Fig. 6

HuR regulates telomerase activity in mouse cells. a Schematic representation depicting the variants of mTERC. The point mutation sites are marked in red. b RNA pull-down assays were performed by using NIH3T3 cell lysates and in vitro-transcribed TERC variants depicted in Fig. 6a. The presence of HuR in the pull-down materials was assessed by western blot analysis. A 5-µg aliquot (input, Inp.) and proteins bound to GAPDH were included. Data represent the means ± SD of the band intensity from three independent experiments; significance was analyzed by Student’s t-test (*p < 0.05; **p < 0.01). c NIH3T3 cells were transfected with a siRNA targeting HuR. Forty-eight hours later, TRAP assays were performed to assess the telomerase activity. d RNA prepared from cells described in Fig. 6c was subjected to bisulfite RNA sequencing to analyze the methylation of C64 in mTERC. e A vector expressing mouse TERT (mTERT) was used for expressing mTERT in rabbit reticulocyte in vitro translation system. In vitro-transcribed mTERC or its variant bearing U15A + U58A or C64G was added into the system and used for TRAP assays to assess the telomerase activity. Data in (c, d), and (e) represent the means ± SD from three independent experiments; significance was analyzed by using Student’s t-test (**p < 0.01)