Fig 2: Proposed model of regulation of trophoblast stem cell proliferation and endoreduplication by Hippo. In TSCs, a substantial pool of YAP remains unphosphorylated and translocate to the nucleus. Nuclear YAP sequesters CDX2, thereby controlling trophoblast proliferation by reducing CYCLIN D1 levels. LATS1 in TSCs form LATS1-LIMK2 complex resulting in reduced activation of LIMK2 followed by reduced COFILIN inactivation. On the other hand, phosphorylation of YAP at ser127 prevents it from translocating into the nucleus when TSCs are induced to differentiate. Here, scarcity of LATS1 activates LIMK2 by enhancing pLIMK2Thr505 which phosphorylate COFILIN and inhibit it from depolymerizing actin. As a result, endoreduplicated trophoblast giant cell formation occurs

Fig 3: Inhibition of LIMK2 impedes TGC formation. A Western blot analysis of pLIMK2Thr505, COFILIN, pCOFILINSer3 in trophoblast cells treated with the indicated doses of BMS-3. Control cells received treatment with equivalent amount of vehicle (DMSO). Cells were harvested after 72 h of treatment. B Densitometry-based quantification of the protein bands from A using ImageJ. Band intensities of COFILIN were normalized using GAPDH as endogenous control. The level of pLIMK2Thr505 and pCOFILINSer3 was normalized relative to the basal level of the respective proteins. C Quantitative real-time PCR analysis of Prl2c2 transcript in trophoblast cells treated with 5 µM and 10 µM BMS-3. D Representative confocal photomicrographs of trophoblast cells under similar experimental conditions. Counterstaining of the nuclei was done using Hoechst (blue). DyLight™ 554 Phalloidin (red) was used to stain the F-actin filaments. Disintegration of the actin filaments has been represented in the boxed areas which have been magnified in the rightmost panel. Scale bar: 10 µm. Magnification: × 72 D ImageJ-based quantification of the nuclear surface area/cell of trophoblast cells upon LIMK2 inhibition. Error bars represent the SEM of 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 4: Endoglin silencing affects cofilin dynamics. (a,b) Western blot performed on n = 5 different clones of ECFC at passage 3–4, <30 days. A representative experiment is shown. Eng silencing in ECFC reduces P-Cofilin, suggesting an increased activity of cofilin when Eng is downregulated. Actin and tubulin were used as loading controls. The last row displays the reduced expression of Eng after siRNA silencing. (b) Quantification of five different ECFC clones shows a significant difference (*** p < 0.001) between ctr-siRNA and Eng-siRNA in the P-Cofilin/Total-Cofilin ratio. (c). Using a real-time impedance-based cell analyzer (iCELLigence system, ACEA Biosciences), ECFC controls (DMSO) and ECFC treated by LIMKi were analyzed in basal condition and under 20 ng/mL TNFa stimulation (gray and black lines, respectively). As shown by the quantification in (d), a significant difference between control ECFC (gray bar) and ECFC treated with LIMKi (black bar) 12 h after TNFa stimulation (** p < 0.01). (e,f) Immunofluorescence for F-actin in ECFC controls vs. ECFC Eng-siRNA +/-TNFa (e) and ECFC controls vs. ECFC LIMKi +/-TNFa (f). (scale bar, 22 µm). (g) The quantification of (e) and (f) confirms that ECFC Eng-siRNA and ECFC LIMKi display the same modifications in terms of F-actin (* p < 0.05).

Fig 5: Association between CFL-1 protein levels and local dissemination in CRC patients. a Representative IHC images for CFL-1 staining in adjacent normal tissues, in tumor stages (I–IV), and in hepatic metastatic lesions. Scale bar = 100 µm. b Quantification of the IHC score data; this value was calculated using the intensity × area of staining. The P value was derived from the paired Student’s t-test (*P < 0.05). IHC and immunohistochemistry of 49 primary tumors (stages I, n = 13; II, n = 12; III, n = 11; and IV, n = 13), and 10 hepatic metastases