Figure 4.
Collagen Transport Defects with ARCN1 KD
(A) Reduction of ARCN1 caused accumulation of type I collagen in total cellular lysates and reduction of type I collagen secretion. The type I collagen antibody (AB758) was from Millipore.13 For the measurement of culture supernatant type I collagen, skin fibroblast cell numbers were counted, and equal numbers were plated in culture flasks. The cells were changed to serum-free culture media for the final 24 hr, the time point at which the medium was harvested. One-tenth the volume of TCA was added and placed on ice for 30 min. The samples were centrifuged and precipitates were washed with ethanol. After the ethanol wash, the supernatants were dried and the remaining cell pellets were dissolved with SDS sample buffer for immunoblotting. TCA precipitation was performed four times, and consistent results were obtained. Two bands of type I collagen represent the bands for alpha 1 and alpha 2 type I collagen. Top larger band represents alpha 1 type I collagen, and lower smaller band represents alpha 2 type I collagen.
(B) Brefeldin A (BFA; B5936, Sigma-Aldrich) treatment caused the intracellular accumulation of type I collagen. 10 uM of BFA was used. DMSO was added for control. The BFA treatment experiment was performed three times, and consistent results were obtained.
(C) COL1A1 and COL1A2 expression with ARCN1 KD. Slight reduction of COL1A1 expression was elicited with ARCN1 KD; however, the expression of COL1A2 remained unchanged. Gene expression was normalized against GAPDH expression. Gene expression relative to that of the control sample is shown. Data represent mean ± 2 SD. ∗p < 0.05, ∗∗∗p < 0.001, two-tailed t test. n = 3 technical replicates per group.