Fig 1: SP600125 or PMA induce Prss14/epithin ectodomain shedding. A, diagram of Prss14/epithin domain structure and processed forms. Epi-S', Epi-S, aEpi-S are indicated. B, effects of MAP kinase inhibitors in Prss14/epithin shedding. 427.1.86 cells were treated with 10 μm PD98059 (PD), 20 μm SB203580 (SB), and 5 μm SP600125 (SP) for 30 min and then with or without 0.5 μm PMA for an additional 2 h. C, dose- and time-dependent profiles of Epi-S' and Epi-S. 427.1.86 cells were treated with the indicated concentration of SP600125 for 2 h (left panel) and 5 μm SP600125 up to 2 h (right panel). D, 427.1.86 cells were pretreated with 10 μm TAPI-0 (TPI) for 30 min, and then cells were treated with 5 μm SP600125 or 0.5 μm PMA for an additional 2 h. The TACE inhibitor abolished the appearance of Epi-S' while retaining Epi-S in the cell, regardless of shedding induction methods, PMA, and SP600125. E, removal of TACE with siRNA abolished shedding of Prss14/epithin. 427.1.86 cells were transfected with 200 nm TACE siRNA for 48 h, starved of serum for 4 h, and then treated with 5 μΜ SP600125 or 0.5 μΜ PMA for 2 h. The control samples were treated exactly the same way except for transfection with nontargeting control siRNA. F, SP600125 dose-dependently induced Epi-S' and aEpi-S in T47D cells. SP600125 was treated for 2 h. G, SP600125 time-dependently (with 5 μm) and dose-dependently (for 2 h) induced Epi-S' and aEpi-S in 4T1 cells. In all panels, Epi-S' collected from culture medium and other proteins, including Epi-S, from cell lysates were detected by Western blot analysis. Tubulin or β-actin was used for normalization. TM, transmembrane domain; SEA, sperm protein, enterokinase, and agrin domain; CUB1, CUB2, complement subcomponent C1r/C1s domain; 1, 2, 3, 4 LDLRA, low-density lipoprotein receptor class A repeats.
Fig 2: NSCLC cells promoted CX3CL1 secretion of VBMECs by promoting MAPK14/ADMA17-dependent protein release and enhancing NF-κB-dependent CX3CL1 synthesis. (A) Quantification of CX3CL1 concentration in culture media of the indicated co-cultures composed of VBMECs and H1975 or A549 cells by ELISA. Data represent the mean ± SEM (n = 3). **P < 0.01. (B) RT-qPCR analysis of CX3CL1 mRNA levels in VBMECs co-cultured with culture media of NSCLC cell lines, A549 and H1975. Data represent the mean ± SEM (n = 3). **P < 0.01. (C) ADMA17 mRNA levels in VBMECs co-cultured with culture media of NSCLC cell lines by RT-qPCR. Data represent the mean ± SEM (n = 3). *P < 0.05 and **P < 0.01. (D) Western blotting analysis of ADMA17 protein levels in VBMECs co-cultured with the indicated culture media of NSCLC cell lines. (E) Genetic silencing of ADAM17 in VBMECs. (F) Quantification of CX3CL1 secretion in culture media of indicated co-cultures combining ADAM17-KD VBMECs and H1975 or A549 cells by ELISA. Data represent the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, and NSP > 0.05. (G) RT-qPCR analysis of CX3CL1 in the indicated VBMECs co-cultured with the indicated culture media of NSCLC cell lines. Data represent the mean ± SEM (n = 3). **P < 0.01. (H) Western blotting analysis of CX3CL1, MAPK14, p-MAPK14, ADAM17, and p-ADAM17 protein levels in VBMECs treated with conditioned media from A549 or H1975 cells in the presence or absence of the MAPK14 inhibitor SB203580. (I) Quantification of CX3CL1 concentration in culture media of the indicated co-cultures composed of VBMECs and H1975 or A549 cells treated with SB203580 by ELISA. Data represent the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, and NSP > 0.05. (J) RT-qPCR analysis of CX3CL1 mRNA levels in VBMECs co-cultured with culture media of NSCLC cell lines and treated with SB203580. (K) Western blotting analysis of NF-κB, p-NF-κB, and CX3CL1 protein levels in VBMECs treated with conditioned media from A549 or H1975 cells in the presence or absence of the NF-κB inhibitor Bay11-7085. (L) RT-qPCR analysis of CX3CL1 in the indicated VBMECs co-cultured with the indicated culture media of NSCLC cell lines and treated with Bay11-7085. Data represent the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, and NSP > 0.05. (M) Quantification of CX3CL1 concentration in culture media of the indicated co-cultures composed of VBMECs and H1975 or A549 cells treated with Bay11-7085 by ELISA. Data represent the mean ± SEM (n = 3). *P < 0.05 and **P < 0.01.
Fig 3: JNK inhibition increases PKCβII activity, translocation into the membrane, and TACE phosphorylation. A, phosphorylation of PKCβII by the JNK inhibitor. 427.1.86 cells were pretreated with 10 μm anisomycin (AN) for 30 min and then treated with 5 μm SP600125 (SP) for an additional 1 h. SP600125 treatment induced PKCβII phosphorylation. Relative values of band intensity are expressed as means ± S.D. of four independent experiments. ***, p < 0.001. B, kinetics of SP600125- or PMA-induced PKCβII activity. 427.1.86 cells were incubated with 5 μm SP600125 or 0.5 μm PMA for 0, 30, 60, and 120 min. After immunoprecipitation with PKCβII antibody, PKCβII activities were determined using the ADP-GloTM kinase assay kit. All values are expressed as means ± S.D. **, p < 0.01; #, p < 0.05; ##, p < 0.01; n = 3. C, Enzymatic activity of PKCβII was induced by SP600125 alone not by anisomycin combination to SP600125. 427.1.86 cells were pretreated with 1 μm anisomycin for 30 min and then threated with 5 μm SP600125 for an additional 1 h. All values are expressed as means ± S.D. **, p < 0.01; n=3. D, immunofluorescent staining of PKCβII-overexpressing 427.1.86 cells. Cells were transfected with 1 μg/ml of PKCβII WT cDNA for 48 h and then treated with 5 μm SP600125 or 0.5 μm PMA for 1 h. Immunofluorescence staining was performed with anti PKCβII polyclonal antibody (1:50) followed by FITC-conjugated anti rabbit IgG antibody (1:200). For nucleus staining, cells were incubated with DAPI for 10 min. Membrane localization of PKCβII is indicated by arrows. Images of two cells treated with SP600125 were stylized by embossing the appearance of the signal intensities using Adobe Photoshop. The graph indicates the percentage of cells with PKCβII localized in the membrane from four independent experiments. All values are expressed as means ± S.D. **, p < 0.01; ***, p < 0.001. E, membrane localization of PKCβII by cellular fractionation. 427.1.86 cells were treated with 5 μΜ SP600125 up to 60 min, and then PKCβII in cytosolic and membrane fractions was examined. Relative values of band intensity are expressed as means ± S.D. for three independent experiments. The arrowhead indicates PKCβII. *, p < 0.05. F, phosphorylation of TACE by shedding inducers. In the cells treated with 0.5 μm PMA or 5 μm SP600125 for 1 h, phosphorylation of TACE was analyzed by Western blot analysis using an antibody specific for phosphorylated TACE (Thr-735).
Fig 4: HDAC6 promotes sIL-6R release by activating the TAK1–p38–ADAM17 axis.A Western blot analysis of the total protein expression and phosphorylation of ADAM17, ERK1/2 and p38. GAPDH was used as an internal reference protein. B Schematic of the experimental procedure used to analysis of HDAC6 interacting proteins. The unique peptides and peptide-spectrum matches of TAK1 are 4, which is credible. C. Stable knockdown of TAK1 in HCT116 cells. D, E Analysis of sIL-6R levels in the cell media by ELISA D and detection of ADAM17 activity E in HCT116 cells after TAK1 knockdown. F Flow cytometry assessed CD11b, CD86 and CD206 expression of cocultured macrophages in shCtrl- and shTAK1-treated HCT116 cells. G QPCR detected the expression of M1 and M2 polarization related genes of cocultured macrophages in shCtrl- and shTAK1-treated HCT116 cells. H Treatment with the TAK1 selective inhibitor Takinib decreased TAK1 phosphorylation in HCT116 cells. I, J Analysis of sIL-6R levels in the supernatants by ELISA I and detection of ADAM17 activity J in HCT116 cells after treated with Takinib. K Western blot analysis of TAK1 total protein expression and phosphorylation in HCT116 and HT29 cells with stable HDAC6 knockdown. Data were shown as the mean ± SD of three independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 5: TAK1 phosphorylation is positively correlated with HDAC6 expression in colon cancer.A Protein was extracted from 38 fresh postoperative colon cancer tissues for western blot analysis of p-TAK1, TAK1, and HDAC6 protein levels. Relative protein levels were normalized by GAPDH. Thirteen samples are shown. B Quantification of the intensities of the indicated proteins was performed using ImageJ software, followed by Pearson’s correlation coefficient analysis. C, D M2 macrophage infiltration in the 38 colon cancer tissues was evaluated using an mIHC platform (panel: CD68/CD163). Patients were divided into two groups according to p-TAK1/TAK1 or HDAC6/GAPDH expression based on western blot analyses of tissue proteins, and then the proportion of CD163+CD68+/CD68+ cells were calculated. Representative mIHC images are shown, scale bar, 50 μm. E Western blot analysis of the phosphorylated and total protein expression of TAK1, ADAM17, and p38 in xenograft mouse models (BALB/c nude) originating from HCT116-shCtrl and HCT116-shHDAC6 cells. F Working model. WCL is the abbreviation of whole-cell lysate. Data were shown as the mean ± SD, *P < 0.05; **P < 0.01.
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