Fig 2: GSN frameshift mutations failed to reduce the Aß level and apoptosis in SH-SY5Y-APPswe cells. (A) Immunofluorescence images of SH-SY5Y-APPswe cells transfected with GSN plasmids. SH-SY5Y-APPswe cells were characterised with GFP (green). SH-SY5Y cells transfected with GSN plasmids were characterised with DsRed (red). SH-SY5Y-APPswe cells transfected with GSN plasmids were characterised with both GFP (green) and DsRed (red), which appeared yellow in merged images. Scale bar: 20 µm. (B) Western blot analyses of the flag tag to show the expression of different GSN mutant proteins. GSN-WT was used as the negative control; GSN-D214N was the positive control (reported to be associated with FAF); GSN-K346fs and GSN-P3fs were frameshift mutations identified in patients with AD; GSN-R188I, GSN-Y301C and GSN-R525H were VUS identified in patients with AD and distributed in different domains of GSN (C) GSN K346fs mutation failed to reduce Aß42 levels or the ratio of Aß42/40 in SH-SY5Y-APPswe cells, as shown by ELISA analysis. n=3 per group. (D) ELISA analysis of Aß42 and Aß40 in SH-SY5Y-EV cells transfected with GSN plasmids. n=3 per group. (E) Immunostaining of Hoechst 33 342 (light blue) in SH-SY5Y-APPswe and SH-SY5Y-EV cells transfected with GSN plasmids. Scale bar: 50 µm. (F) Quantification of Hoechst-positive cells in (E). GSN K346fs and P3fs mutations failed to reduce apoptosis in SH-SY5Y-APPswe and SH-SY5Y-EV cells. n=6 per group. (G) ROS levels in SH-SY5Y-APPswe and SH-SY5Y-EV cells transfected with GSN plasmids. #P<0.05 vs the control group, *P<0.05 vs the WT group; #/*P<0.05, ##/**P<0.01, ###/***P<0.001, ####/****P<0.0001. Aß, ß-amyloid; AD, Alzheimer’s disease; DAPI, 4',6-diamidino-2'-phenylindole; DsRed: discosoma sp. red fluorescent protein; EV, empty lentivirus; FAF, familial amyloidosis of the Finnish type; GFP: green fluorescent protein; GSN, gelsolin; ROS, reactive oxygen species; VUS, variants of undetermined significance; WT, wild type.

Fig 3: GSN expression is associated with TMB, MSI, TME, and immune checkpoints in 33 cancer types. Relationship between GSN expression and TMB (A), MSI (B) in 33 cancers. (C) Relationship between GSN expression and StromalScore, ImmuneScore, and ESTIMATEScore in 33 cancers. (D) Relationship between GSN expression and immune checkpoint expression in 33 cancers.

Fig 4: Correlation of GSN expression with different tumor stages, molecular subtypes, and immune subtypes. Correlation between GSN expression and different tumor stages, including BLCA (A), THCA (B), SKCM (C), KIRC (D). Correlations between molecular subtypes and GSN expression across TCGA tumors, including (E) ACC; (F) BRCA; (G) COAD; (H) GBM; (I) HNSC; (J) KIRP; (K) LGG; (L) LIHC; (M) LUSC; (N) OV; (O) PCPG; (P) PRAD; (Q) SKCM; (R) STAD; (S) UCEC. (T) Correlations between immune subtypes and GSN expression across TCGA tumors, including ACC, BLCA, BRCA, COAD, HNSC, KICH, KIRC, KIRP, LIHC, LUAD, LUSC, OV, PCPG, PRAD, STAD, THCA, UCEC and UVM. C1 (wound healing), C2 (IFN-g dominant), C3 (inflammatory), C4 (lymphocyte deplete), C5 (immunologically quiet), and C6 (TGF-b dominant).

Fig 5: GSN frameshift mutations failed to inhibit the toxicity of Aß40 in SH-SY5Y cells. (A) Toxicity analysis of SH-SY5Y cells by CCK8 suggested that the concentration of 5 µM Aß40 was a suitable concentration for intervention. n=3 per group. (B) Calcein-AM/PI staining images of SH-SY5Y cells transfected with GSN plasmids and then treated with 5 µM Aß40. Live cells were characterised with calcein-AM (green). Dead cells were characterised with PI (red). Live cells transfected with GSN plasmids appeared yellow in the merged images. Scale bar: 50 µm. (C) Quantification analysis of dead cells in (B). n=6 per group. GSN K346fs and P3fs mutations failed to inhibit the toxicity of Aß40 in SH-SY5Y cells.?p<0.05 vs the 0 µM Aß40 group, #P<0.05 vs the control group, *P<0.05 vs the WT group; ???P<0.001, ####/****P<0.0001. GSN, gelsolin; PI, propidium iodide; WT, wild type.