Journal: Frontiers in Molecular Biosciences

Article Title: Innate immunity mediator STING modulates nascent DNA metabolism at stalled forks in human cells

doi: 10.3389/fmolb.2022.1048726

Figure Lengend Snippet: cGAS contributes to the activation of STING, which is required for STING effect on replication forks. (A) Depletion of STING and induction of cGAS-GFP expression in the cGAS knockout (KO) HeLa cells. Cells were transfected with the indicated siRNAs and in 24 h doxycycline was added to all cells (KO and complemented) at 400 ng/ml for another 16–24 h. A dotted line marks a spliced lane. (B) qPCR measurements of IL6 mRNA in cGAS KO and cGAS KO/cGAS-GFP HeLa incubated with doxycycline as in (A) to induce cGAS-GFP, and treated with 5 mM HU for 5 h. Error bars are standard deviations. (C,D) Boxplots of first label (EdU) track length distributions in doxycycline-treated cGAS KO cells (C) and cGAS KO + cGAS-GFP cells (D) . The experimental design and HU treatment was as in , and track length values in restarted and stalled forks after HU are combined due to the low fraction of restarted forks. The results represent two independent experiments. (E) A modification to the experimental design to include a STING agonist SR-717 treatment. (F) Boxplots of first label (EdU) track length distributions in cGAS KO cells with and without an incubation with SR-717 (4 μM). The results represent two independent experiments. p values in (C–F) are determined in KS tests.

Article Snippet: Doxycycline stock solution was at 100 μg/ml in PBS and SR-717 (MedChemExpress) was at 10 mM in DMSO.

Techniques: Activation Assay, Expressing, Knock-Out, Transfection, Incubation, Modification

Journal: Developmental cell

Article Title: Essential role of MESP1-RING1A complex in cardiac differentiation.

doi: 10.1016/j.devcel.2022.10.009

Figure Lengend Snippet: Figure 4. Overexpression of MESP1 variants at days 2 to 4 post hESC differentiation impaired cardiomyocyte differentiation (A) Graph showing the timescale of Dox-induced FLAG-MESP1 overexpression during hESCs cardiac-directed differentiation. Dox was added from days 2 to 4. (B) RT-qPCR from day 30 cardiomyocytes with MESP1 or MESP1 variants (E11D, E104K, or L120P) induced at the cardiac mesoderm stage. Relative expression of MESP1 (at day 4) induced by varying doxycycline concentrations (mg/mL) was shown on the right side. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. (C) Immunostaining and quantification of irregular organized sarcomeric a-ACTININ in day 30 cardiomyocytes with or without MESP1 (WT, E11D, E104K, or L120P) overexpression induced by Dox at the cardiac mesoderm stage. n = 96 (OE MESP1 Dox), n = 38 (OE MESP1 + Dox 0.5 mg/mL, 26-fold overexpression), n = 14 (OE MESP1 E11D Dox), n = 25 (OE MESP1 E11D + Dox 0.5 mg/mL, 40-fold overexpression), n = 27 (OE MESP1 E104K Dox), n = 52 (OE MESP1 E104K + Dox 0.1 mg/mL, 5-fold overexpression), n = 34 (OE MESP1 L120P Dox), and n = 33 (OE MESP1 L120P + Dox 0.2 mg/mL, 17-fold overexpression). Scale bars, 50 mm. ns, p > 0.05, ***p < 0.001 by Fisher’s exact test.

Article Snippet: For FLAG-MESP1 ChIP, cells with Dox-inducible FLAG-MESP1 were treated with 0.03 mg/ml doxycycline (Selleck Chemicals, Houston, TX) during day 3 - day 4 of the cardiomyocyte differentiation process.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Immunostaining