Fig. 7: Stimulating Adenosine signaling through Adora2b reversed PAN-induced injury in NHP podocytes.

a Adora2b was efficiently knocked-down by siRNAs in NHP podocytes. A total of 50 nM siRNAs were transfected into the cells using Lipofectamine RNAiMax (Life Technologies). Total RNAs were isolated for RT-qPCR at 48 h post transfection. Data were normalized to GAPDH. Error bar represents data from two replicates (Left panel). Protein expression of Adora2b was also significantly decreased in siA2B transfected cells (Right panel). Total proteins were isolated for western blotting at 48 h post transfection. Tubulin was used as the loading control. b Knockdown of Adora2b promoted PAN-induced injury in NHP podocytes. Cells were transfected with siControl, miR-27b mimic, and siA2B at the final concentration of 50 nM. PAN treatment (10 µg/ml) was initiated 1 day after transfection. Cells were subject to viability test using CellTiter-96 (Promega) at day 5 post PAN treatment. Relative cell viability was calculated by normalizing absorbance readings from PAN-treated samples to non-treated controls. Error bar represents data from two independent experiments with eight replicates. *p < 0.05 by Student's t-test. c Stimulating adora2b had a protective role in PAN-induced injury in NHP podocytes. Cells were stimulated with 5 µM NECA and PAN treatment (10 µg/ml) was initiated 1 day after. Cell viability test was the same as in b. Error bar represents data two independent experiments with eight replicates. *p < 0.05 by Student's t-test. d Adora2b was efficiently knocked-down by shRNAs in NHP podocytes. Total RNAs were isolated for RT-qPCR at 72 h post lentivirus infection. Data were normalized to GAPDH. Error bar represents data from two technical replicates. e Protein expression of Cleaved Caspase-3 was elevated in shRNA infected NHP podocytes cells. Cells were infected with shRNA and PAN treatment (50 µg/ml) was initiated 1 day after. Total proteins were isolated for western blotting at 48 h post PAN treatment. Actin was used as the loading control. f Quantification of cleaved caspase-3 expression. Relative quantification was performed by normalizing signal of cleaved caspase-3 to actin. g Overexpression of Adora2b by lentiviral vectors. Coding sequence of Adora2b was cloned into pCMV-MCS-EF1-copGFP vector (System Biosciences). Lentivirus transduced cells were spotted by GFP imaging. Scale: 100 µm. h Overexpression of Adora2b by lentiviral vectors. Total RNAs were isolated for RT-qPCR at 72 h post lentivirus infection. Data were normalized to GAPDH. Error bar represents data from two technical replicates. i Overexpression of Adora2b had protective effects against PAN-induced injury in NHP podocytes. Equal number of empty or Adora2b transduced cells were seeded in 96-well plates and treated with 10 µg/ml PAN and with or without NECA (5 µM). Data represent two independent experiments with eight replicates. **p < 0.01 by Student's t-test. j Protein expression of Cleaved Caspase-3 was modestly suppressed in pLenti-A2B infected NHP podocytes cells (Left panel). Cells were infected with empty or Adora2b and PAN treatment (50 µg/ml) was initiated 1 day after. Total proteins were isolated for western blotting at 48 h post PAN treatment. Actin was used as the loading control. Relative quantification was performed for western results (Right panel). k Stimulation of Adora2b by NECA decreased the expression of Cleaved Caspase-3 in NHP podocytes (Left panel). Cells were stimulated with 5 µM NECA and PAN treatment (50 µg/ml) was initiated 1 day after. Total proteins were isolated for western blotting at 48 h post PAN treatment. Actin was used as the loading control. Relative quantitation was performed for western results (Right panel)