tal1 scl  (Proteintech)

Journal: The Journal of Biological Chemistry

Article Title: The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase

doi: 10.1016/j.jbc.2021.101531

Figure Lengend Snippet: Inhibition of DHFR decreases cytokine-stimulated STAT3 transcriptional activity and STAT3 target gene expression. A , U3A cells were transfected with siRNA targeting DHFR and three other hits from the PISA screen, namely CHCHD2, STIL, and GARNL1. siRNA targeting STAT3 was used as a positive control. About 48 h later, the cells were stimulated with 10 ng/ml interleukin-6 (IL-6) or 10 ng/ml OSM for 6 h. Luciferase was measured and normalized to siConA DMEM. Statistical comparisons were performed between the indicated mean values and that of siConA IL-6/OSM. B , MDA-MB-468 cells were transfected with siRNA targeting DHFR and analyzed by quantitative RT–PCR. mRNA levels were normalized to GAPDH and nontargeting siCon2. C , U3A cells were pretreated with pyrimethamine (PYR) and/or methotrexate (MTX) for 1 h and stimulated with 10 ng/ml OSM for 5 h. Luciferase was measured and normalized to the unstimulated vehicle control. Statistical comparisons were performed between the indicated mean values and that of DMSO OSM. D , U3A cells were pretreated with 1 μM MTX for 1 h, stimulated with 10 ng/ml OSM for 90 min, and analyzed by quantitative RT–PCR. mRNA levels were normalized to GAPDH and unstimulated vehicle control. ns p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001 with two-tailed unpaired Student's t test used in ( A ), two-tailed one-sample Student's t test used in ( B ), and two-tailed unpaired Student's t test (with or without Welch's correction depending on F test) used in ( C ) and ( D ). CHCHD2, coiled-coil helix–coiled-coil helix domain–containing 2; DHFR, dihydrofolate reductase; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; GARNL1, GTPase-activating Rap/Ran-GAP domain–like 1; ns, not significant; OSM, oncostatin M; PISA, proteome integral solubility alteration; STAT3, Signal transducer and activator of transcription 3; STIL, SCL/TAL1 interrupting locus.

Article Snippet: Cells were transfected with 10 nM of siRNA targeting CHCHD2 (catalog no.: 89755; Santa Cruz Biotechnology), DHFR (catalog no.: 37078; Santa Cruz Biotechnology), STAT3 (catalog no.: D-003544-03-0010; Dharmacon), SCL/TAL1 interrupting locus (catalog no.: 4775; Santa Cruz Biotechnology), GTPase-activating Rap/Ran-GAP domain–like 1 (catalog no.: 92345; Santa Cruz Biotechnology), or nontargeting siRNAs designated as siConA (catalog no.: 37007; Santa Cruz Biotechnology), and siCon2 (catalog no.: D-001210-02-05; Dharmacon) using Lipofectamine RNAiMax (Invitrogen).

Techniques: Inhibition, Activity Assay, Expressing, Transfection, Positive Control, Luciferase, Quantitative RT-PCR, Two Tailed Test, Modification, Solubility