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. 2017 May 17;37(3):BSR20170105. doi: 10.1042/BSR20170105

Figure 1. GLT25D1 and LH3 are required for HMW adiponectin secretion.

Figure 1

Adipo-HEK cells were transfected with siRNA for 48 h, then changed to serum-free media containing 56.8 µM ascorbic acid for adiponectin secretion for 24 h. RNA was isolated from cells to confirm successful knockdown of the genes using RT-qPCR. (A) GLT25D1 siRNA-mediated knockdown caused a significant decrease in GLT25D1 mRNA (t-test, n=16, *P=0.0025, P<0.0001), with no effects on adiponectin mRNA (P>0.05). (B) Similarly, LH3 siRNA-mediated knockdown caused a significant decrease in LH3 mRNA (t-test, n=12, *P<0.001), with no effects on adiponectin mRNA (P>0.05). Conditioned media was analysed by Western blot for changes in total and HMW adiponectin secreted. Total adiponectin secreted was determined by reduction and heat denaturation forming adiponectin monomers, followed by separation on SDS/PAGE and Western blot. Western blots were scanned using the Odyssey Infrared Imaging System with quantitation calculated using the integrated intensity of the Western blots. (C) Knockdown of GLT25D1 did not significantly alter the secretion of total adiponectin relative to the scrambled control (P>0.05). (D) Whereas knockdown of LH3 caused a significant decrease in the secretion of total adiponectin relative to the scrambled control (t-test, n=6, *P=0.0172, P<0.05). The adiponectin trimer was analysed for changes in PTMs. (E) Fifty micrograms of protein lysate was loaded onto the gel. Samples were reduced with DTT and not boiled (n=3). Knockdown of GLT25D1 and LH3 caused an increase in the mobility of the adiponectin trimer by electrophoresis indicating that knockdown of LH3 and GLT25D1 decreased the PTM on adiponectin. Changes to the adiponectin multimers were analysed using sucrose gradients and Western blot analysis. (F) Representative images of adiponectin multimers separated on sucrose gradients. There was a decrease in HMW adiponectin when GLT25D1 and LH3 were knocked down. (G) Quantitation of the ratio of HMW to total adiponectin (Sa) indicated that there was a significant decrease in the proportion of HMW adiponectin caused by GLT25D1 knockdown (t-test, n=7, *P=0.0421, P<0.05). (H) Knockdown of LH3 also caused a significant decrease in the Sa of adiponectin (t-test, n=5,*P=0.0249, P<0.05).