Fig 2: Immunohistochemical characterization of MARK1-4 protein expression in the hippocampus. (A-H) Photomicrographs of immunohistochemical staining from the CA2 area of representative NDE and AD cases (#9, #7, #13, #18 and #34) with specific antibodies for each MARK isoform. Neurons containing granulovacuolar degeneration bodies (GVDs) are marked with arrowheads and magnified in insets. MARK1 and MARK2 antibodies reveal a diffuse cytoplasmic staining but no positive staining in GVDs (red arrowheads). Black arrowheads indicate positive staining in GVDs as this is observed in all GVDs with the MARK4 antibody and in some but not all GVDs with the MARK3 antibody. Scale bar: 50 μm. (I) Quantification of the number of MARK3, MARK4 or CK1δ neurons with granular immunoreactivity in the CA1-CA2 area or CA3-CA4 area. n = 4–5 NDE; 7–10 AD. Data depicts means ± SEM. *p < 0.05, **p < 0.01 by Student’s unpaired t-test. (J) Data in (I) plotted against Braak stage. n = 1(Br0); 2-3(Br1); 1(Br2); 2(Br4); 2-5(Br5); 3(Br6).

Fig 3: MARKs mediate phosphorylation at S502 and 14-3-3 binding to TNK1, which restrains the movement of TNK1 into heavy membrane-associated clusters.a Radiometric protein kinase assays were performed to identify the kinase responsible for phosphorylation of TNK1 at S502. Corrected kinase activity (raw value minus sample peptide background) was measured in biological triplicate. Graph shows mean kinase activity in counts per minute (cpm) with error bars representing SEM. b Radiometric protein kinase assays were performed with recombinant MARK3 incubated with TNK1 WT or S502A peptides. Corrected kinase activity (raw value minus sample peptide background) was measured in biological triplicate. Graph shows mean kinase activity in counts per minute (cpm) with error bar representing SEM. P value is calculated using a two-tailed student t-test (n = 3). c HEK293Ts expressing FLAG-TNK1 were treated with 1uM of indicated inhibitor for 4 h (MARKi: MRT 67307 dihydrochloride, CaMKKi: STO-609, CaMKIIi: KN-92, KN-93, RSKi: LJI-308, IKKi: BMS 345541). FLAG-TNK1 was immunoprecipitated and blotted for pS502 and 14-3-3. Graphs show quantitation of n = 3 biological replicates normalized to WT TNK1. Error bars represent SEM. P-values were calculated using a two-tailed student t-test. d MARK kinases were individually depleted with siRNA (100 nM) in A549 cells stably expressing FLAG-TNK1. FLAG-TNK1 was immunoprecipitated on FLAG resin and immunoblotted for pS502 and 14-3-3. Blot shows the representative image from n = 5 biological replicates. (Bottom left) Alignment of TNK1 sequence encompassing pS502 to a MARK-phosphorylated 14-3-3 binding site on PAR3a. (Bottom right) Schematic model of MARK-mediated phosphorylation of TNK1 at S502 and 14-3-3 binding. e HEK-293A cells expressing TNK1-GFP (WT or indicated mutants) were analyzed by confocal imaging for diffuse or punctate TNK1 localization and deconvolved with Hyugens software. TNK1 ?UBA-expressing cells were treated with 1 µM MARK inhibitor for 2 h (MRT 67307) (n = 50). The scale bar is 10 µm. f Cells from panel e were analyzed for TNK1 puncta volume using Leica 3D analysis software. Graph shows mean TNK1 puncta volume with error bars representing SEM between individual cells. (WT n = 10, AAA n = 6). P-value was calculated using a two-tailed student t-test. g Cells from panel e were scored for diffuse and punctate TNK1 localization as in Fig. 1b (n = 50). h HEK-293T cells expressing WT or the indicated mutants of FLAG-TNK1 were biochemically fractionated into the cytosol and heavy membrane fractions, followed by immunoblotting for FLAG - TNK1 and TNK1 phosphorylation at Y277. Fractions were also immunoblotted for Bak and actin as loading controls. The graph shows the relative HM/C ratio normalized to WT TNK1. Quantitation is from multiple biological replicates (n = 4) with error bars representing SEM. P-values were calculated using a two-tailed student t-test. Source data are provided as a Source Data file.

Fig 4: Expression of MARK1-3 in hippocampal brain sections. Low magnification micrographs of three adjacent hippocampal sections from an NDE case (#7), with antibodies towards MARK1-3. MARK1 expression is particularly strong in the CA2-layer, with apical dendrites and cell bodies intensely labeled in this area. Less expression is detectable in the CA3, CA4 and DG. MARK2 expression is more homogenously expressed in the CA layers of the hippocampus, particularly strong in CA2 and dentate gyrus (DG), although considerable inter-individual difference existed for this isoform. MARK3 expression on the cytoplasmic level is only detectable in the CA2 layer. Inset images are boxed areas in the CA2 layer. High magnification images are included in Figure 2. Scale bar: 1 mm.

Fig 5: Downregulation of MARK3 is associated with poor clinical outcome in patients with HGSOC.a Schematic of in silico integrative analysis to identify HGSOC-associated dysregulated genes. MARK3 is downregulated in HGSOCs across two microarray datasets and one RNA-seq data set, and low MARK3 mRNA expression is associated with poor clinical outcomes in the TCGA HGSOC cohort. b–d mRNA expression levels of MARK3 (b), MAF (c), and BNC1 (d) in two independent microarray datasets. GSE18521 and GSE26712 offer microarray data of human ovarian surface epithelial cell (HOSE) samples and HGSOC samples [GSE18521(HOSE: n = 10, HGSOC: n = 53) and GSE26712 (HOSE: n = 10, HGSOC: n = 185)]. Statistical analysis was performed using an unpaired Student’s t-test. e Kaplan–Meier survival curves classified by high (n = 128) or low (n = 297) MARK3 mRNA expression in the TCGA HGSOC cohort. Low MARK3 group exhibits poor overall survival (left) and poor disease-free survival (right). f Platinum-resistant HGSOCs (n = 90) have lower MARK3 mRNA expression compared to platinum-sensitive HGSOCs (n = 197). Error bars represent mean ± standard deviation (SD). Statistical analysis was performed using an unpaired Student’s t-test. g, h RT-qPCR shows that MARK3 expression is downregulated in ovarian cancer cell lines [comparison between control (n = 3) and ovarian cancer cell line (n = 9)] (g) and the HGSOC tissues [ovary (n = 6), fallopian tube (n = 5) and HGSOC (n = 49)] (h). Ovarian cancer cell lines with HGSOC-like genetic profile, such as TP53 mutation and hyper CNA are included in this experiment. Error bars represent mean ± SD from three biological replicates. Statistical analysis was performed using an unpaired Student’s t-test. i Immunoblotting shows that MARK3 expression is downregulated in ovarian cancer cell lines compared to that in 293T cells (non-OvCa; non-Ovarian Carcinoma). j Immunohistochemistry displays that MARK3 is ubiquitously expressed in human fallopian tube secretory epithelial cells (HFTSECs) and serous tubal intraepithelial carcinomas (STICs), whereas its expression is diminished in primary HGSOCs. The expression of LKB1 is clearly decreased in STICs (arrow). p53 is used as a positive marker for STICs (arrow) and HGSOCs. Scale bars, 100 µm.