Fig 1: Proteasomal, lysosomal and SIK inhibitors stabilize Par3A. HEK293T cells were co-transfected with increasing amount of WT SIK and constant amount of Par3 in the presence and absence of MG132 and chloroquine (CQ) to inhibit proteasomal and lysosomal protease activity respectively, followed by immunoblotting of total cell lysates. α-Tubulin was used as a loading control. B. HEK293T cells were co-transfected with WT SIK and Par3 in the presence and absence of the pan-SIK inhibitor HG-9-91-01 for a total period of 36 h, followed by immunoblotting of total cell lysates. GAPDH was used as a loading control. Representative immunoblots out of at least three repeats are shown in each panel. Densitometric quantification is provided on top of the Par3 and SIK immunoblot lanes in each panel; the values indicate relative expression of each protein normalized to the corresponding level of α-tubulin (panel A) or GAPDH (panel B). The basal levels corresponding to endogenous expression of each protein show small variability from one panel to the other, which reflects protein expression and stability in each independent experiment. Molecular size markers in kilo Dalton (kD) are also marked.
Fig 2: Yap and Taz deletion suppresses ectopic RGP generation and heterotopia formation in the Pard3-deficient cortex. (A) Representative confocal images of E14.5 control, Pard3 cKO, Yap;Taz cDKO, and Pard3;Yap;Taz conditional triple-knockout (cTKO) cortices stained for PAX6 (green) and counterstained for DAPI (blue). Asterisk indicates the ectopic PAX6+ RGPs in the Pard3 cKO cortex. Bar, 30 µm. (B) Quantification of the number of PAX6+ cells per 200-µm radial column in E14.5 control, Pard3 cKO, Yap;Taz cDKO, and Pard3;Yap;Taz cTKO cortices. n = 6 brains per genotype; unpaired two-tailed t-test with Welch's correction. (C) Representative nissl staining images of P12 control, Pard3 cKO, Yap;Taz cDKO, and Pard3;Yap;Taz cTKO brain sections. (Asterisks) Giant heterotopia; (arrowheads) defective VZ surface and hydrocephalus. Bar, 2.5 mm. (D) Representative confocal images of P12 control, Pard3 cKO, Yap;Taz cDKO, and Pard3;Yap;Taz cTKO cortices stained for layer V/VI neuronal marker CTIP2 (green) and layer II–IV neuronal marker CUX1 (red) and counterstained for DAPI (blue). Bar, 70 µm. (E) Quantification of the number of CTIP2+ (top) and CUX1+ (bottom) cells per 300-µm radial column in control, Pard3 cKO, Yap;Taz cDKO, and Pard3;Yap;Taz cTKO cortices at P12. n = 6 brains per genotype; unpaired two-tailed t-test with Welch's correction. For all box and whisker plots, the center line indicates the median, the box indicates the interquartile range, and the whiskers indicate minimum and maximum.
Fig 3: SIK induces degradation of Par3A. Schematic overview of the in silico predicted phosphorylation site in Par3. The underlined serine corresponds to Ser885, while the grey serines correspond to Ser884 and Ser888. The Par3 domain organization is also shown. B.-C. SIK induces degradation of Par3. Increasing amounts of SIK plasmid were co-transfected with full-length Par3 (B) or a single amount of SIK WT and K56R mutant was co-transfected with full length Par3 in HEK293T cells. α-Tubulin was used as a loading control. D.-F. Mutation of Ser885 to alanine (S885A) in Par3 prevents SIK-mediated degradation (D), while mutation of Ser888 (E) or Ser884 (F) to alanine has no effect. WT SIK was co-expressed with either WT or mutated full-length Par3 in HEK293T cells. α-Tubulin was used as a loading control (D, F), and protein levels in (E) were normalized with a Bradford assay. Densitometric quantification is provided on top of the Par3 and SIK immunoblot lanes in each panel; the values indicate relative expression of each protein normalized to the corresponding level of α-tubulin (panels B, C, D and F) or absolute densitometric values after subtraction of background density (panel E). The basal levels corresponding to endogenous expression of each protein show small variability from one panel to the other, which reflects protein expression and stability in each independent experiment.
Fig 4: SIK forms a complex with Par3A. Co-immunoprecipitation of SIK with Par3. HEK293T cells were transfected with SIK and the three isoforms of Par3 (termed 180 kD, 150 kD and 100 kD according to their molecular size). Total cell lysates (TCL) are shown, with α-tubulin as loading control. Rabbit IgG was used in control immunoprecipitations. B. Co-immunoprecipitation of Par3 with SIK. The same Par3 constructs as in (A) were transfected, together with a kinase-dead mutant of SIK (K56R). An in-house made SIK-specific antibody was used for immunoprecipitation. A non-related antibody was used in control immunoprecipitations. C. Semi-endogenous co-immunoprecipitation between transfected SIK and endogenous Par3. Wild-type (WT) and K56R mutant SIK were transfected in NMuMG cells and the immunoprecipitation was performed with either a Par3- or a SIK-specific antibody. The cells were treated with 5 ng/ml TGFβ or vehicle overnight. Dotted lines in panels B and C indicate that intervening lanes of the same membrane have been cut out. D. Detection of endogenous phosphorylated forms of Par3. NMuMG cells were transfected with 10 nM of control siRNA or siSIK for 24 h. Afterwards, the growth medium was changed and a second siRNA transfection identical to the first was performed for another 24 h. After lysing, endogenous Par3 was immunoprecipitated and after washing, the beads were incubated with radioactive [32P]ATP for 1 h. Following addition of SDS-containing Laemmli buffer and SDS-PAGE, radioactive bands were visualized by autoradiography. The top band corresponds to 180 kD. Representative immunoblots out of at least three repeats are shown in each panel. Molecular size markers in kilo Dalton (kD) are also marked.
Fig 5: Expression of SIK and Par3 negatively correlate in human tumorsA. Immunohistochemical analysis of expression of SIK (brown staining) in ductal breast carcinoma, Gleason grade 3 prostate carcinoma, cervical carcinoma and colorectal carcinoma. Tumor tissue was counterstained with hematoxylin. B. Table of SIK and Par3 protein expression in various human tumors analyzed by immunohistochemistry. The data are derived from the human protein atlas (https://www.proteinatlas.org/) and the number of individual patient samples per tumor type (left column) is plotted under four different categories, undetectable, low, medium and high. Note that in all 10 tumor types relatively high (medium and high) SIK expression corresponds to significantly low (low and medium) Par3 expression. The average number of patient specimen analyzed were between 10 and 12 for most tumors except for thyroid carcinoma where 4 independent patient samples were scored. C. Kaplan-Meier survival curves of breast, stomach (gastric) and ovarian carcinoma patients with low (black curves) and high (red curves) levels of SIK mRNA expression according to TCGA dataset. Statistical significance of the difference between the two curves is also indicated in terms of the corresponding hazard ratio (HR) with 95% confidence interval and associated probability value, which is smaller than 0.02, based on a χ-squared statistic. D. Overview of SIK-mediated effects on Par3. TGFβ acts on epithelial cells causing SIK expression, which then promotes Par3 phosphorylation (P symbol in circle), causing degradation of both Par3 and SIK1. During the same course of TGFβ signaling epithelial cells transdifferentiate (thick arrow) to mesenchymal.
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