Fig 2: STK25 acts through LATS1/2 to inhibit YAP, independent of MST/MAP4Ks. a HEK293A cells stably expressing STK25-WT, STK25-KD, or vector were stained for YAP (red) and DNA (white). Scale bar, 20 µm. b YAP intensities from a were quantified and nuclear:cytoplasmic ratios were calculated (n = 225 per group over three biological replicates; ****p < 0.0001, Mann–Whitney test). c YAP localization from a was quantified (n = 3 biological replicates; N>C, YAP is enriched in the nucleus; N=C, YAP is evenly distributed between the nucleus and the cytoplasm; NYAP is enriched in the cytoplasm). d Wild-type and LATS dKO HEK293A were transfected with a vector encoding FLAG-STK25-WT and were stained for YAP (green), FLAG (red), and DNA (white). Scale bar, 20 µm. Arrows indicate representative cells selected for quantification that were positive for FLAG signal (indicating expression of transfected wild-type STK25) as well as an immediately adjacent cell negative of FLAG signal also selected for quantification to serve as controls. e YAP intensities from d and nuclear:cytoplasmic ratios were calculated (n = 200 per group over four biological replicates; ****p < 0.0001, Kruskal–Wallis test with Dunn’s post-test; N.S. indicates “not significant”). f YAP localization from d were quantified as before (n = 4 biological replicates). g Wild-type and LATS dKO HEK293A were transfected with the indicated siRNA, grown to confluence, then stained for YAP (green), Tubulin (red), and DNA (white). Scale bar, 20 µm. h YAP intensities from g were quantified and nuclear:cytoplasmic ratios were calculated (n = 225 over three biological replicates; ****p < 0.0001, Kruskal–Wallis test with Dunn’s post-test; N.S. indicates “not significant.”) i YAP localization from g were quantified as before (n = 3 biological replicates). j Immunoblot and quantification of phosphorylated YAP in MM8KO 293A cells transfected with the indicated siRNA (n = 3 biological replicates; *p < 0.05, paired t-test). k Immunoblot and quantitation of YAP phosphorylation in confluent MM8KO 293A cells stably expressing either the pWZL vector, STK25-WT, or STK25-KD. Quantitation corresponds to levels of phosphorylated YAP as measured via phos-tag electrophoresis (n = 4 biological replicates; *p < 0.05, **p < 0.01, one-way ANOVA with Dunnett’s post-hoc analysis). All data are presented as mean ± SEM

Fig 3: Loss of STK25 promotes activation of YAP. a Control and STK25 KO HEK293A were stained for YAP (green) and DNA (white). Scale bar, 20 µm. b Nuclear:cytoplasmic YAP ratios of control and STK25 KO HEK293A were quantified (n = 225 per group over three biological replicates; ****p < 0.0001, Kruskal–Wallis test). c YAP localization in control and STK25 KO HEK293A was quantified (n = 3 biological replicates; N>C, YAP is enriched in the nucleus; N=C, YAP is evenly distributed between the nucleus and the cytoplasm; NYAP is enriched in the cytoplasm). d HEK293A cells transfected with either control siRNA or STK25 siRNA stained for YAP (green), Actin (red), and DNA (white) following treatment with 5 µM DCB. Scale bar, 20 µm. e YAP localization was quantified (n = 3 biological replicates; N>C, YAP is enriched in the nucleus; N=C, YAP is evenly distributed between the nucleus and the cytoplasm; NYAP is enriched in the cytoplasm). f YAP intensity was quantified and nuclear:cytoplasmic ratios were calculated (n = 225 per group over three biological replicates; ****p < 0.0001, Mann–Whitney test). g qPCR analysis of YAP-target gene expression in IMR90 fibroblasts transfected with the indicated siRNA (n = 4; **p < 0.01, unpaired t-test). h qPCR analysis of YAP-target gene expression in wild-type, STK25+/-, and STK25-/- mouse embryonic fibroblasts (n = 3 biological replicates; **p < 0.01, ****p < 0.0001, one-way ANOVA with Dunnett’s post-hoc analysis). i Expression of the TEAD luciferase reporter in HEK293A cells transfected with the indicated siRNA. Cells were transfected with siRNA, followed by transfection with 8X GTIIC TEAD luciferase reporter and pRL-TK renilla luciferase. Reporter luciferase activity was normalized to Renilla luciferase (n = 3 biological replicates; *p < 0.05, one-way ANOVA with Dunnett’s post-hoc analysis). j An expression signature of genes most upregulated upon loss of STK25 was constructed and GSEA was performed against a curated list of publicly available active YAP/TAZ gene sets. The top three most enriched gene sets are shown here. All data are presented as mean ± SEM

Fig 4: STK25 loss is common in human cancers and adversely affects patient survival. a Graphical representation of human cancers with the highest frequencies of STK25 deletion. Data was accessed using the cBioPortal online program (http://www.cbioportal.org/). b Survival data of sarcoma patients from the TCGA dataset were accessed using the Xenabrowser online program (https://xenabrowser.net/) and overall survival rates and times were assessed for patients with and without deletions of STK25 (*p = 0.0172, n = 215, log-rank test). c Proposed model of STK25 in Hippo tumor suppressor signaling

Fig 5: STK25 directly promotes phosphorylation of LATS activation loop. a LATS2 was immunoprecipitated from HEK293A cells co-transfected with HA-LATS2 and either vector control or FLAG-STK25. Co-precipitation of FLAG-STK25 with HA-LATS2 was assessed by immunoblotting. b HEK293A cells were transfected with Myc-LATS1 or HA-LATS2. LATS1 and LATS2 were immunoprecipitated using antibodies directed against their tags and co-precipitation of endogenous STK25 was assessed by immunoblotting. c Schema of the in vitro kinase assay set-up. d Immunoprecipitation (IP)-purified wild-type LATS2 (HA-LATS2-WT) was co-incubated with IP-purified wild-type STK25 (FLAG-STK25-WT), kinase-dead STK25 (FLAG-STK25-KD), or wild-type MAP4K1 (FLAG-MAP4K1), and assessed for phosphorylation of its hydrophobic motif (P-LATS-HM) or activation loop (P-LATS-AL). e IP-purified kinase-dead LATS2 (HA-LATS2-KD) from transfected LATS1/2-STK25 triple KO 293A cells (LS tKO 293A) was co-incubated with IP-purified FLAG-STK25-WT, FLAG-STK25-KD, or FLAG-MST1, all from transfected LS tKO 293A. Levels of phosphorylated LATS at the activation loop (P-LATS-AL) were assessed via immunoblotting. Levels of phosphorylated LATS2-KD at the activation loop were then quantitated via densitometry (n = 4 biological replicates; ****p < 0.0001, N.S. indicates “not significant,” unpaired t-test). f Immunoblot and quantitation of LATS activation loop phosphorylation (P-LATS-AL) following treatment with 10 µM DCB in HEK293A cells transfected with the indicated siRNA (n = 4 biological replicates; *p < 0.05, paired t-test). g HEK293A cells were co-transfected with HA-tagged wild-type LATS2 (HA-LATS2-WT) and either vector control (Vector), wild-type STK25 (FLAG-STK25-WT), or kinase-dead STK25 (FLAG-STK25-KD). LATS2 was immunoprecipitated and used to assess levels of activation loop phosphorylation by immunoblotting. Input lysates were assessed by immunoblotting for assessing protein loading and verification of transfected protein expression. h Immunoblot and quantitation of YAP phosphorylation in LATS dKO 293A cells transfected with the indicated expression plasmids. LATS2-TA indicates hydrophobic motif mutant LATS2 T1041A (n = 4 biological replicates; **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Dunnett’s post-hoc analysis). All data are presented as mean ± SEM