uncorrected fisher’s lsd test  (GraphPad Software Inc)

Journal: Scientific Reports

Article Title: Tissue-specific endothelial cell heterogeneity contributes to unequal inflammatory responses

doi: 10.1038/s41598-020-80102-w

Figure Lengend Snippet: Chemokine and cytokine production by cytokine-activated endothelial cells. Endothelial monolayers were stimulated with TNFα (20 ng/mL) or IL-1β (20 ng/mL). ( a ) After 4 h, stimulated endothelial cells were lysed in RLT buffer, and mRNA for immune response genes was measured by Nanostring. Results are presented in the heat map, with hierarchical clustering and color scale representing relative mRNA counts of each chemokine across conditions. Heat maps is transformed by row min/max with hierarchical clustering using one minus Pearson correlation and grouped by stimulus. ( b ) After 18 h, conditioned media were tested for secreted factors by Luminex and ELISA. Results are presented in the heat map, with hierarchical clustering and color scale normalized to represent relative protein concentrations (pg/mL) of each secreted chemokine protein across conditions (n = 5). ( c ) Normalized mRNA counts for CCL5 (RANTES) gene expression across endothelial cells stimulated for 4 h with TNFα or IL-1β. ( d ) Fold increase in the concentration of secreted RANTES (CCL5) protein after 18 h across endothelial cells (n = 3). Results are presented as mean ± SEM. TNFα-induced RANTES was significantly lower from HAEC and HCAEC compared with HCMVEC, HPMVEC, and HLSEC (all p < 0.001, by two way ANOVA followed by Tukey’s multiple comparison’s test). IL-1β-induced RANTES was significantly lower from HAEC than microvascular EC ( p < 0.05). ( e ) Normalized mRNA counts for CX3CL1 (fractalkine) gene expression across endothelial cells stimulated for 4 h with TNFα or IL-1β. ( f ) Concentration of secreted fractalkine (CX3CL1) protein after 18 h across endothelial cells measured by Luminex (n = 4). Results are presented as mean ± SEM. IL-1β-induced fractalkine was significantly lower from HAEC and HLSEC compared with HCAEC and HPMVEC (all p < 0.05, by two way ANOVA followed by uncorrected Fisher’s LSD).

Article Snippet: Backtransformed data were also analyzed for differences in individual gene expression by one way ANOVA followed by uncorrected Fisher’s LSD t test (GraphPad).

Techniques: Transformation Assay, Luminex, Enzyme-linked Immunosorbent Assay, Gene Expression, Concentration Assay

Journal: Scientific Reports

Article Title: Tissue-specific endothelial cell heterogeneity contributes to unequal inflammatory responses

doi: 10.1038/s41598-020-80102-w

Figure Lengend Snippet: Adhesion molecule expression by cytokine-activated endothelial cells. ( a ) After 4 h activation with TNFα (20 ng/mL) or IL-1β (20 ng/mL), stimulated endothelial cells were lysed in RLT buffer, and mRNA for immune response genes was measured by nanostring. Absolute mRNA counts for endothelial adhesion molecule genes are presented in the heat map. ( b , c ) After 4 h or 18-24 h, stimulated endothelial cells were detached and TNFα ( b ) and IL-1β ( c ) induced cell surface E-selectin was measured by flow cytometry. Results are presented as fold increase in the median fluorescence intensity of cell surface E-selectin, normalized within each experiment to the untreated condition for each cell type.( d , e ) After 4 h or 18-24 h, stimulated endothelial cells were detached and TNFα ( d ) and IL-1β ( e ) induced cell surface ICAM-1 was measured by flow cytometry. Results are presented as fold increase in the median fluorescence intensity of cell surface ICAM-1, normalized within each experiment to the untreated condition for each cell type. ( f , g ) After 4 h or 18-24 h, stimulated endothelial cells were detached and TNFα ( f ) and IL-1β ( g ) induced cell surface VCAM-1 was measured by flow cytometry. Results are presented as fold increase in the median fluorescence intensity of cell surface VCAM-1, normalized within each experiment to the untreated condition for each cell type. (TNFα and IL-1β 4 h, n = 4; 18-24 h, n = 5–6 donors per endothelial cell type). # p < 0.1, * p < 0.05, ** p < 0.001 comparing HAEC and HCAEC to HLSEC by two way ANOVA followed by uncorrected Fisher’s LSD. ( h ) mRNA and ( i ) fold increase in the MFI of cell surface expression of VCAM-1 was measured on aortic HAEC and liver HLSEC endothelial cells stimulated with IL-4 (20 ng/mL) for 4 h or 24 h (n = 3 donors per endothelial cell type). Results are presented as mean ± SEM. # p < 0.1 comparing HAEC to HLSEC, by two way ANOVA and uncorrected Fishers LSD.

Article Snippet: Backtransformed data were also analyzed for differences in individual gene expression by one way ANOVA followed by uncorrected Fisher’s LSD t test (GraphPad).

Techniques: Expressing, Activation Assay, Flow Cytometry, Fluorescence

Journal: Scientific Reports

Article Title: Tissue-specific endothelial cell heterogeneity contributes to unequal inflammatory responses

doi: 10.1038/s41598-020-80102-w

Figure Lengend Snippet: Endothelial cell activation by HLA antibody and complement. Endothelial cell monolayers were treated with a polyclonal mixture of anti-HLA I antibodies in the presence of 25% intact human serum complement for 4 h or 24 h. ( a ) Stimulated endothelial cells were lysed in RLT buffer, and mRNA for immune response genes was measured by nanostring. Volcano plots demonstrate changes in gene expression across the 6 primary endothelial cells stimulated with HLA + C’ for 4 h (left panel) and 24 h (right panel). ( b ) After 4 h and 24 h exposure to HLA + C, mRNA expression of chemokines was measured by Nanostring (n = 1). Results are presented in the heat map, with hierarchical clustering and color scale representing relative mRNA counts of each chemokine across conditions. ( c ) After 4 h or 24 h treatment with DSA + C, conditioned media were tested for secreted factors by Luminex and ELISA. Results are presented in the heat map, with color scale representing fold increase in protein concentrations (pg/mL) of each chemokine compared to untreated. ( d ) Mean fold increase in the MFI of cell surface E-selectin, ICAM-1 and VCAM-1 across HAEC, HCAEC and HLSEC is shown, after stimulation with HLA monoclonal antibodies and intact human complement for 4 h (n = 3 donors per endothelial cell type). Results are presented as mean ± SEM. * p < 0.05, *** p < 0.0001 comparing HLSEC to HAEC, by two way ANOVA and uncorrected Fisher’s LSD. E-selectin: HAEC versus HLSEC p < 0.001; HCAEC versus HLSEC p = 0.038. VCAM-1: HAEC versus HLSEC p = 0.0212; HCAEC versus HLSEC p = 0.0175. ( e ) Mean fold increase in the MFI of cell surface E-selectin, ICAM-1 and VCAM-1 across HAEC, HCAEC and HLSEC is shown, after stimulation with HLA monoclonal antibodies and intact human complement for 24 h (n = 3 donors per endothelial cell type). Results are presented as mean ± SEM. * p < 0.05, *** p < 0.0001 comparing HLSEC to HAEC, by two way ANOVA and uncorrected Fisher’s LSD.

Article Snippet: Backtransformed data were also analyzed for differences in individual gene expression by one way ANOVA followed by uncorrected Fisher’s LSD t test (GraphPad).

Techniques: Activation Assay, Gene Expression, Expressing, Luminex, Enzyme-linked Immunosorbent Assay, Bioprocessing