Samples were mixed and loaded in NC-Slide A8 (ChemoMetec A/S, Denmark), and then fluorescence at the single cell level in the slide was analyzed and quantified with all the NucleoCounter NC-3000 (ChemoMetec A/S, Denmark). HepG2, MCF7 and Hepa-1c1c7 cells have been cultured at eight 105, eight 105 and six 105 cells respectively per 6-cm dish overnight, then were STAT3 Source treated with the test compounds. Total RNA of cells treated with the test compound was extracted using the NucleoZOL (MACHEREY AGEL GmbH Co. KG, German). The complementary (c)DNA was reverse-transcribed from RNA employing Magic RT cDNA synthesis kit (Bio-Genesis Technologies Inc., Taiwan) with oligo-dT (18) and random hexamer. The cDNA was amplified inside the quantitative PCR with distinct oligonucleotide primers for human CYP1A1 (GenBank: NM_000499), human GAPDH (GenBank: NM002046.5), mouse Cyp1a1 (GenBank: NM_009992), and mouse -actin (GenBank: NM_007393), as described previously41. GAPDH and -actin mRNA was also analyzed to normalize differences in sample uptake. The quantitative (q) PCR were performed employing IQ2 SYBR Green Fast qPCR System Master Mix (Bio-Genesis Technologies, Taiwan) and CFX96 Real-Time PCR Detection Method (Bio-Rad, CA), as described previously42.Vitality assay (analysis of the level of cellular thiols). Cell vitality is evaluated by the alterations in theReversetranscription polymerase chain reaction (RTPCR) and quantitative PCR.Scientific Reports |(2021) 11:5457 | Vol.:(0123456789) cells and Hepa-1c1c7, c4 and c12 cells have been seeded at 8 105 cells/6-cm dish overnight. Afterwards, cells had been cultured with test compounds for proper time periods. At the finish from the preferred treatment occasions, cell lysates were prepared, and Western blots were performed as described previously43. The blots had been cut as well as the area of blot corresponding to CYP1A1 or GAPDH was selected for hybridization individually and shown within the figures (Supplementary Data).Western blotting.Reporter plasmids and reporter activity assay. The pAhRDtkLuc3 comprises 3 AHRE motifs linked to the HSV-TK minimum promoter44,45 within the pGL3-basic vector. The RSV-lacZ plasmid consists of a lacZ gene-encoded -galactosidase, having a Rouse sarcoma virus (RSV) LTR because the promoter. MCF cells, HepG2 cells and Hepa-1c1c7 cells were subcultured at 6 104, six 104, and two.5 104 cells/well respectively, inside a 24-well plate overnight. Afterwards, the luciferase reporter plasmid and RSV-lacZ plasmids have been transfected into cells applying the liposome for six h, followed by therapy using the test compounds as described previously34. Cell lysates were harvested at the suitable time points after treatment with test compounds and were respectively assayed for each luciferase and -galactosidase activities using Britelite (PerkinElmer) and the Galacto-Star Method (Tropix, Bedford, MA) as described previously34,41. Transcription activity from the promoter was indicated by luciferase activity, and -galactosidase activity of RSV-lacZ was utilized to normalize the luciferase activity. Immunocellular fluorescence staining. To analyze in situ CYP1A1 MMP Species expression, Hepa-1c1c7 cells were seeded at three 105 cells/well in 6-well plates with microscope cover glasses in the properly for greater than 14 h and after that treated with test compounds, followed by washing with phosphate-buffered saline (PBS) and being fixed with ethanol, as described previously46. The detection on the in situ CYP1A1 expression.