Journal: Nature Communications

Article Title: Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin

doi: 10.1038/s41467-023-42714-4

Figure Lengend Snippet: a Activity of 3033 on JAK family enzymes. Human HEL 92.1.7 cells were treated with siRNA at 1.5 μM for 72 h, and mRNA levels of JAK1, JAK2, JAK3, and TYK2 were measured using the QuantiGene 2.0 assay ( n = 7 biologically independent samples, mean ± s.d.; one-way ANOVA, **** P < 0.0001, ns: not significant). b Ruxolitinib inhibits JAK1 and JAK2 to prevent the pathogenesis of vitiligo driven by IFN-γ signaling. c mRNA expression of IFN-γ-inducible chemokines CXCL9, 10, and 11. Cells were first treated with siRNA or ruxolitinib at 1.5 μM for 72 h, and then stimulated with recombinant human IFN-γ for 24 h ( n = 7 biologically independent samples, mean ± s.d.; one-way ANOVA, ** P < 0.01, **** P < 0.0001). d , e Chemical engineering of 3033 for skin delivery. Terminal nucleotides were stabilized with phosphorothioate backbone modifications for nuclease stability ( d ). Human HeLa cells were transfected with DCA-siRNAs for 72 h through lipofectamine RNAiMax-mediated uptake and mRNA levels were measured using the QuantiGene 2.0 assay ( n = 3 biologically independent samples, mean ± s.d.; two-way ANOVA multiple comparison, * P < 0.05). f Molecular modeling (PyMOL 2) of DCA-siRNA 3033 in scaffold 2, si3033. g – i ruxolitinib and si3033 both reduce luciferase activity in IFN-γ-JAK1/2-STAT1 HeLa reporter cells ( n = 7 biologically independent samples, mean ± s.d.; one-way ANOVA, ** P < 0.01, **** P < 0.0001). Cells were treated with ruxolitinib or siRNA for 72 h and then stimulated with IFN-γ for 18 h. si3033 was transfected to the HeLa reporter cells using lipofectamine RNAiMax. j JAK1 and JAK2 mRNA expression in si3033-treated and untreated HeLa reporter cells ( n = 7 biologically independent samples, mean ± s.d.; two-sided unpaired t test, **** P < 0.0001, ns: not significant). Source data are provided as a Source Data file.

Article Snippet: GAS reporter (Luc)-HeLa cell line (IFN-γ/JAK/STAT1 pathway) was purchased from BPS Bioscience (#79041).

Techniques: Activity Assay, Expressing, Recombinant, Transfection, Comparison, Luciferase

Journal: Nature

Article Title: Experimental and computational framework for a dynamic protein atlas of human cell division

doi: 10.1038/s41586-018-0518-z

Figure Lengend Snippet: Quantitative imaging of mitotic proteins. (a) Automatic calibrated 3D live confocal imaging pipeline. Cells in prophase were identified by online classification, imaged through mitosis in the landmarks and protein of interest channels, and measured by FCS at selected positions. (b) The local protein concentrations determined by FCS fitting linearly correlate with the background subtracted image intensities at the corresponding positions (shown are data acquired on the same day). (c) Example cell showing concentration map resulting from FCS-based intensity calibration (mean z-projection). Scale bar: 10 μm. Data shown in (a)-(c) is for H2B-mCherry mNEDD1-LAP (EGFP) and is representative of n = 92 independent experiments performed with 28 different cell lines.

Article Snippet: HeLa Kyoto cell lines with H2B-mCherry were imaged live using an excitation laser at 561 nm every 5 min for about 16 hours on the Zeiss 780 microscopy system.

Techniques: Imaging, Concentration Assay