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. 2025 Aug 22;11(34):eadt4814. doi: 10.1126/sciadv.adt4814

Fig. 4. AltSFPQ exhibits reduced phase separation compared to wtSFPQ and is up-regulated during neuronal stress.

Fig. 4.

(A) AlphaFold-predicted wtSFPQ protein structure showing the isoform-specific low-complexity C-terminal region (red). (B) Schematic depiction of wtSFPQ and altSFPQ protein domains and IUPred disorder prediction graphs. aa, amino acids. (C) SFPQ protein purification strategy for LLPS assays. (D) Representative images of the eGFP-(wt/alt)SFPQ protein homotypic liquid droplet formation through increasing PEG and fixed protein (3 μM) concentrations, in 150 mM salt buffer; zoomed insets demonstrate spherical forms. (E) Quantification of the LLPS droplet number for 3 μM eGFP-(wt/alt)SFPQ proteins in 150 mM salt buffer and 1% PEG (n = 3; unpaired t test). (F) Ratio of GFP intensity inside/outside LLPS droplets for 3 μM eGFP-(wt/alt)SFPQ proteins in 150 mM salt buffer and 1% PEG (n = 3; unpaired t test). (G) Representative image of the sedimentation assay, gel loaded with supernatant and pellet fractions of TEV cleaved eGFP-SFPQ proteins ± PEG. (H) Quantification of the pellet/supernatant ratio of cleaved eGFP-SFPQ proteins, for samples processed without PEG (n = 3; unpaired t test). (I) AltSFPQ expression (qPCR) in nuclear fractions of motor neurons (DIV6) in response to sorbitol-induced osmotic (OSM) and sodium arsenite–induced oxidative (OX) stresses, normalized over Nit1 and NFX1 fraction housekeeping genes (Frac_HKGs). Data are expressed as FC over untreated samples per line and presented as means ± SEM from three control lines; ANOVA with Dunnett’s multiple comparisons. (J) As for (I) but in cytoplasmic fractions. (K) Representative images of untreated and sodium arsenite–treated motor neurons (DIV6), with BaseScope altSFPQ RNA-FISH, DAPI, and bright-field differential interference contrast (DIC) overlay; arrowheads show RNA puncta within axons. (L) Quantification of BaseScope RNA-FISH puncta per cell in untreated versus sodium arsenite–induced oxidative stress condition in motor neurons (DIV6); data points reflect fields of view (n = 5 to 7), multiple unpaired t tests.