Fig 2: MARK4 blocks mitochondrial oxidative respiration in pig primary trophoblast cells challenged with 400 µM NEFA. (A) Mitochondrial ATP production after transfection with Myc-MARK4, sh-MARK4 for 48 h in cytotrophoblasts. Before assessment of ATP generation, cells were incubated with 400 µM NEFA for 24 h (n = 3); (B) Mitochondrial respiratory parameters were measured in pig cytotrophoblasts after transfection with Myc-MARK4, sh-MARK4 for 48 h. Cells were then treated with 400 µM NEFA for 24 h (n = 3). Oxygen consumption rate (OCR) measurements were normalized to total protein level of whole cell lysates extract (pmol O2/µg protein); (C) Impaired mitochondrial function was estimated by JC-1 fluorescent staining using flow cytometry in cytotrophoblasts (n = 3). The upper right (UR) quadrant depicts the percentage of FL-2 (red channel)-positive cells (high mitochondrial membrane potential) and the lower right (LR) quadrant depicts the percentage of FL-2 (red channel)-negative cells (depolarized mitochondrial membrane potential); (D,E) The activity of mitochondrial complexes I (D) and III (E) in pig trophoblast cells after transfection with Myc-Mark4, sh-Mark4 for 48 h. Cells were then treated with 400 µM NEFA or 2 mM VE for 24 h (n = 3); (F) mRNA levels of mitochondrial complexes I (NDUFB8), II (SDHB), III (UQCRC2) and V (ATP5a) (n = 3). Values are expressed as mean ± SEM. ** p < 0.01; * p < 0.05 compared with the control group. NEFA: non-esterified fatty acid; VE: Vitamin E; Myc-MARK4 group: over expression of MARK4 group; sh-MARK4 group: knock down of MARK4 group; Control: empty vector (EV) group.

Fig 3: Western blot of MARK3 and MARK4 in hippocampal homogenates. (A) Western blots showing MARK3 (top) and MARK4 (bottom) immunoreactivities in hippocampal homogenates from 4 NDE and 3 AD cases. HEK cells transfected with either MARK3 or MARK4, respectively, served as positive controls, whereas non-transfected HEK cells served as negative control. ß-actin served as a normalizing control for protein content in the homogenates. (B) Quantification of full-length (84 respective 82 kDa) and shorter (40–65 kDA) band densities normalized against ß-actin band density. No full-length MARK4 band could be identified or measured. Data depicts means ± SEM. *p < 0.05, ns non-significant by Student’s unpaired two-tailed t-test.

Fig 4: Colocalization analysis of MARK3 and MARK4 with GVD-marker CK1d. Double-immunofluorescence with (A) MARK3 (FITC) or (B) MARK4 (FITC) and GVD-marker CK1d (Alexa Fluor 594, red) in GVD-containing neurons in the CA1-CA2 area. Images are compressions of a Z-stack (first three images) and a 3D-reconstruction of a Z-stack imaged by confocal microscopy showing colocalization in the GVD indicated by arrowheads. Bar graphs show the percentage of intracellular granules that were single stained with MARK3 (MARK3+), MARK4 (MARK4+) or CK1d (CK1d+), or double-stained with MARK3 and CK1d (MARK3+CK1d+) or MARK4 and CK1d (MARK4+ CK1d+), as well as the average size of these granules. MARK4+ granules were significantly larger than MARK4+CK1d+ and CK1d+ granules. MARK3+, MARK3+CK1d+ or CK1d+ granules size was not significantly different. Data depict means ± SEM. *p < 0.05, by 1-way ANOVA with Bonferroni’s Multiple Comparison test, (n = 4–6 cases). Scale bars: 10 µm.

Fig 5: A knockdown of Dvl3 in Sertoli cells cultured in vitro on the TJ-permeability barrier function.a Regimen used in this study to monitor the effects of Dvl3 knockdown by RNAi to assess: (i) changes in the steady-state level of target gene expression by RT-PCR or protein level by immunoblotting (IB) and also protein expression by immunofluorescence analysis (IF); or (ii) changes in Sertoli cell TJ-permeability barrier function by quantifying transepithelial resistance (TER) across the Sertoli cell epithelium on the bicameral units. Transfection of Sertoli cells with siRNA duplexes (including negative non-targeting control siRNA duplexes) was performed on day 3 for 12 h prior to their removal by washing (thrice) with F12/DMEM medium, based on pilot experiments. b Representative findings of an RT-PCR experiment, illustrating specificity of Dishevelled protein knockdown, in which knockdown of Dvl1, Dvl2, or Dvl3 using corresponding specific siRNA duplexes (Table 2) only downregulated the expression of the intended Dvl gene expression. c Representative IB results illustrating a knockdown of Dvl3 by almost ~ 80% (see bar graph on the right panel also) failed to perturb the expression of multiple BTB-associated proteins except a considerably down-regulation of Eps8 (an actin barbed end capping and bundling protein) and up-regulation of MARK4 (a Ser/Thr protein kinase known to stabilize MT45) (see also right panel for composite data of n = 3 independent experiments), thereby perturbing the corresponding actin- and MT-based cytoskeletal function. Uncropped IBs were shown in Figure S2. Each bar in the histogram shown in the right panel is a mean ± SD of n = 3 independent IB experiments. *P < 0.01, compared to the corresponding control by Student’s t test. d Fluorescent staining of Dvl3 (green fluorescence) at the Sertoli cell–cell interface (and some in cell cytosol and in cell nuclei), illustrating a ~ 80% knockdown of Dvl3 following transfection of Sertoli cells with Dvl3-specific siRNA duplexes vs. non-targeting negative control siRNA duplexes (see also bar graph on the right panel for the composite data). Successful transfection was indicated by siGLO (red fluorescence) transfection reagent. Scale bar, 40 µm, which applies to all other micrographs. Each bar in the bar graph on the right panel is a mean ± SD of n = 3 independent experiments. *P < 0.01, by Student’s t test. e A knockdown of Dvl3 (vs. Dvl1 and Dvl2 when compared with control) was considerably more effective to perturb the Sertoli cell TJ-permeability barrier function than Dvl1 or Dvl2 alone, and it was just as effective as Dvl1/2/3 triple knockdown. Each data point is a mean ± SE of n = 4 quadruple bicameral units of a representative experiment. *P < 0.05, when compared to the corresponding control by Student’s t-test. This experiment was repeated thrice with n = 3 independent experiments and yielded similar results