Fig. 1.

Targeted siRNA knockdown of nucleotide kinases in PBMC, colorectal tissue, and vaginal tissue and the resulting impact on TFV-MP and TFV-DP intracellular formation.

PBMC, colorectal tissue, and vaginal tissue were electroporated with 500 nM non-targeting siRNA or siRNA targeting AK2, GUK1, PKM, PKLR, and CKM and incubated for 24 h or 48 h for tissue or PBMC, respectively. Representative immunoblots demonstrate decreased nucleotide kinase expression with each targeted siRNA treatment relative to the non-targeting siRNA control. siRNA treated (a) PBMC, (b) colorectal tissue, and (c) vaginal tissue were incubated with 10 μM TFV for 12 h (n = 3 per treatment). Intracellular anabolites were extracted from which TFV-MP and TFV-DP were detected using uHPLC–MS/MS as depicted in the corresponding bar graphs showing mean ± standard deviation for each treatment. Statistical analyses were performed using a two-tailed unpaired t test to compare relative levels of anabolite production between non-targeted and targeted siRNA conditions; ** = p ≤ 0.01; *** = p ≤ 0.001.