature stage of hypertrophic scar. Scale bar, 200 µm, 20 µm. (d) Quantification of the relative positive cells. Data are presented as mean values ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. Significance levels are indicated as * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001.

Article Snippet: Cells were washed and incubated with secondary fluorescence antibodies (1:2000, 4408S, 4409S, 4412S, 4413S, Cell signalling Technology) for 1 h and DAPI for 10 min at room temperature.

Techniques: Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Epidermal Stem Cell‐Derived Extracellular Vesicles Induce Fibroblasts Mesenchymal‐Epidermal Transition to Alleviate Hypertrophic Scar Formation via miR‐200s Inhibition of ZEB1 and 2

doi: 10.1002/jev2.70160

Figure Lengend Snippet: ESC‐EVs induced MET of HSFs via miR‐200s/ZEBs axis. (a) The multiplex CRISPR/Cas9 in a single lentiviral vector. (b) Five sgRNAs are indicated by scissors targeting two genomic loci of human miR‐200 family host genes. (c) RT‐qPCR analysis of EVs secreted from wild‐type ESC‐EVs (WT), ESCs infected with scramble vectors (Scramble), or miR‐200s knockdown vectors (KD). Data are presented as mean values ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. (d) RT‐qPCR analysis of COLI, α‐SMA, N‐cad, K1, K15 and E‐cad in HSFs. Graph represented the expression of the markers relative to that of GAPDH. Data are presented as mean values ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. (e) Western blotting analysis of COLI, α‐SMA, N‐cad, K1, K15 and E‐cad in HSFs. (f) Quantification of the relative band density compared to α‐tubulin. Data are presented as mean values ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. (g) Representative images of COLI, α‐SMA, N‐cad, K1, K15 and E‐cad (green) immunofluorescence staining in ACTIN‐labeled HSFs (red) incubated with WT‐EVs, Scramble‐EVs, KD‐EVs, or PBS for 24 h. Scale bar, 50 µm. (h) Quantification of the relative fluorescence ratio. Data are presented as mean values ± SD ( n = 4). Statistical analysis was performed using one‐way ANOVA. (i) Representative images of ZEB1 and ZEB2 (green) immunofluorescence staining in ACTIN‐labelled HSFs (red). Scale bar, 50 µm. (j) Quantification of the relative fluorescence ratio. Data are presented as mean values ± SD ( n = 4). Statistical analysis was performed using one‐way ANOVA. (k) RT‐qPCR analysis of the expression of ZEB1 and ZEB2 in HSFs relative to GAPDH. Data are presented as mean values ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. (l) Western blotting analysis of ZEB1 and ZEB2 in HSFs. (m) Quantification of the relative band density compared to α‐tubulin. Data are presented as mean values ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. Significance levels are indicated as * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001.

Article Snippet: Cells were washed and incubated with secondary fluorescence antibodies (1:2000, 4408S, 4409S, 4412S, 4413S, Cell signalling Technology) for 1 h and DAPI for 10 min at room temperature.

Techniques: Multiplex Assay, CRISPR, Plasmid Preparation, Quantitative RT-PCR, Infection, Knockdown, Expressing, Western Blot, Immunofluorescence, Staining, Labeling, Incubation, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Epidermal Stem Cell‐Derived Extracellular Vesicles Induce Fibroblasts Mesenchymal‐Epidermal Transition to Alleviate Hypertrophic Scar Formation via miR‐200s Inhibition of ZEB1 and 2

doi: 10.1002/jev2.70160

Figure Lengend Snippet: ESC‐EVs inhibited the activation of HSFs via miR‐200s. (a) CCK8 assay to detect the impact of both ESC‐EVs, FB‐EVs, or PBS on HSFs cell viability. Data are presented as mean values ± SD ( n = 6). Two‐sided unpaired t ‐test. (b) Representative images of Ki67 (green) immunofluorescence staining in ACTIN‐labelled HSFs (red). Scale bar, 100 µm. (c) Quantification of the relative fluorescence ratio. Data are presented as mean values ± SD ( n = 4). Statistical analysis was performed using one‐way ANOVA. (d) Scratch tests showed the comparison of relative migration areas among ESC‐EVs, FB‐EVs or PBS at 0 and 24 h. Scale bar, 100 µm. (e) Quantification of the migration rate at 6, 12, 24 and 48 h. Data are presented as mean values ± SD ( n =6). Statistical analysis was performed using Two‐sided unpaired t ‐test. (f) Collagen gel contraction test illustrated the contraction of ESC‐EVs, FB‐EVs or PBS populated collagen gels at 0, 24 and 48 h. (g) Quantification of contraction surface areas at 24, 48 and 72 h. Data are presented as mean values ± SD ( n = 4). Statistical analysis was performed using Two‐sided unpaired t ‐test. (h) Scratch tests showed the comparison of relative migration areas among WT‐EVs, Scramble‐EVs, KD‐EVs or PBS at 0 and 24 h. Scale bar, 100 µm. (i) Quantification of the migration rate at 12 and 24 h. Data are presented as mean values ± SD ( n = 6). Statistical analysis was performed using two‐sided unpaired t ‐test. (j) Collagen gel contraction test illustrated the contraction of WT‐EVs, Scramble‐EVs, KD‐EVs or PBS populated collagen gels at 0 and 72 h. (k) Quantification of contraction surface areas at 24, 48 and 72 h. Data are presented as mean values ± SD ( n = 4). Statistical analysis was performed using Two‐sided unpaired t‐test. Significance levels are indicated as * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001.

Article Snippet: Cells were washed and incubated with secondary fluorescence antibodies (1:2000, 4408S, 4409S, 4412S, 4413S, Cell signalling Technology) for 1 h and DAPI for 10 min at room temperature.

Techniques: Activation Assay, CCK-8 Assay, Immunofluorescence, Staining, Fluorescence, Comparison, Migration

Journal: Journal of Extracellular Vesicles

Article Title: Epidermal Stem Cell‐Derived Extracellular Vesicles Induce Fibroblasts Mesenchymal‐Epidermal Transition to Alleviate Hypertrophic Scar Formation via miR‐200s Inhibition of ZEB1 and 2

doi: 10.1002/jev2.70160

Figure Lengend Snippet: ESC‐EVs alleviated the RHS through the miR‐200s/ZEBs axis. (a) FISH stains of miR‐200a (red), miR‐200b (green), miR‐200c (pink), miR‐141 (red), miR‐429 (green) in PBS, RESC‐EVs, RFB‐EVs treated RHS. Scale bar, 20 µm. (b) Quantification of the relative fluorescence ratio. Data are presented as mean values ± SD ( n = 4). Statistical analysis was performed using one‐way ANOVA. (c) Representative images of COLI, α‐SMA, N‐cad, K1, K15, E‐cad, ZEB1 and ZEB2 immunofluorescence staining. Scale bar, 200 µm, 20 µm. (d) Quantification of the relative positive cells. Data are presented as mean values ± SD ( n = 4). Statistical analysis was performed using one‐way ANOVA. Significance levels are indicated as * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001.

Article Snippet: Cells were washed and incubated with secondary fluorescence antibodies (1:2000, 4408S, 4409S, 4412S, 4413S, Cell signalling Technology) for 1 h and DAPI for 10 min at room temperature.

Techniques: Fluorescence, Immunofluorescence, Staining