Human and murine Mks express collagen VI. (A) RT-PCR of COL6A1, COL6A2, and COL6A3 messenger RNAs in human (left panel) and murine (right panel) Mks. Factor XIII (F13A1) and β2-microglobulin (B2m) were used as positive controls. The migration of the DNA size marker (bp) is shown. (B) Western blot for α1, α2, and α3 chains of collagen VI in human (left panel) and murine (right panel) Mks. Membranes were probed with monoclonal antibodies raised against each single chain. β-Actin was used as a loading control. Where indicated, Mks were treated with 100 μM the prolyl-4-hydroxylase inhibitor EDHB. In parallel, Mks were treated with vehicle alone (0.1% ethanol) as control. (C) Confocal microscopy immunofluorescence of WT mouse Mks stained with a polyclonal antibody against collagen VI (red) and a monoclonal antibody against CD41 (green). Where indicated, Mks were cultured in the presence of 50 μg/mL ascorbic acid for 48 hours. Scale bar, 10 μm. (D) Western blot analysis of collagen VI secretion in the culture medium by Mks. Differentiated WT Mks were cultured for 48 hours in the absence or presence of 50 μg/mL ascorbic acid (AA). Then, the culture medium was collected, centrifuged, and resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and denaturing conditions. Membranes were probed with a monoclonal antibody recognizing collagen VI α2 chain. CTRL, control; NTC, no template control; VI, collagen VI.