sirna targeting scl/tal1 interrupting locus  (Santa Cruz Biotechnology)

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

Journal: Lab on a chip

Article Title: Multiplexed analysis of neural cytokine signaling by a novel neural cell-cell interaction microchip.

doi: 10.1039/d0lc00401d

Figure Lengend Snippet: Fig. 3 Immunocytology of neural rosettes formed on the NCCIM. (a) EB-derived rosettes in the dish and in the NCCIM. Cells are stained with DAPI, tight junction protein ZO1, NSC multipotency marker Nestin, neural ectoderm (Pax6), proliferation marker ki67, and neural progenitor (Sox1, Sox2) biomarkers as indicated. (b) Monolayer rosettes in the dish are dissociated and seeded into the NCCIM and form uniformly sized NSC rosette neurospheres by day 2. Cells are evaluated with similar biomarkers as (a). (c) Phase and fluorescence microscopy images of EB-derived rosettes in the PDMS microchambers of the NCCIM. Spontaneously formed rosettes in microwells results in three main size distribution categories: one large rosette, 3–4 medium size rosettes, and >5 small size rosettes. Cells are stained with ZO-1 (green) and Sox1 (red) antibodies. Scale bar is 50 μm.

Article Snippet: The antibodies, including Sox1 (R&D systems AF3360), Sox2 (R&D systems MAB2018), ZO-1 (ThermoFisher Scientific 33-9100), Ki67 (Abcam 15580), Nestin (R&D systems MAB1259), Pax6 (DSHB PAX6, Pax6 was deposited to the DSHB by Kawakami, A.) were added (1 : 1000) and incubated at 4 °C overnight.

Techniques: Derivative Assay, Staining, Marker, Fluorescence, Microscopy

Journal: Lab on a chip

Article Title: Multiplexed analysis of neural cytokine signaling by a novel neural cell-cell interaction microchip.

doi: 10.1039/d0lc00401d

Figure Lengend Snippet: Fig. 5 Viability and multipotency of rosettes on the NCCIM. (a) Flow diagram from seeding of cells into the PFMA that is combined with the MIST array to generate the assembled NCCIM. Cells were maintained within the NCCIM for 8 hours then evaluated by (b) propidium iodide (PI) staining of EB-derived rosettes and rosette neurospheres to visualize dead cells at hour 0 and hour 8. In (c) neural progenitor marker SOX1 is shown with adherens junction protein ZO-1 that highlights the center of polarized rosette cells. Cells maintain their multipotent state after NCCIM incubation for 8 hours. Scale bar is 50 μm.

Article Snippet: The antibodies, including Sox1 (R&D systems AF3360), Sox2 (R&D systems MAB2018), ZO-1 (ThermoFisher Scientific 33-9100), Ki67 (Abcam 15580), Nestin (R&D systems MAB1259), Pax6 (DSHB PAX6, Pax6 was deposited to the DSHB by Kawakami, A.) were added (1 : 1000) and incubated at 4 °C overnight.

Techniques: Staining, Derivative Assay, Marker, Incubation