Journal: Scientific Reports

Article Title: GPER activation protects against epithelial barrier disruption by Staphylococcus aureus α-toxin

doi: 10.1038/s41598-018-37951-3

Figure Lengend Snippet: 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 Hla H35A (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 (Hla H35A ) 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.

Article Snippet: ECIS 96W10idf disposable electrode arrays (Applied Biophysics) were coated with 25 μl of 0.01% poly-L-lysine (Millipore Sigma, Burlington, MA, USA) and incubated at least 15 min. Poly-L-lysine was then removed and replaced with media A (250 μl DMEM low without phenol red, with sodium pyruvate, L-glutamine, 1% MEM-NEAA and 10% charcoal-stripped FBS (JR Scientific, Woodland, CA, USA)) and TER was measured every 10 min at 1000 Hz.

Techniques: Permeability, Disruption, Control, MANN-WHITNEY, Western Blot, Incubation, Molecular Weight, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Expressing, Staining, Flow Cytometry