Fig 2: Role of LATS1 in trophoblast giant cell differentiation. A Western blot analysis of LATS1 and pLATS1Thr1079 in TSCs (Stem cells) and differentiated trophoblast cells. GAPDH was used as an internal control. B Densitometric quantification of the protein bands from A. The level of pLATS1Thr1079 was quantified relative to the basal level of LATS1. C Western blotting showing the coimmunoprecipitation of endogenous LATS1 with LIMK2 in TSCs and differentiated trophoblast cells. Immunoprecipitation (IP) with anti-LATS1 antibody was followed by immunoblot with anti-LIMK2 antibody. Immunoprecipitation using an isotype matched IgG was used to ensure the specificity of the capturing antibody. D Immunoprecipitation demonstrates that LATS1 exists as a protein complex pulled down by anti-LIMK2 antibody. E Western blot analysis showing the probing of LIMK2, pLIMK2Thr505, COFILIN, pCOFILINSer3 and CHRONOPHIN in TSCs and differentiated trophoblast cells. D Densitometric analysis of the protein bands in E using GAPDH as an endogenous control. LIMK2, COFILIN and CHRONOPHIN are normalized relative to the level of the housekeeping gene, whereas the level of pLIMK2Thr505 and pCOFILINSer3 was normalized relative to the basal level of LIMK2 and COFILIN, respectively. Values are represented as mean ± SEM from three independent biological replicates. Statistical analysis was performed using Student’s unpaired t-test, *p < 0.05; **p < 0.005; ***p < 0.0005, ns, nonsignificant

Fig 3: Limited LATS1 potentiates trophoblast endoreduplication. A Quantitative real-time PCR analysis of LATS1 overexpression in trophoblast cells. Differentiation was induced 6 h post-transfection and the cells were harvested after 72 h. B Quantitative real-time PCR analysis of Prl2c2 in LATS1 overexpressing cells under similar conditions described in A. C Confocal photomicrographic images of LATS1 overexpressing cells 72 h post-differentiation induction. The nuclei have been stained using Hoechst (blue) and counterstaining of the cytosolic actin filaments has been done using DyLight™ 554 Phalloidin (red). Boxed areas represent disintegration of the actin filaments which has been magnified in the rightmost panel. Scale bar: 10 µm. Magnification: × 88 D Quantitative assessment of the nuclear surface area/cell of trophoblast cells upon LATS1 overexpression using ImageJ software. E FACS profile showing the DNA content histogram (top) of the gated population in cells transiently transfected with LATS1 or empty vector control and stained with Hoechst. The percentage of cells with DNA content > 4 N is shown in the right panel. E The percentage of polyploid nuclei has been plotted from an average of six independent biological flow cytometric replicates. Values are represented as mean ± SEM from three independent biological replicates. Statistical analysis was performed using Student’s unpaired t-test, *p < 0.05; **p < 0.005; ***p < 0.0005

Fig 4: 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

Fig 5: The impact of modulating cellular YAP activity on MCP-1 expression.A Cellular expression of LATS1, B YAP, p-YAP(ser127), TEAD, and C MCP-1 in HK-2 cells with RNA interference (Lats1 knockdown). Shown are representative blots from at least three separate experiments with similar results. D Representative images of immunofluorescent staining for YAP in HK-2 cells with LATS1 silence. E Semi-quantification of nuclear YAP expression in cells with LATS1 silence (n = 10 randomly captured images per group). F Cellular expression of YAP, p-YAP (ser127), TEAD in HK-2 cells treated with LPA or VP. G Cellular expression of YAP, p-YAP (ser127), TEAD, and H MCP-1 in HK-2 cells with RNA interference (Yap knockdown). Shown are representative blots from at least three separate experiments with similar results. I Representative images of immunofluorescence staining for YAP in HK-2 cells with Yap knockdown. J Semi-quantification of nuclear YAP expression in HK-2 cells after silencing YAP expression (n = 10 randomly captured images per group). Scale bar = 20 µm in all images of D and I. All values are means ± SEM. #p < 0.001 defined as significant.