Journal: Frontiers in Oncology

Article Title: Axitinib and HDAC Inhibitors Interact to Kill Sarcoma Cells

doi: 10.3389/fonc.2021.723966

Figure Lengend Snippet: Axitinib interacts with HDAC inhibitors to kill sarcoma cells. (A–F) Sarcoma cells human (HT1080, MES) and mouse (SAL, SAR180, TSC2, SAL/N) were treated with vehicle control, axitinib (50 nM), entinostat (50 nM), vorinostat (500 nM), sodium valproate (250 μM) alone or in combination as indicated in the graphical panels. Twenty-four h later, cells were isolated, and viability determined by trypan exclusion assays in triplicate (n = 3 +/-SD). # p < 0.05 greater than axitinib alone value.

Article Snippet: Entinostat, vorinostat, sodium valproate and axitinib were purchased from Selleck Chem (Houston, TX).

Techniques: Control, Isolation

Journal: Frontiers in Oncology

Article Title: Axitinib and HDAC Inhibitors Interact to Kill Sarcoma Cells

doi: 10.3389/fonc.2021.723966

Figure Lengend Snippet: Axitinib combined with HDAC inhibitors inactivates AKT, mTORC1, mTOR2 and Raptor. (A) Human sarcoma cells were treated with vehicle control, axitinib (50 nM), entinostat (50 nM), vorinostat (500 nM), sodium valproate (250 μM) alone or in combination as indicated in the graphical panels for 6h. Cells were fixed in place and in-cell immunoblotting performed to determine the total protein levels of AKT, mTOR and Raptor, and the phosphorylation of AKT T308, mTOR S2448, mTOR S2481, Raptor S792 and Raptor S722. The percentage phosphorylation is stated for each site corrected for total protein loading with vehicle control defined as 100% (n = 3 +/-SD). *p < 0.05 less than vehicle control; **p < 0.05 less than HDAC inhibitor alone; # p < 0.05 greater than vehicle control; ## p < 0.05 greater than HDAC inhibitor alone. (B) HT1080 cells were transfected with a scrambled control siRNA (siSCR) or with validated siRNA molecules to knock down the expression of either ATM or AMPKα. Twenty-four h after transfection, cells were treated with vehicle control, axitinib (50 nM), entinostat (50 nM), vorinostat (500 nM), sodium valproate (250 μM) alone or in combination as indicated in the graphical panels for 6h. Cells were fixed in place and in-cell immunoblotting performed to determine the total protein levels of ATM and AMPKα, and the phosphorylation of ATM S1981 and AMPKα T172 determined. The percentage phosphorylation is stated for each site corrected for total protein loading with vehicle control of each transfection being defined as 100% (n = 3 +/-SD). # p < 0.05 greater than vehicle control value; *p < 0.05 less than corresponding value in siSCR cells. (C) Sarcoma cells were transfected with a plasmid to express LC3-GFP-RFP. Twenty-four h after transfection, cells were treated with vehicle control, [axitinib (50 nM) + entinostat (50 nM)], [axitinib (50 nM) + vorinostat (500 nM)], [axitinib (50 nM) + sodium valproate (250 μM)] in combination as indicated in the graphical panels for 4h and 8h. The mean number of intense green and red punctae from a minimum of 40 cells are defined and plotted (n = 3 +/-SD).

Article Snippet: Entinostat, vorinostat, sodium valproate and axitinib were purchased from Selleck Chem (Houston, TX).

Techniques: Control, Western Blot, Phospho-proteomics, Transfection, Knockdown, Expressing, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: Axitinib and HDAC Inhibitors Interact to Kill Sarcoma Cells

doi: 10.3389/fonc.2021.723966

Figure Lengend Snippet: Signaling by ATM-AMPK plays a key role in regulating both mTOR and Raptor function. (A) HT1080 and MES sarcoma cells were transfected with a scrambled control siRNA (siSCR) or with validated siRNA molecules to knock down the expression of either ATM or AMPKα. Twenty-four h after transfection, cells were treated with vehicle control, [axitinib (50 nM) + entinostat (50 nM)], [axitinib (50 nM) + vorinostat (500 nM)], [axitinib (50 nM) + sodium valproate (250 μM)] in combination as indicated in the graphical panels for 6h. Cells were fixed in place and in-cell immunoblotting performed to determine the total protein levels of mTOR and Raptor, and the phosphorylation of mTOR S2448, mTOR S2481, Raptor S722 and Raptor S792. The percentage phosphorylation is plotted for each site corrected for total protein loading with vehicle control of each transfection being defined as 100% (n = 3 +/-SD). # p < 0.05 greater than corresponding value in siSCR control; *p < 0.05 less than corresponding value in siSCR cells; § p < 0.05 less than corresponding value in siATM cells. (B) Sarcoma cells were either transfected with an empty vector plasmid (CMV) or with plasmids to express BCL-XL, c-FLIP-s or dominant negative caspase 9; or were transfected with a scrambled siRNA control or with validated siRNA molecules to knock down the expression of ATM, AMPKα, ULK1, ATG5 and Beclin1. Twenty-four h after transfection, cells were treated with vehicle control, axitinib (50 nM), entinostat (50 nM), vorinostat (500 nM), sodium valproate (250 μM) alone or in combination as indicated in the graphical panels. Twenty-four h later, cells were isolated, and viability determined by trypan exclusion assays in triplicate (n = 3 +/-SD). Data are rounded to the nearest whole number. *p < 0.05 less than corresponding value in siSCR/CMV transfected cells.

Article Snippet: Entinostat, vorinostat, sodium valproate and axitinib were purchased from Selleck Chem (Houston, TX).

Techniques: Transfection, Control, Knockdown, Expressing, Western Blot, Phospho-proteomics, Plasmid Preparation, Dominant Negative Mutation, Isolation

Journal: Frontiers in Oncology

Article Title: Axitinib and HDAC Inhibitors Interact to Kill Sarcoma Cells

doi: 10.3389/fonc.2021.723966

Figure Lengend Snippet: Alterations in ULK1 phosphorylation require ATM-AMPK signaling; autophagy is required for cell killing. (A) HT1080 cells were transfected with a scrambled control siRNA (siSCR) or with validated siRNA molecules to knock down the expression of either ATM or AMPKα. Twenty-four h after transfection, cells were treated with vehicle control, axitinib (50 nM), entinostat (50 nM), vorinostat (500 nM), sodium valproate (250 μM) alone or in combination as indicated in the graphical panels for 6h. Cells were fixed in place and in-cell immunoblotting performed to determine the total protein levels of ULK1 and the phosphorylation of ULK1 S317 and ULK1 S757. The percentage phosphorylation is stated for each site corrected for total protein loading with vehicle control of each transfection being defined as 100% (n = 3 +/-SD). # p < 0.05 greater than vehicle control; *p < 0.05 less than vehicle control. (B, C) HT1080 and MES cells were transfected with a scrambled control siRNA (siSCR) or with validated siRNA molecules to knock down the expression of either ATM or AMPKα. Twenty-four h after transfection, cells were treated with vehicle control, axitinib (50 nM), entinostat (50 nM), vorinostat (500 nM), sodium valproate (250 μM) alone or in combination as indicated in the graphical panels for 6h. Cells were fixed in place and in-cell immunoblotting performed to determine the total protein levels of ATG13 and the phosphorylation of ATG13 S318. The percentage phosphorylation is plotted for ATG13 S318, corrected for total ATG13 protein loading with vehicle control of each transfection being defined as 100% (n = 3 +/-SD). # p < 0.05 greater than vehicle control; § p < 0.05 less than corresponding value in siSCR cells; *p < 0.05 less than vehicle control.

Article Snippet: Entinostat, vorinostat, sodium valproate and axitinib were purchased from Selleck Chem (Houston, TX).

Techniques: Phospho-proteomics, Transfection, Control, Knockdown, Expressing, Western Blot

Journal: Frontiers in Oncology

Article Title: Axitinib and HDAC Inhibitors Interact to Kill Sarcoma Cells

doi: 10.3389/fonc.2021.723966

Figure Lengend Snippet: Axitinib combined with HDAC inhibitors reduces the expression of multiple HDAC proteins in HT1080 cells via ATM-AMPK signaling and ULK1-autophagy. HT1080 cells were transfected with a scrambled control siRNA (siSCR) or with validated siRNA molecules to knock down the expression of ATM, AMPKα, ULK1, ATG5 or Beclin1. Twenty-four h after transfection, cells were treated with vehicle control, axitinib (50 nM), entinostat (50 nM), vorinostat (500 nM), sodium valproate (250 μM) alone or in combination as indicated in the graphical panels for 6h. Cells were fixed in place and in-cell immunoblotting performed to determine the total protein levels of HDACs 1, 2, 3, and 6, and of invariant ERK2. The percentage expression is presented for each HDAC protein corrected for total ERK2 protein loading with vehicle control of each transfection being defined as 100% (n = 3 +/-SD). *p < 0.05 less than HDAC inhibitor alone exposure.

Article Snippet: Entinostat, vorinostat, sodium valproate and axitinib were purchased from Selleck Chem (Houston, TX).

Techniques: Expressing, Transfection, Control, Knockdown, Western Blot

Journal: eLife

Article Title: Spatiotemporal control of cell cycle acceleration during axolotl spinal cord regeneration

doi: 10.7554/eLife.55665

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , Venus-fwd , This paper , PCR primer , TCAT GCTAGC ATGGTGAGCAAGGGCGAGGForward primer to amplify mVenus sequence from addgene plasmid #27793. Bold indicates a NheI restriction site..

Techniques: Transgenic Assay, Generated, Derivative Assay, Recombinant, Plasmid Preparation, Sequencing, Imaging, Refractive Index, Software, Fluorescence

Journal: Current biology : CB

Article Title: Microtubule-Dependent Confinement of a Cell Signaling and Actin Polymerization Control Module Regulates Polarized Cell Growth.

doi: 10.1016/j.cub.2018.05.076

Figure Lengend Snippet: Figure 4. Microtubules Confine SPK1 Signaling Nodules to the Branch Apex (A) YFP:SPK1 punctae are clustered within a microtubule-depletion zone. Right: representative intensity plot of YFP:SPK1 and mRUBY2:MBD signal along the cell periphery below the yellow line. (B) Microtubule organization in developing tri- chomes expressing GFP:MBD with and without oryzalin treatment. Microtubules are completely depolymerized after 1.5 hr of oryzalin treatment, but recovered after inhibitor washout. (C–E) SPK1 localization becomes progressively unfocused following oryzalin treatment, and the effect is reversible following inhibitor washout (C). Perimeter length of the SPK1 signal, normalized by tip radius of curvature, as a function of oryzalin treatment time and inhibitor washout (D). Signal intensity of the cortical SPK1 signal normalized to local cytosolic signal intensity as a function of oryzalin treatment time and inhibitor washout (E). Mean ± SD (n = 22). *p < 0.05, **p < 0.01 (ANOVA with Tukey HSD test). Scale bars represent 5 mm. See also Figure S4.

Article Snippet: The MYB5 promoter, mRUBY2 and MBD-nos terminator were amplified from pEGADMYB5pro (provided by D Marks, University of Minnesota, St. Paul, MN), pFA6a-link-yomRuby2-Kan (Addgene, Cambridge, MA) and GFP:MBD [36], respectively.

Techniques: Expressing