Journal: bioRxiv

Article Title: Differential regulation of histone H1 subtypes by N6-methyladenosine RNA methylation

doi: 10.1101/2025.01.22.634368

Figure Lengend Snippet: A. m6A fold change of the immunoprecipitated mRNA from cells treated with cycloleucine, with respect to the untreated cells. B. mRNA fold change of the cells treated with cycloleucine, with respect to the untreated cells. C. Nascent RNA fold change of the immunoprecipitated BrdU labeled RNA after the run-on of cells treated with cycloleucine, with respect to the control. D, log2 fold change of the transcript levels of the region surrounding the translation start site obtained by targeted ribosome profiling of cells treated with cycloleucine with respect to the untreated cells. E, Western blots of total protein extracts after m6A inhibition. F, Quantification of the Western blot images, relative to control and normalized by tubulin. In all cases, HeLa cells were treated with 50 mM cycloleucine for 24h. Error bars correspond to the standard deviation of three biological replicates. Untreated cells were used as a negative control in all experiments. UNTR, untreated.

Article Snippet: For m6A inhibition, cells were treated with 25, 50, or 100 mM cycloleucine (TCI, A1063) for 24h or with 20 µM STM2457 (Merck, SML3360) for 48h.

Techniques: Immunoprecipitation, Labeling, Control, Western Blot, Inhibition, Standard Deviation, Negative Control

Journal: bioRxiv

Article Title: Differential regulation of histone H1 subtypes by N6-methyladenosine RNA methylation

doi: 10.1101/2025.01.22.634368

Figure Lengend Snippet: A, B. Western blots of m6A readers after pull-down experiments using H1.2 (A) and H1.4 (B) specific probes to capture ribonucleoprotein complexes in HeLa cells untreated or treated with cycloleucine (CYC). C. RT-qPCR after RNA immunoprecipitation using YTHDF2 antibody of HeLa cells untreated, treated with cycloleucine and STM2457 (STM). D I, input. CP, control probe. PD, pull-down with the specific probe. D. Changes in mRNA levels after partial depletion of m6A readers with siRNA in HeLa cells. E. Western blots of H1 subtypes after partial depletion of m6A readers. F. Quantification of the Western blot images, relative to control and normalized by histone H3. Untreated cells were used as a negative control in all experiments. HeLa cells were treated either with 50 mM cycloleucine for 24h or with 20 μM STM for 48h. Error bars correspond to the standard deviation of three biological replicates. The differences between the mRNA immunoprecipitated in the untreated cells and after treatment with m6A inhibitors were analyzed with a two-tailed Student’s t-test. n.s. not significant. ** p-value < 0.01. *** p-value< 0.001.

Article Snippet: For m6A inhibition, cells were treated with 25, 50, or 100 mM cycloleucine (TCI, A1063) for 24h or with 20 µM STM2457 (Merck, SML3360) for 48h.

Techniques: Western Blot, Quantitative RT-PCR, RNA Immunoprecipitation, Control, Negative Control, Standard Deviation, Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: Near-infrared fluorogenic RNA for in vivo imaging and sensing

doi: 10.1038/s41467-024-55093-1

Figure Lengend Snippet: a Modular design of the Squash-based NIR fluorescent sensor for small molecule targets. The target binding aptamer (blue) was fused to Squash (beige) through a transducer module (black). Target binding induces the allosteric conformation of tetracycline aptamer, then stabilizes the helix structure of the transducer domain, thus enabling Squash to bind NIR fluorophore. b , c Optimization of the transducer for small molecules sensor. The optimal tetracycline sensor ( b ) containing transducer 2 domain (black box), or SAM sensor ( c ) was chosen with the highest signal-to-background of 14-fold or 5-fold, respectively. Data represent mean values ± s.d. for n = 3 independent experiments. d Using Squash-based NIR fluorescent sensors to image tetracycline in living cells. We expressed the tetracycline sensor in HEK293T cells. Cells treated with tetracycline (0.1 mM) showed increased NIR fluorescence. Image acquisition time, 500 ms. Scale bar, 20 µm. Data represent mean values ± s.d. for n = 3 independent experiments. e Squash-based NIR fluorescent sensor for tetracycline imaging in vivo. We incubated sensor-expressing cells (10 8 cells) in the presence or absence of tetracycline (0.1 mM). Then, we transplanted these cells to mice by subcutaneous injection. After 2 h, DFQL-1T (10 µM) was injected in situ for imaging. Mice transplanted with tetracycline-treated cells exhibit higher NIR fluorescence (bottom row). Image acquisition time, 500 ms. Data represent mean values ± s.d. for n = 3 independent experiments. f Imaging SAM in live cells in the NIR channel. We readily detected NIR fluorescence in cells expressing SAM sensors. After being treated with cycloleucine (30 mM), NIR fluorescence decreased significantly. Scale bar, 20 µm. Data represent mean values ± s.d. for n = 3 independent experiments. g Monitoring SAM metabolites in vivo. Engineered HEK 293T cells expressing SAM sensor (10 8 cells) were incubated with cycloleucine (30 mM) for 1 h. Cells were then transplanted into mice by subcutaneous injection. About 2 h after transplant, DFQL-1T (10 µM) was injected in situ for imaging. Sensor-expressing cells display pronounced skin-permeable NIR fluorescence in mice. Cycloleucine treatment induced a significant decrease in fluorescence. Image acquisition time, 500 ms. Data represent mean values ± s.d. for n = 3 independent experiments.

Article Snippet: After adding cycloleucine (30 mM, Ark Pharm AK-29341), we imaged the cells for 2 h at 15-min intervals.

Techniques: Binding Assay, Fluorescence, Imaging, In Vivo, Incubation, Expressing, Injection, In Situ