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. 2019 Feb 4;9:1343. doi: 10.1038/s41598-018-37951-3

Figure 4.

Figure 4

G-1 reduces Hla-mediated keratinocyte permeability barrier disruption. HaCaTs were grown to confluence in the presence of vehicle or 100 nM G-1 prior to challenge (t = 0) with 1 µg/mL Hla or HlaH35A (indicated by arrow in (a)). Changes in permeability barrier resistance at 1000 Hz were measured by ECIS. (a) Representative ECIS recording of HaCaT monolayer treated as described above. (b) Change in HaCaT permeability barrier resistance at 12 h after Hla exposure. Data are mean ± SEM from two (HlaH35A) to six (Control and Hla) independent experiments each with 6–8 technical replicates per condition. Mann-Whitney U test. (c) HaCaT cells were grown in G-1, G15, G-1 + G15 or vehicle control. Shown is the change in permeability barrier resistance at 12 h after Hla exposure. Data are mean ± SEM of a representative experiment of two independent experiments each with a minimum of 8 technical replicates. ANOVA < 0.0001. (d) Western blot analysis of full-length (FL) E-cadherin and the cleaved C-terminal fragment (CTF) in vehicle- and G-1-treated (100 nM) HaCaT cell monolayers after eight hours incubation with 0 or 0.5 µg/mL Hla. MW, molecular weight markers. Four replicates for each group are shown. Image Studio Lite was used to invert luminescent image to that shown to uniformly enhance contrast for quantification of CTF. (e) Relative quantification of E-cadherin FL (left) and CTF (right) of samples in (c) and based on band intensity relative to vehicle-treated cells in the absence of Hla. (f) Quantitative PCR analysis of CDH1 and (g) ADAM10 transcription by vehicle- and G-1-treated (100 nM) HaCaT cells. Expression is shown relative to GAPDH and normalized to vehicle-treated control cells. (h) Surface expression of ADAM10 on vehicle- and G-1-treated (100 nM) HaCaT cells measured by immunofluorescent staining and flow cytometry. Shown is percent expression relative to vehicle-treated controls. Data are mean ± SEM from two independent experiments each with three technical replicates per condition. Unpaired t-test: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.