Journal: Genes, Brain, and Behavior

Article Title: Transcriptomics analysis reveals potential regulatory role of nSMase2 ( Smpd3 ) in nervous system development and function of middle‐aged mouse brains

doi: 10.1111/gbb.12911

Figure Lengend Snippet: Brain cortex deficient in nSMase2 has a different transcriptome and proteome. (A) Flow chart of the RNA‐seq experiment and data analysis. (B) The top 10 most increased transcripts and (C) decreased transcripts. The transcripts in red color were selected to be analyzed either by immunoblotting or RT‐PCR. (D) Immunoblots and quantification of protein levels of Grin2b, Map1b (antibody also detects Map1b light chain), phosphorylated Gsk3α/β (p‐Gsk3α/β) and Gapdh in fro/fro versus fro/+ cortex. (E) qRT‐PCR to confirm differences in the amounts of Naa38, Hapln2, Mrpl27, Rnaset2a, Atp5k mRNAs in fro/fro versus fro/+ cortex.

Article Snippet: Non‐specific binding sites were blocked with 5% fat‐free dry milk in PBS/0.1% Tween‐20 (PBST) followed by overnight incubation with primary antibodies, such as Map1b (Santa Cruz Biotechnology, sc‐8970), Gsk3α/β (Santa Cruz Biotechnology, sc7291), phospho‐Gsk3α/β (Santa Cruz Biotechnology, sc135653) and GAPDH (Cell signaling tech, #5174) and secondary anti‐rabbit (Jackson Immune Research, 715‐035‐152) and anti‐mouse (Jackson Immune Research, 715‐035‐151) horseradish peroxidase (HRP) antibodies for 1 h at room temperature.

Techniques: RNA Sequencing Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Single-molecule live-cell RNA imaging with CRISPR-Csm

doi: 10.1101/2024.07.14.603457

Figure Lengend Snippet: a , Fixed-cell image of individual MAP1B mRNAs labeled by GFP-tagged Csm complex and 48 MAP1B -targeting spacers (left), image of individual MAP1B mRNAs labeled by smFISH probes (middle), and their overlay (right). Scale bar, 10 μm. b , Enlarged view of the red boxed region in a . Scale bar, 1 μm. c, Violin plot showing distance to the nucleus from individual NOTCH2 or MAP1B mRNA molecules. Median indicated by solid line; quartiles indicated by dashed lines. d, Same as c, but showing distance to cell edge. e , Temporal-color coded trajectory of MAP1B mRNA in live U2OS cell. Scale bar, 10 μm. White boxed regions indicate 4 directional transport MAP1B mRNA molecules f , Enlarged views of the white boxed regions 1 and 2 in e . Full movie shown in Supplementary Movie 2. g . Kymograph of boxed regions 1 and 2 in e showing the directed movement of the indicated MAP1B mRNAs. h , A proposed directional transport mechanism directing MAP1B mRNA to cell periphery based on motor protein trafficking along microtubules.

Article Snippet: Next, cells were incubated for 5 min in wash buffer comprising 2×SSC (Thermo Fisher Scientific), 30% (vol/vol) formamide (Thermo Fisher Scientific), and then stained with NOTCH2 or MAP1B mRNA FISH probes in hybridization buffer containing 30% (vol/vol) formamide, 0.1% (wt/vol) yeast tRNA (Thermo Fisher Scientific), 1% (vol/vol) murine RNase inhibitor (New England Biolabs), 10% (wt/vol) dextran sulfate (Sigma), and 2×SSC in a humidity-controlled 37 °C incubator overnight.

Techniques: Labeling

Journal: bioRxiv

Article Title: Single-molecule live-cell RNA imaging with CRISPR-Csm

doi: 10.1101/2024.07.14.603457

Figure Lengend Snippet: a , Single snapshot of individual MAP1B mRNAs in live U2OS cell treated with puromycin. Scale bar, 10 μm. b , Temporal-color coded trajectory of MAP1B mRNA in cell shown in a . c , Kymograph of boxed regions 1 and 2 in b showing the directed movement of the indicated MAP1B mRNAs. Full movie shown in Supplementary Movie 3. d , Directed movement speeds of MAP1B mRNAs under untreated (n=39 events) and puromycin-treated (n=35 events) conditions. Each dot represents a directed movement event. **p < 0.01, t-test. e , Temporal-color coded trajectory of MAP1B mRNA at cell edge. Yellow arrows mark stationary MAP1B RNAs at the cell edge. Scale bar, 2 μm. f , Fixed-cell image of MAP1B mRNAs labeled by GFP-tagged Csm complex after puromycin treatment (left), immunostaining for the P-body marker DCP1A (middle), and their overlay (right). Scale bar, 10 μm. g , Quantification of P-body number with or without puromycin treatment. In both conditions, cells are seeded at the same density; each dot represents P-body number per field of view (FOV) with the same area. ****p < 0.0001, t-test. h , Time-lapse micrographs of MAP1B RNA granule formation after puromycin treatment in live U2OS cell. Yellow arrows mark two small puncta fusing into one larger RNA granule. Full movie is shown in Supplementary Movie 4. Scale bar, 1μm.

Article Snippet: Next, cells were incubated for 5 min in wash buffer comprising 2×SSC (Thermo Fisher Scientific), 30% (vol/vol) formamide (Thermo Fisher Scientific), and then stained with NOTCH2 or MAP1B mRNA FISH probes in hybridization buffer containing 30% (vol/vol) formamide, 0.1% (wt/vol) yeast tRNA (Thermo Fisher Scientific), 1% (vol/vol) murine RNase inhibitor (New England Biolabs), 10% (wt/vol) dextran sulfate (Sigma), and 2×SSC in a humidity-controlled 37 °C incubator overnight.

Techniques: Labeling, Immunostaining, Marker

Journal: bioRxiv

Article Title: Single-molecule live-cell RNA imaging with CRISPR-Csm

doi: 10.1101/2024.07.14.603457

Figure Lengend Snippet: a , Fixed-cell image of individual MAP1B mRNAs labeled by GFP-tagged Csm complex and 48 MAP1B -targeting spacers (left), image of individual MAP1B mRNAs labeled by smFISH probes (middle), and their overlay (right). Scale bar, 10 μm. b , Enlarged view of the red boxed region in a . Scale bar, 1 μm. c, Violin plot showing distance to the nucleus from individual NOTCH2 or MAP1B mRNA molecules. Median indicated by solid line; quartiles indicated by dashed lines. d, Same as c, but showing distance to cell edge. e , Temporal-color coded trajectory of MAP1B mRNA in live U2OS cell. Scale bar, 10 μm. White boxed regions indicate 4 directional transport MAP1B mRNA molecules f , Enlarged views of the white boxed regions 1 and 2 in e . Full movie shown in Supplementary Movie 2. g . Kymograph of boxed regions 1 and 2 in e showing the directed movement of the indicated MAP1B mRNAs. h , A proposed directional transport mechanism directing MAP1B mRNA to cell periphery based on motor protein trafficking along microtubules.

Article Snippet: The next day, 0.8 μg cytoplasmic-targeting Csm complex plasmid and two MAP1B crRNA array plasmids (0.7 μg each) were transfected into cells using 5 μl TransIT-LT1 transfection reagent (Mirus Bio).

Techniques: Labeling

Journal: bioRxiv

Article Title: Single-molecule live-cell RNA imaging with CRISPR-Csm

doi: 10.1101/2024.07.14.603457

Figure Lengend Snippet: a , Single snapshot of individual MAP1B mRNAs in live U2OS cell treated with puromycin. Scale bar, 10 μm. b , Temporal-color coded trajectory of MAP1B mRNA in cell shown in a . c , Kymograph of boxed regions 1 and 2 in b showing the directed movement of the indicated MAP1B mRNAs. Full movie shown in Supplementary Movie 3. d , Directed movement speeds of MAP1B mRNAs under untreated (n=39 events) and puromycin-treated (n=35 events) conditions. Each dot represents a directed movement event. **p < 0.01, t-test. e , Temporal-color coded trajectory of MAP1B mRNA at cell edge. Yellow arrows mark stationary MAP1B RNAs at the cell edge. Scale bar, 2 μm. f , Fixed-cell image of MAP1B mRNAs labeled by GFP-tagged Csm complex after puromycin treatment (left), immunostaining for the P-body marker DCP1A (middle), and their overlay (right). Scale bar, 10 μm. g , Quantification of P-body number with or without puromycin treatment. In both conditions, cells are seeded at the same density; each dot represents P-body number per field of view (FOV) with the same area. ****p < 0.0001, t-test. h , Time-lapse micrographs of MAP1B RNA granule formation after puromycin treatment in live U2OS cell. Yellow arrows mark two small puncta fusing into one larger RNA granule. Full movie is shown in Supplementary Movie 4. Scale bar, 1μm.

Article Snippet: The next day, 0.8 μg cytoplasmic-targeting Csm complex plasmid and two MAP1B crRNA array plasmids (0.7 μg each) were transfected into cells using 5 μl TransIT-LT1 transfection reagent (Mirus Bio).

Techniques: Labeling, Immunostaining, Marker