one step reverse transcriptase quantitative polymerase chain reaction pcr  (Bio-Rad)

Journal: Acta Pharmacologica Sinica

Article Title: Triptolide suppresses the growth and metastasis of non-small cell lung cancer by inhibiting β-catenin-mediated epithelial–mesenchymal transition

doi: 10.1038/s41401-021-00657-w

Figure Lengend Snippet: The effect of triptolide on the morphology of NCI-H1299 cells. NCI-H1299 cells were exposed to various concentrations of triptolide for 48 h, and morphological changes were observed with a microscope (×100 magnification) ( a ) and with a laser scanning confocal microscope (×40 magnification) after staining using rhodamine-phalloidin ( b ). c , d The effect of triptolide on EMT markers in NCI-H1299 cells. The cells were exposed to various concentrations of triptolide for 48 h or 1 nM triptolide for 24, 36, and 48 h. The cells were harvested and subjected to Western blotting and RT-qPCR assays to detect the expression of ZEB1, N-cadherin, vimentin, slug, snail, ZO-1, and E-cadherin. β-Actin and GAPDH were used as internal controls in Western blotting and RT-qPCR assays. The results of Western blotting and RT-qPCR assays are presented in c and d , respectively. The data are presented as the mean ± SEM, n = 3. * P < 0.05, *** P < 0.01 and *** P < 0.001 vs. the vehicle group. e Triptolide modulated the expression of EMT markers, as indicated by immunofluorescence staining. NCI-H1299 cells were treated with or without triptolide for 48 h, and then, E-cadherin, ZO-1, vimentin, and ZEB1 expression was detected using an immunofluorescence staining assay. DAPI was used to visualize the nuclei. Images were taken using a Zeiss LSM 800 confocal microscope with a ×63 oil immersion lens. TPL triptolide.

Article Snippet: The Transcriptor First Strand cDNA Synthesis kit and One-Step SYBR PrimeScript Plus quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kit were purchased from TaKaRa (Kyoto, Japan).

Techniques: Microscopy, Staining, Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence

Journal: Acta Pharmacologica Sinica

Article Title: Triptolide suppresses the growth and metastasis of non-small cell lung cancer by inhibiting β-catenin-mediated epithelial–mesenchymal transition

doi: 10.1038/s41401-021-00657-w

Figure Lengend Snippet: a – c The effect of triptolide on the protein and mRNA levels of β-catenin in NCI-H1299 and NCI-H460 cells. The cells were treated with triptolide for the indicated times and then collected for Western blotting assays and RT-qPCR assays. Representative images and quantitative Western blotting data are presented in a and b . The RT-qPCR results are shown in c . d , e The effect of triptolide on the nuclear translocation of β-catenin. The cells were lysed by using a NE-PERTM Nuclear and Cytoplasmic Extraction Kit and subjected to Western blotting assays. β-Actin and Lamin B were used as loading controls. Representative images and quantitative data are shown in d and e , respectively. f , g The effect of triptolide on β-catenin translocation from the cytoplasm to the nucleus, as indicated by immunofluorescence assays. After treatment with triptolide for 24 h, the cells were fixed with 4% paraformaldehyde, blocked using 5% BSA, and incubated with β-catenin antibody, followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG and DAPI. TPL triptolide. Representative images and quantitative data are shown in f and g , respectively. Images were taken using a Zeiss LSM 800 confocal microscope with a 63× oil immersion lens. TPL triptolide. Quantitative data are presented as the mean ± SEM, n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. the vehicle group.

Article Snippet: The Transcriptor First Strand cDNA Synthesis kit and One-Step SYBR PrimeScript Plus quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kit were purchased from TaKaRa (Kyoto, Japan).

Techniques: Western Blot, Quantitative RT-PCR, Translocation Assay, Immunofluorescence, Incubation, Microscopy

Journal: Acta Pharmacologica Sinica

Article Title: Triptolide suppresses the growth and metastasis of non-small cell lung cancer by inhibiting β-catenin-mediated epithelial–mesenchymal transition

doi: 10.1038/s41401-021-00657-w

Figure Lengend Snippet: a , b β-Catenin vector transfection increased β-catenin expression, as detected by Western blotting and RT-qPCR assays. c , d The effects of β-catenin overexpression on the triptolide-mediated inhibitory effect on the migration and invasion of NCI-H1299 and NCI-H460 cells. NCI-H1299 and NCI-H460 cells were transfected with NC vector or β-catenin vector and then subjected to Transwell migration assays ( c ) and invasion assays ( d ). The images were taken at ×100 magnification. e , f The effect of triptolide on EMT in NCI-H1299 and NCI-H460 cells was weakened by β-catenin overexpression. After transfection, the cells were collected and used for Western blotting and RT-qPCR assays. The results of Western blotting and RT-qPCR assays are shown in e and f , respectively. TPL triptolide. The data are presented as the mean ± SEM, n = 3. *** P < 0.001 compared with the vector group, ### P < 0.001 vs. the β-catenin vector group.

Article Snippet: The Transcriptor First Strand cDNA Synthesis kit and One-Step SYBR PrimeScript Plus quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kit were purchased from TaKaRa (Kyoto, Japan).

Techniques: Plasmid Preparation, Transfection, Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Migration

Journal: Acta Pharmacologica Sinica

Article Title: Triptolide suppresses the growth and metastasis of non-small cell lung cancer by inhibiting β-catenin-mediated epithelial–mesenchymal transition

doi: 10.1038/s41401-021-00657-w

Figure Lengend Snippet: a , b β-Catenin siRNA transfection decreased β-catenin expression, as measured by Western blotting and RT-qPCR assays. c , d The effect of triptolide combined with β-catenin siRNA on the migration and invasion of NCI-H1299 and NCI-H460 cells as indicated by Transwell migration and invasion assays. The images were taken at ×100 magnification. e , f The effect of triptolide combined with β-catenin siRNA on EMT as indicated by Western blotting and RT-qPCR assays. TPL triptolide. The data are presented as the mean ± SEM, n = 3. *** P < 0.001 vs. the siNC group, ### P < 0.001 vs. the β-catenin vector group.

Article Snippet: The Transcriptor First Strand cDNA Synthesis kit and One-Step SYBR PrimeScript Plus quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kit were purchased from TaKaRa (Kyoto, Japan).

Techniques: Transfection, Expressing, Western Blot, Quantitative RT-PCR, Migration, Plasmid Preparation