Anap labeling of TDP-43 and G3BP1 in HeLa cells.

A, B. HeLa cells expressing G3BP1-Anap and TDP-43-Anap under basal conditions or 250 μM sodium arsenite treatment. Anap and antibody signals are shown in blue and green, respectively; for merged panels, Anap was pseudo-colored red. Scale bars: 10 μm (overview), 3 μm (zoom). C, D. FRAP of G3BP1-Anap, G3BP1-GFP, and TDP-43-Anap, following 250 μM sodium arsenite treatment. ROI signal intensities are displayed in rainbow RGB (red–high, blue– low). Scale bars: 5 μm (cells), 1 μm (ROI). E. Comparison of HeLa cells expressing TDP-43-Anap and TDP-43-YFP under basal conditions or 250 μM sodium arsenite treatment. Here, Anap labeling and YFP labeling yield a blue signal and a yellow to green signal, respectively. F, G. Relative fluorescence recovery of each time point after photobleaching for G3BP1-Anap, G3BP1-GFP, and TDP-43-Anap. H. Immunoblotting of wild-type TDP-43 and TDP-43-Anap. The Rabbit anti-TDP-43 antibody was used. KO: TDP-43 knockout; V100Stop/Anap: the expression of TDP-43V100TAG via Anap labeling without or with the addition of Anap. I. Survival of TDP-43 knockout HeLa cells expressing TDP-43-Anap after being treated with 12.5 μM sodium arsenite for 24h. Here, the calcien EM staining was used to detect the survival rate of cells, and the relative survival rate= sodium arsenite treatment/nontreatment for each group. OE: overexpression of TDP-43. J. Survival of iTDPKO inducible mouse ES cells expressing TDP-43-Anap. Here, cell counting-Lite 2.0 Luminescent cell viability assay kit was used to detect the survival rate of ES cells. TDP-43 knockout was induced by 4-HT (300ng/ml) for 5 days. The relative survival rate= 4-HT induction/DMSO for each group. All quantitative data (F, G, I, J) are shown as mean ± SEM. ***P = 0.0001; ****P < 0.0001; n.s., not significant.

Anap labeling of TDP-43 and G3BP1 in neurons.

A, C. Primary mouse cortical neurons expressing G3BP1-Anap and TDP-43-Anap under basal conditions or 250 μM sodium arsenite treatment. Cells were stained with anti-G3BP1 (human-specific) or anti-TDP-43 (human-specific) antibodies with chicken anti-Tuj1 as a neuron marker. Signals: Anap (blue, pseudo-colored red in merged images), antibody (green), Tuj1 (gray). Scale bar, 10 μm. B, D. The colocalization level of each region of interest for G3BP1 and TDP-43. Colocalization threshold analysis in Fuji ImageJ was used to analyze the R value of each region (a higher R value means a higher colocalization level). Scale bar, 1 μm.

Schematic of the Anap labeling system for G3BP1 and TDP-43 using genetic code expansion.

Briefly, two plasmids were required to express the protein with site-specific Anap incorporation, one for Anap incorporation and one for the mutated protein of interest (TAG introduction). As the plasmids were transfected into cells, the orthogonal Anap-tRNA synthetase would charge the Anap to its cognate tRNA, and the tRNA would incorporate the Anap site-specifically into the protein of interest in response to the TAG stop codon. Cells expressing Anap-labeled TDP-43 and G3BP1 were subsequently imaged by confocal microscopy, either after fixation or in live-cell conditions.