Fig 1: TBX3 represses proliferation of fibrosarcoma cells by activating key cell cycle regulators. (a, b) Protein extracts from indicated cell lines were analysed by western blotting using an antibody specific to TBX3 and p38 was used as a loading control. (b) Right panel, immunocytochemistry with an antibody specific to FLAG shows TBX3 overexpression in HT1080 FLAG-Tbx3 cells and FLAG-Tbx3+2a. Hoechst was used to stain the nuclei. Representative images are shown (scale bars, 50 μm). (c–f) Growth curve analyses and BrdU incorporation assays for (c, e) shCtrl and shTBX3 and (d, f) FLAG-empty and FLAG-Tbx3 cells. For the BrdU incorporation assays cells were pulsed with BrdU and processed for immunocytochemistry using an antibody specific to BrdU and visualized by fluorescence microscopy. Bar graphs show the average percentage of BrdU-positive cells in 20 fields of view. (c–f) Data are the mean±s.d. of three independent experiments, *P<0.05; **P<0.01; ***P<0.001. (g, h) Western blotting with antibodies specific to TBX3, p53 and p21. p38 was used as a loading control.
Fig 2: AJ-5 activates the DNA damage and the p38 MAPK pathways.a γH2AX protein levels detected by western blotting in RH30 (aRMS) and RD (eRMS) cells treated with vehicle (V), 0.1 µM or IC50 AJ-5 for 24 and 48 h. p38 was used as a loading control. Densitometry readings were obtained using ImageJ and protein expression levels are represented as a ratio of protein of interest/p38 normalized to the vehicle control sample (where possible). Blots are representative of at least two independent repeats. b Representative confocal immunofluorescence maximum intensity projection images (×630; Carl Zeiss LSM 510) of RH30 and RD cells treated with IC50 AJ-5 or vehicle for 24 h and γH2AX detected with a fluorophore-conjugated Cy3 secondary antibody. Nuclei of cells were stained with DAPI. Scale bar is 20 µM. Box plots represent quantification of γH2AX levels per treatment condition as mean nuclear Cy3 fluorescence from 20 fields of view from three independent repeats. Data were analysed using GraphPad Prism 6.0 and a parametric unpaired t-test was performed where *p < 0.05, **p < 0.01, ***p < 0.001. c Western blot analyses with antibodies to key DNA damage and stress signalling pathway proteins: p-ATM, p-Chk2, p-p38, p53, and p21. RH30 and RD cells were treated with AJ-5 and protein expression quantified as described above in a. Broken lines in western blots shown in this figure indicate where lanes not relevant were removed
Fig 3: Model for aberrant activation of p38 MAP kinase(s) by mutant and misfolded ALS-associated proteins. Available data (from this study and others10, 12, 20) support a model whereby mutant and misfolded forms of FUS (mFUS; left) and SOD1 (mSOD1; right) induce the aberrant activation of the p38 MAPK pathway. Hsp110 likely synergizes with other chaperones to ameliorate the effects of mSOD1 and mFUS, possibly upstream of ASK1 and additional unidentified factors. While mSOD1 and mFUS converge on p38 MAPK activation, perfusion of these proteins into squid axoplasm have differential effects on FAT; mFUS inhibits both anterograde (←) and retrograde (→) FAT (Figs 1 and 2) whereas mSOD1 only inhibits anterograde FAT10, 12, 20, consistent with inhibition of the beta and alpha isoforms of P38 MAPK, respectively12. In addition to FAT inhibition in squid (solid lines), mFUS- and mSOD1-induced activation p38 MAPK can manifest different phenotypes in mammalian systems (dashed lines), including but not limited to inhibition of axon outgrowth (Fig. 4) and enhanced susceptibility to cell stress12. This figure is adapted from Song et al. 20.
Fig 4: Summary figure of our findings: oxidized LDL (oxLDL) activates the oxLDL receptor. Expression of the receptor by cardiomyocytes was shown in this study and silencing of the receptor by siRNA attenuated all subsequent steps. oxLDL induces oxidative stress as indicated by oxidative modifications of tropomyosin (Trp-ox). Oxidative stress activates p38 MAP kinase as indicated by western blot. Inhibition of p38 MAP kinase activation by SB20190 attenuates this effect. Subsequently, oxLDL causes increased expression of PCSK9 as indicated by western blots and inhibition of transcription by actinomycin D (ActD) or translation by cycloheximide (chx) block all subsequent steps. Silencing of PCSK9 upregulation attenuates future steps
Fig 5: Phosphorylation of p38 during NGF treatment is increased in PPM1A underexpressed PC6-3 cells.PC6-3 PPM1A knock-down cells and control cells were incubated with NGF for the indicated times. The cells were harvested and lysed. Cell extracts were analyzed by western blot using the indicated antibodies. The graph represents quantification of the phospho-p38 levels. The results shown are from representative experiments out of 3 independent that were performed.
Supplier Page from MilliporeSigma for Anti-p38 MAP Kinase antibody produced in rabbit