Fig 1: Antiandrogen treatment increases pMK5 Ser354, and TLK1 inhibition reduces it. (A) pMK5‐S354, total MK5 and TLK1/1B level in different androgen‐dependent and androgen‐independent PCa cell lines determined by WB. GAPDH was used as a loading control. (B) pMK5 Ser354 level in LNCaP cells after two different concentrations of bicalutamide treatment compared with the vehicle control (VC) determined by WB. (C) pMK5‐S354 and TLK1 level in LNCaP cells after treatment with two different TLK1 inhibitors (THD and J54) at 10 µm concentration. (D) Densitometric quantification of pMK5‐S354 level normalized to total MK5 in different PCA cell lines. (E) Densitometric quantification of pMK5‐S354 level normalized to total MK5 upon bicalutamide treatment. (F) Densitometric quantification of pMK5‐S354 level normalized to loading control upon two different TLK1 inhibitor treatments. Each figure is representative of three replicates. One‐way ANOVA followed by Tukey’s post hoc analysis was used for multiple group comparison. Error bar represents standard error of the mean (SEM).
Fig 2: MK5 inhibition reduces wound healing in PCa cell lines. Scratch wound repair assay was conducted to determine the 2D migration rate by plotting relative wound density against different time points. (A) LNCaP vehicle control (VC), MR: 1.08 ± 0.05; LNCaP GLPG 0.1 µm, MR: 1.00 ± 0.05; LNCaP GLPG 1 µm, MR: 0.60 ± 0.04 cells; and LNCaP GLPG 3 µm, MR:0.21 ± 0.04. (B) C4‐2B VC, MR: 4.47 ± 0.14; C4‐2B GLPG 0.1 µm, MR: 2.72 ± 0.34; C4‐2B GLPG 1 µm, MR: 1.87 ± 0.31; and C4‐2B GLPG 3 µm, MR: 0.83 ± 0.14. (C) PC3 VC, MR: 9.98 ± 0.08; PC3 GLPG 0.1 µm, MR:9.65 ± 0.08; PC3 GLPG 1 µm, MR: 8.98 ± 0.11; and PC3 GLPG 5 µm, MR:5.68 ± 0.21. (D) DU145 VC, MR: 9.07 ± 0.13; DU145 GLPG 0.1 µm, MR: 8.26 ± 0.23; DU145 GLPG 1 µm, MR: 7.00 ± 0.25; and DU145 GLPG 5 µm, MR: 2.60 ± 0.12. (E) 22Rv1 VC, MR: 1.32 ± 0.05; 22Rv1 GLPG 0.1 µm, MR: 1.34 ± 0.05; 22Rv1 GLPG 1 µm, MR: 0.85 ± 0.05; and 22Rv1 GLPG 5 µm, MR: 0.47 ± 0.03. *=P < 0.05, ***= P < 0.0005, ****=P < 0.0001 and n.s. = not significant. Each data point contains 8‐12 technical replicates and n = 3 independent experiments. One‐way ANOVA followed by Tukey’s post hoc analysis was used for multiple group comparison. Error bar represents standard error of the mean (SEM).
Fig 3: TLK1 interacts and phosphorylates MK5 both in vitro and in cultured cells. (A) Co‐immunoprecipitation of endogenously expressed MK5 with TLK1 antiserum from LNCaP cells and blotted sequentially for TLK1 and MK5. (B) GFP‐MK5 was overexpressed in HEK 293 cells. MK5 was immunoprecipitated using GFP‐specific antibody and blotted for both MK5 (upper panel) and TLK1 (lower panel). (C) His pulldown assay using purified his‐tagged TLK1B incubated with GFP‐MK5‐overexpressing HEK 293 cell lysate. Upper panel, WB showing both GFP‐MK5 and endogenous MK5 level. Lower panel, Ponceau S image showing TLK1B band. (D) GST pulldown assay using purified GST‐tagged MK5 incubated with GFP‐MK5 and TLK1 co‐expressing HEK 293 cell lysates. Left panel, WB detection of TLK1. Right panel, WB detection of both GFP‐MK5 and GST‐MK5. (E) An in vitro kinase (IVK) assay using recombinant his‐tagged HSP27 incubated with either his‐tagged TLK1B or his‐tagged MK5 or altogether. [ɣ‐32P] ATP was used as a radioactive source. Top panel, an autoradiograph showing the intensity of the exposed bands of the corresponding protein. Bottom panel, Coomassie‐stained gel showing equal amount of protein loading. (F) Relative densitometry of the fold change of HSP27 phosphorylation. One‐way ANOVA followed by Tukey’s post hoc analysis was used. Error bar represents standard error of the mean (SEM). (G) HEK 293 cells transfected with either GFP‐MK5 alone, or GFP‐MK5+TLK1, or GFP‐MK5+TLK1 KD. Lambda protein phosphatase (LPP) treatment was done in the 4th lane. WB showing hyperphosphorylated GFP‐MK5 band in the 2nd lane, which is reduced by LPP treatment (4th lane). GAPDH was used as a loading control. Each figure is representative of n = 3 experiments.
Fig 4: Copy‐number increase and upregulation of TLK1 and MK5 in metastatic PCa. Bioinformatic analysis of PCa data sets to determine: (A) genomic alteration of MK5; (B) mRNA expression of TLK1 based on nodal metastasis; (C) mRNA expression of MK5 based on nodal metastasis; (D) mRNA expression of TLK1 based on tumour Gleason score; (E) mRNA expression of MK5 based on tumour Gleason score. CRPC= castration‐resistant prostate cancer, PRAD = prostate adenocarcinoma, NEPC = neuroendocrine prostate cancer. Box‐and‐whisker plots represent interquartile range (IQR) including minimum, 25th percentile, median, 75th percentile and maximum values. N0 = no regional lymph node metastasis and N1 = metastases in 1 to 3 axillary lymph nodes. GS = Gleason score. *=P < 0.05, **=P < 0.005 and ****=P < 0.0001.
Fig 5: Both MK5 and TLK1 regulate cell migration. A, B, E, F, G and H) Scratch wound repair assay was conducted to determine the 2D migration rate by plotting relative wound density against different time points. (A) Wild‐type MEF (MEF WT), mean rate (MR): 10.97 ± 0.45; MEF MK5−/−, MR: 9.40 ± 0.35; and MEF GFP‐MK5, MR: 15.32 ± 1.2. (B) MEF MK5−/−, MR: 4.23 ± 0.30; MEF MK5−/− TLK1, MR: 5.19 ± 0.22; and MEF MK5−/− GFP‐MK5, MR: 13.69 ± 0.57. (E) LNCaP WT, MR:3.97 ± 0.10; LNCaP TLK1 kinase dead (KD), MR:3.47 ± 0.11; and LNCaP GFP‐MK5, MR: 5.10 ± 0.16. (F) MEF wild‐type vehicle control (MEF WT VC, MR: 12.18 ± 0.71); MEF WT J54 10 µm, MR: 9.65 ± 0.30; and MEF WT siTLK1, MR: 10.51 ± 0.37. (G) MEF MK5−/− GFP‐MK5 VC, MR: 17.40 ± 0.40; MEF MK5−/− GFP‐MK5 J54 10 µm, MR: 14.39 ± 0.22. (H) MEF MK5−/−, MR: 5.62 ± 0.12; MEF MK5−/− GFP‐MK5‐S354A, MR: 5.79 ± 0.55; MEF MK5−/− GFP‐MK5 K51E, MR: 4.96 ± 0.31; and MEF MK5−/− GFP‐MK5, MR: 15.63 ± 0.22. (C) 3D chemotactic trans‐well migration assay among MEF MK5−/−, MEF MK5−/− TLK1 and MEF MK5−/− GFP‐MK5 cells at different time points using fibronectin coating. Migration rate was determined by plotting total object phase area against time among MEF MK5−/−, MR: 0.04 ± 0.005; MEF MK5−/− TLK1, MR: 0.07 ± 0.014; and MEF MK5−/− GFP‐MK5, MR: 0.16 ± 0.009. (D) Proliferation assay between MEF MK5−/− and MEF MK5−/− GFP‐MK5 cells by plotting confluence percentage over time. MEF MK5−/−, MR: 1.93 ± 0.08; MEF MK5−/− GFP‐MK5, MR: 1.97 ± 0.006. 2‐tailed Student’s t‐test was used for two group comparison, and one‐way ANOVA followed by Tukey’s post hoc analysis was used for multiple group comparison. *=P < 0.05, **=P < 0.005, ***=P < 0.0005, ****=P < 0.0001 and n.s. = not significant. Each data point contains 8‐12 replicates. Error bar represents standard error of the mean (SEM).
Supplier Page from Sino Biological, Inc. for Human MAPKAPK5 Protein (His & GST Tag)