Journal: The FASEB Journal
Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs
Figure Lengend Snippet: Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of DYX1C1 and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.
Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).
Techniques: Expressing, Binding Assay, Incubation, Quantitative RT-PCR