Intracellular TLR-regulated pathways and ISGs are inhibited in oncogenic HPV cell lines. (a) Flow cytometry of TLRs. Protein levels of TLRs 3, 7, 8, and 9 in Caski, HeLa or C33A cells were shown in bar charts as mean fluorescence intensity (MFI). (b) qRT-PCR of ISGs. Total RNA was extracted from Caski, HeLa or C33A cells. After reverse transcription, qRT-PCR was performed using ISG15 or Mx-1 primers. As positive control, GAPDH was amplified at exponential phase of 20 cycles. (c) Functionality of TLR-regulated pathways. CaSki, HeLa and C33A cells were stimulated for 24 h with ligands of TLRs 3, 7, 8, and 9 as indicated in the Figure. Ex vivo production of cytokines including interleukin (IL-6), IL-8, IL-10 and IL-12p70, tumor necrosis (TNF)-α and IL-1β in culture supernatant was measured by Cytometric Bead Array. Experiments were repeated three times in triplicate.