Fig. 3: Mechanisms of NSG assembly driven by hnRNPC recruitment.

A Venn diagram of proteins associated with canonical SGs and NSGs, with corresponding gene ontology analysis of unique components in the NSGs group shown below. B Protein-protein interaction (PPI) network elucidating the key components that interact with G3BP1 in NH₂ and 60% CLS groups. Created in BioRender. Huo (2025) https://BioRender.com/0k8dayr. C The petal Venn diagram identifies the ubiquitous protein hnRNPC critical for NSGs formation. D Radar chart shows the LLPS capacity of hnRNPC. E Immunofluorescence graphs showing the colocalization of hnRNPC with NSGs and free SGs (denoted by f-SGs). 3D-reconstructed information can be found beneath the fluorescence graphs, with certain components pseudo-colored as noted. Normalized fluorescent intensity (FL.Int.) of hnRNPC in f-SGs and NSGs was quantified as presented in (F, n > 100 hnRNPC puncta per group from 3 independent biological replicates, arbitrary units: a.u.). G Colocalization efficacy of G3BP1 and hnRNPC in origination-differentiated SGs (n ≥ 200 cells per group, biological triplicates). For the color-coding, please refer to (E). Randomly depicted regions (marked by a red dashed square) were enlarged (upper panel in the middle) and reconstructed in 3D (lower panel in the middle). The percentage of colocalization is plotted in a pie chart on the right. Legends of Coloc and Free stand for hnRNPC colocalized with or away from G3BP1, respectively. H and I Condensation of G3BP1 and cytoplasmic translocation extent of hnRNPC upon exposure to 60% CLS-AuNPs, AS, and their combination. The proportion of outer nucleus hnRNPC can be found in (J). K Expression of SGs in wild-type and hnRNPC KO cells received stimulation of AS with (w/) or without (w/o) 60% CLS-AuNPs (60% CLS). Quantified condensation results (with fluorescence intensity of each droplet normalized by its volume) can be found in (L, arbitrary units: a.u.). M A schematic illustration revealing the proposed mechanism underlying the formation of NSGs, in which a tri-phase separation proceeded sequentially. Created in BioRender. Huo (2025) https://BioRender.com/jswqnld. Statistical differences were calculated using a two-tailed t-test or one-way ANOVA. Data represents mean ± SD (Independent biological replicates for J and L, 3 and 6, respectively). Source data are provided as a Source Data file.