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. 2024 Jun 12;15:5033. doi: 10.1038/s41467-024-49416-5

Fig. 2. METTL3 downregulation reduces the number of stress granules in ALS cellular models.

Fig. 2

a Representative images of the indicated stressed SK-N-BE cells. G3BP1 antibody staining is depicted in green, FUS-Flag in red. The nuclei are stained with DAPI (blue). The merge of the signal is shown. The scale bar is 10 µm. (n = 3 biologically independent replicates.) b Representative single focal planes captions of mFISH for CLSTN1 transcript (red), immunofluorescence for stress granule marker G3BP1 (green) and DAPI (blue) in the indicated SK-N-BE cells (top). In the right panel, the digital magnification of G3BP1 granules is highlighted by dashed squares. All scale bars correspond to 3 µm. Bar plot showing representing the percentage of CLSTN1 smFISH signals colocalizing with stress granules as mean of replicates with standard deviation. Dots represent independent replicates. From 356 to #498, cells were analyzed for each condition. n = 3 biologically independent replicates. The ratio of each sample versus its control was tested by a two-tailed Student’s t-test. P values are indicated. c, d Box plots illustrating the number of stress granules per cell in the indicated SK-N-BE cell lines (n = 3 biologically independent replicates.) or iPS-derived MN (n = 4 biologically independent replicates). The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum (See Source Data file for values). The red dot indicates the average number of granules in each sample after 1 h stress. Each black dot represents the number of granules in a single field. Seven fields were acquired for each biological replicate. The ratio of each sample versus its control was tested by a two-tailed Student’s t-test. P values are indicated. e Box plots illustrating the number of stress granules per cell in SK-N-BE cells either overexpressing METTL3 (“oeMETTL3”) or downregulated for ALKBH5 (“ALKBH5 KD”) with respect to a control condition (“Ctrl”). The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum (See Source Data file for values). The red dot indicates the average number of granules in each sample after 1 h stress. Each black dot represents the number of granules in a single field. Seven fields were acquired for each biological replicate. n = 3 biologically independent replicates. The ratio of each sample versus its control was tested by a two-tailed Student’s t-test. P values are indicated. Source data are provided as a Source Data file.