Figure 1
Figure 1. Modulation of Resto properties under TNF/LT stimulation. (A) Membrane expression of TNFR1 and LTβR on Resto cells. Blue lines indicate immunoglobulin control, red lines indicate specific staining. (B) Induction of an extracellular meshwork. Resto cells and HFFs were cultured for 7 days with or without TNF/LT stimulation. Microscopic visualization of a dense meshwork of extracellular matrix fibers was performed with fibronectin and transglutaminase (TG) extracellular staining. Bar represents 20 μm. (C) Differential effects of TNF and LT treatment. Resto cells were cultured with TNF, LT, TNF/LT or without stimulation for 7 days before transglutaminase (TG), CD54, and CD106 staining. TG was detected by microscopic analysis. Bar represents 20 μm. CD54 and CD106 were revealed by flow cytometry. Blue lines indicate immunoglobulin control, red lines indicate specific staining. Ratio of mean fluorescence intensity (mean fluorescence intensity of CD54 or CD106/mean intensity fluorescence of immunoglobulin-specific control) is indicated on the top right of each panel. (D) Adhesion assay. Tonsil leukocytes or monocyte-derived immature DCs (iDCs) were incubated for 2 hours with Resto cells pretreated or not with TNF/LT for 7 days. Unbound cells were then removed and adherent CD45+ cells and stromal CD45− cells were collected using trypsin and quantified by flow cytometry. Results are the mean values of the ratio of CD45+ to CD45− cells (n = 4). The error bars indicate the SD about the mean. *Mean value is statistically different from that of coculture with unstimulated Resto cells (P < .01). (E) Real-time PCR quantification of chemokine expression. Resto cells were cultured for 3 days with TNF/LT and were then analyzed for CXCL9, CXCL10, CCL5, CXCL12, and CCL19 expression. Each sample was normalized to ABL and compared with expression levels in untreated (UT) Resto cells. The arbitrary value of 1 was assigned to UT Resto cells. CCL19 was not detected in UT Resto cells and the ΔΔCT was then calculated with an arbitrary CT value of 40. Results are those of 1 experiment of 3.

Modulation of Resto properties under TNF/LT stimulation. (A) Membrane expression of TNFR1 and LTβR on Resto cells. Blue lines indicate immunoglobulin control, red lines indicate specific staining. (B) Induction of an extracellular meshwork. Resto cells and HFFs were cultured for 7 days with or without TNF/LT stimulation. Microscopic visualization of a dense meshwork of extracellular matrix fibers was performed with fibronectin and transglutaminase (TG) extracellular staining. Bar represents 20 μm. (C) Differential effects of TNF and LT treatment. Resto cells were cultured with TNF, LT, TNF/LT or without stimulation for 7 days before transglutaminase (TG), CD54, and CD106 staining. TG was detected by microscopic analysis. Bar represents 20 μm. CD54 and CD106 were revealed by flow cytometry. Blue lines indicate immunoglobulin control, red lines indicate specific staining. Ratio of mean fluorescence intensity (mean fluorescence intensity of CD54 or CD106/mean intensity fluorescence of immunoglobulin-specific control) is indicated on the top right of each panel. (D) Adhesion assay. Tonsil leukocytes or monocyte-derived immature DCs (iDCs) were incubated for 2 hours with Resto cells pretreated or not with TNF/LT for 7 days. Unbound cells were then removed and adherent CD45+ cells and stromal CD45− cells were collected using trypsin and quantified by flow cytometry. Results are the mean values of the ratio of CD45+ to CD45− cells (n = 4). The error bars indicate the SD about the mean. *Mean value is statistically different from that of coculture with unstimulated Resto cells (P < .01). (E) Real-time PCR quantification of chemokine expression. Resto cells were cultured for 3 days with TNF/LT and were then analyzed for CXCL9, CXCL10, CCL5, CXCL12, and CCL19 expression. Each sample was normalized to ABL and compared with expression levels in untreated (UT) Resto cells. The arbitrary value of 1 was assigned to UT Resto cells. CCL19 was not detected in UT Resto cells and the ΔΔCT was then calculated with an arbitrary CT value of 40. Results are those of 1 experiment of 3.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal