Fig 2: Representative localization of 14-3-3 interacting proteins in the mouse heart. (A). Venn diagram of proteins identified in our cardiac 14-3-3 interactome and the corresponding proteins within three mitochondrial protein databases, IMPI (Q2 2018), MitoCarta (2.0) and Gene Ontology (GO) mitochondrion. (B). Colocalization of 14-3-3 proteins and mitochondria in cardiomyocytes. Immunofluorescence images of indicated proteins and subcellular components and their merged images in HL-1 cells or primary left ventricular cardiomyocytes. Staining color: green, 14-3-3 proteins; red, TOMM20 proteins that localize in the outer membrane of mitochondria or MitoTracker indicating mitochondria; purple, MitoTracker in HL-1 cells; blue, nucleus; yellow or white, localization of 14-3-3 in mitochondria. Bars as shown in each row. (C). Colocalization of 14-3-3 proteins and ribosome in cardiomyocytes. Immunofluorescence images of indicated proteins and their merged images in HL-1 cells or primary left ventricular cardiomyocytes. Staining color: green, 14-3-3 proteins; red, three different ribosomal subunits, RPS6 (C) and RPS3, indicating ribosome; blue, nucleus; yellow, localization of 14-3-3 in ribosome. Bars as shown in each row.

Fig 3: 14-3-3s are required for the interaction between Tctp and Rheb and organ development.(a) Knockdown of 14-3-3s in S2 cells shows depletion of 14-3-3s levels after 6 days of RNAi treatment. ?i, ?i and ?i+?i indicate RNAi for 14-3-3?, 14-3-3? and both isoforms, respectively. Levels of Tctp and Rheb were not noticeably affected by 14-3-3 depletion. The treatment performed to detect Tctp, Rheb, and actin level was double knockdown of both isoforms of 14-3-3. (b) 14-3-3 depletion affects the interaction between V5-Tctp and V5myc-Rheb after 6 days of RNAi treatment. Anti-Myc antibody was used for IP. Tctp was detected by anti-Tctp antibody. Depletion of both isoforms abolished the Tctp–Rheb interaction. (c–j) Double knockdown of 14-3-3s causes loss of targeted tissues. ey-Gal4 control (c) 14-3-3? RNAi (d) and 14-3-3? RNAi (e) show normal pattern of eye-head in pupae. Knockdown of both 14-3-3 isoforms causes pupal lethality with loss of eye and head (f). (g–j) Pattern of Elav staining in larval eye discs from indicated genotypes. Knockdown of 14-3-3? or 14-3-3? has no obvious effect on eye disc development (h,i). Knockdown of both isoforms results in small disc (the area marked with dotted line) with no retinal differentiation (j). (k–n) Double knockdown of 14-3-3s results in reduced proliferation. MS1096-Gal4 control (k) 14-3-3? RNAi (l) and 14-3-3? RNAi (m) show normal level of PH3 staining in wing disc. Knockdown of both 14-3-3 isoforms causes strong reduction of PH3 in the wing pouch (circled area) (n). (o–r) Caspase activation marked by anti-Caspase 3 (Cas-3) staining is not detected in MS1096-Gal4 control (o) 14-3-3? RNAi (p) and 14-3-3? RNAi (q). Double knockdown of both 14-3-3 isoforms results in strong activation of Cas-3 (r). Scale bar, 300 µm (c–f); 100 µm (g–j); 100 µm (k–r).

Fig 4: Tctp and Rheb interact genetically with 14-3-3.(a–j) Genetic interaction between Tctp and 14-3-3 in eye. (a) Adult fly with one copy of ey-Gal4 shows normal eye. (b,c) Knockdown of 14-3-3? (b) or 14-3-3? (c) alone shows no obvious effect on eye. (d,e) Heterozygotes 14-3-3?j2B10/+ (d) or 14-3-3?07103/+ (e) show normal eyes. (f) Knockdown of Tctp results in small and rough eye phenotype. (g,h) Knockdown of both Tctp and 14-3-3? (g) or 14-3-3? (h) strongly enhances the eye phenotype caused by Tctp RNAi alone. (i,j) One copy of 14-3-3?j2B10 (i) or 14-3-3?07103 (j) enhances the Tctp RNAi phenotype. (k–p) Genetic interaction between Tctp and 14-3-3 in wing. (k) Control wing with one copy of nub-Gal4 is normal. (l) Knockdown of 14-3-3? affects the anterior and posterior crossveins, but has no effect on wing size. (m) Knockdown of 14-3-3? does not affect wing development. (n) Tctp RNAi results in mildly reduced and curled wing. (o,p) Double knockdown of Tctp and 14-3-3? (o) or 14-3-3? (p) enhances Tctp RNAi phenotypes. RNAi knockdown was induced by ey-Gal4 (a–c,f–j) and nub-Gal4 (k–p). Scale bar, 200 µm (a–j); 400 µm (k–p).

Fig 5: Functional enrichments from the 14-3-3 interactome using clusterProfiler. IDs of the 52 proteins were transferred into the ENTREZ gene IDs and imported into R package, clusterProfiler to conduct functional enrichment. Top ten terms with adjusted p-value < 0.05 were displayed in bubble charts. Four categories were GO_BP from Biological Process (Gene Ontology) database (A), GO_CC from Cellular Component (Gene Ontology) database (B), GO_MF from Molecular Function (Gene Ontology) database (C), and KEGG from KEGG pathways database (D).