Journal: Frontiers in Genetics

Article Title: Interpreting Gene Expression Effects of Disease-Associated Variants: A Lesson from SNCA rs356168

doi: 10.3389/fgene.2017.00133

Figure Lengend Snippet: The effect of rs356168 on SNCA mRNA expression levels in human brain tissues. The study cohort consisted of unaffected control brain tissues from Caucasian donors. The subjects were genotyped for rs356168. Fold levels of human SNCA -mRNA were assayed by real-time RT-PCR using TaqMan technology, and calculated relative to the geometric mean of SYP- and ENO2- mRNAs reference control, using the 2 -ΔΔ C T method (i.e., results presented are relative to a specific brain RNA sample). The values presented here are means levels ± SE adjusted for age, sex, tissue source and PMI. GLM analysis was used to test the association of rs356168 with SNCA mRNA expression levels in the (A) temporal cortex (TC), and (B) frontal cortex (FC). Significant differences are denoted by ∗ .

Article Snippet: Briefly, duplicates of each sample were assayed by relative quantitative real-time PCR using the ABI ViiA7 to determine the level of SNCA message (ID Hs00240906_m1, 62bp, best coverage for the different SNCA -mRNA isoforms) in brain tissues relative to the geometric mean of mRNAs encoding the human neuronal proteins Enolase 2 ( ENO2 , ID Hs00157360_m1, 77bp) and Synaptophysin ( SYP , ID Hs00300531_m1, 63bp) (Applied Biosystems, Foster City, CA, United States).

Techniques: Expressing, Control, Quantitative RT-PCR

Journal: Cell death & disease

Article Title: POU6F1 cooperates with RORA to suppress the proliferation of lung adenocarcinoma by downregulation HIF1A signaling pathway.

doi: 10.1038/s41419-022-04857-y

Figure Lengend Snippet: Fig. 5 POU6F1 inactivates HIF1A signaling pathway and transcriptionally regulates ENO1, PDK1, and PRKCB expression. A Volcano plot showing differential expression genes (DEGs) in A549 cells stably transfected with POU6F1 compared with empty vector (mock). B Human KEGG pathway analysis of DEGs indicating participated pathways in A549 cells stably transfected with POU6F1 relative to mock. C Venn diagram revealing comprehensive analysis of genes, that were involved with the HIF1A signaling pathway and associated with LUAD progression in various status of death, tumor stage, and metastasis. D Western blotting assay showing the expression of HIF1A, ENO1, ENO2, GAPDH, PDK1, and PRKCB in A549 and NCI-H1299 cells stably transfected with mock or POU6F1. E Real-time qRT-PCR assay indicating the expression of HIF1A, ENO1, PDK1, and PRKCB in A549 cells stably transfected with mock or POU6F1. F Dual-luciferase assay indicating relative activity of HIF1A promoter in A549 and NCI-H1299 cells stably transfected with mock or POU6F1, and those treated with DMSO or ML228 (1.0 μmol/l). G Western blotting assay showing the expression of HIF1A in A549 and NCI-H1299 cells stably transfected with mock or POU6F1, and those treated with DMSO or MG132 (5 μmol/l) for 6 h. H Dual-luciferase assay showing relative activity of ENO1, PDK1, and PRKCB promoter in A549 and NCI-H1299 cells transfected with mock or POU6F1. I ChIP and qPCR assays indicating relative POU6F1 enrichment on ENO1, PDK1, and PRKCB promoter in A549 cells stably transfected with mock or POU6F1. Student t-test and ANOVA compared the difference in E, F and H, I. *P < 0.05, **P < 0.01.

Article Snippet: Thereafter, membranes were blocked with 5% skimmed milk powder and incubated with the primary antibodies specific for POU6F1 (abclonal, A7299), RORA (Proteintech, 10616-1-AP; Santa Cruz Biotechnology, sc-518081), HIF1A (Abcam, ab51608), actin (Abclonal, AC026), ENO1 (Proteintech, 11204-1-AP), ENO2 (Proteintech, 66150-1-Ig), PDK1 (Proteintech, 10026-1-AP), PRKCB (Proteintech, 12919-1-AP), HA (Abcam, ab9110), Histone-H3 (Proteintech, 17168-1-AP), Flag antibody (Abcam, ab205606), and GAPDH (Abcam, ab8245) at 4 °C overnight.

Techniques: Expressing, Quantitative Proteomics, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay