Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: ( A ) Schematic depicting how thermal shifts are measured in a PISA experiment. An increase in melting temperature (thermal stabilization) will result in an increase in the AUC and a positive log 2 fold change in soluble protein abundance. A decrease in melting temperature (thermal destabilization) will result in a decrease in the AUC and a negative log 2 fold change in soluble protein abundance. ( B ) PISA schematic depicting a thermal window vs. a full melting curve. ( C-G ) K562 cells (N=4) were treated with 10 µM palbociclib ( D ), ribociclib ( E ), or abemaciclib ( F ) for 15 min and assayed using PISA. ( C ) The log 2 fold changes for specific proteins are plotted for each treatment using a full melting curve (left) or thermal window (48–58°C; right). ( D–F ). Data is presented as a volcano plot to highlight significant changes in abundance utilizing the thermal window (48–58°C). Significant changes were determined using a permutation-based FDR (FDR – 0.05, S0 – 0.1, N=4). ( G ). Log 2 fold change values for selected proteins following treatment with each compound in D-F.

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Quantitative Proteomics

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: ( A ) Schematic of the cell-based PISA workflow utilized in the large-scale chemical perturbation screen. ( B ) Histogram plotting the log 2 fold change values for all solubility (thermal stability) measurements (main panel). Box plot depicting the %CV of curated targets vs. all other proteins (inset). ( C ) Plots highlighting the significant changes in protein solubility following treatment with OTS167 (top) or GSK-LSD1 (bottom). The mean log 2 fold change of duplicate measurements is plotted on the x-axis and the mean nSD is plotted on the y-axis. Blue boxes contain proteins that exhibit an increase in solubility (increase in melting temperature) and orange boxes contain proteins that exhibit a decrease in solubility (decrease in melting temperature). Orange points represent the known target of each compound. ( D ) The total number of significant solubility changes quantified upon treatment with each compound. The number of stabilizing events (increase in solubility) is plotted on the y-axis and destabilizing events (decrease in solubility) are plotted on the x-axis. ( E ) Plot comparing the log 2 fold changes measurements for each protein in BI 2536 (y-axis)- and volasertib (x-axis)-treated K562 cells. Figure 1—source data 1. This table contains cell-based PISA data for K562 cells treated with ribociclib, palbociclib, and abemaciclib. Figure 1—source data 2. This three part table contains the PISA data for cell- and lysate-based screens (part 1 of 3 for this table). Figure 1—source data 3. This three part table contains the PISA data for cell- and lysate-based screens (part 2 of 3 for this table). Figure 1—source data 4. This three part table contains the PISA data for cell- and lysate-based screens (part 3 of 3 for this table).

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Solubility

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: K562 cells were treated with each of the 96 compounds at 10 µM for 30 min. Three compounds and a DMSO control were assayed in duplicate (batch). All 96 compounds and 64 DMSO controls were assayed using thirty-two treatment batches. Following treatment, an equal number of cells were transferred to 10 PCR tubes. The cells were placed in a thermal cycler and heated across a thermal gradient from 48°C to 58°C for 3 min. The cells were allowed to cool to room temperature for 5 min. An equal volume from each PCR tube was pooled and spun at 300 x g for 3 min to pellet cells. Cells were washed one with PBS and lysed in a buffer containing 0.5% NP-40, which will dissolve membranes without disrupting heat-induced protein aggregates. The lysates were centrifuged for 90 min at 21,000 x g to separate soluble protein from aggregates. An equal volume of each soluble fraction (~20 µg) was prepared for LC-MS/MS analysis. Two treatment batches were combined for each TMTpro 16-plex. Each soluble fraction was reduced and alkylated. Each sample was precipitated onto SP3 carboxylate-coated beads to facilitate a buffer exchange. Proteomes were eluted off the SP3 beads into digestion buffer and digested with a combination of Lys-C and trypsin. Peptides from each sample were labeled with a unique TMTpro reagent. TMT-labelled peptides were pooled into a single sample, which was desalted using a sep-pak. Dried peptides were resuspended in HPLC buffer A and fractionated by basic reverse-phase HPLC. Twelve to 24 fractions were stage-tipped and analyzed on an Orbitrap Eclipse with a FAIMS device enabled (Thermo Fisher). Changes in thermal stability were determined by comparing soluble protein abundance in a compound-treated samples to vehicle-treated controls.

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Buffer Exchange, Labeling, Quantitative Proteomics

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: ( A-B ) K562 cells (N=2) were treated with MK-2206 (top) or CCT128930 (bottom) at 10 µM for 30 min and assayed using PISA. The mean log 2 fold change of duplicate measurements is plotted on the x-axis and the mean nSD is plotted on the y-axis. Blue boxes contain proteins that exhibit an increase in solubility (increase in melting temperature) and orange boxes contain proteins that exhibit a decrease in solubility (decrease in melting temperature). Orange points represent the known target of each compound. ( B ) The plot displays the log 2 fold change values for selected proteins following treatment with each compound in A. ( C ) Plot comparing the log 2 fold changes measurements for each protein in JQ-1 (x-axis)- and volasertib (y-axis)-treated K562 cells. Orange points represent the known targets of the compounds.

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Solubility

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: ( A ) A plot of the mean log 2 fold change (x-axis) and mean nSD (y-axis) of each known compound-target pair that were quantified in the screen. Blue boxes contain proteins that exhibit an increase in solubility (increase in melting temperature) and orange boxes contain proteins that exhibit a decrease in solubility (decrease in melting temperature). ( B and C ) Plots depicting the mean log 2 fold change of p38α ( B ) and PIK3CB ( C ) in response to each of the compound and DMSO treatments. Orange points indicate compounds known to target each protein. Blue points indicate other compounds that induce a significant change in thermal stability for each protein. Green dashed lines mark a SD of 3.5 for each protein. ( D ) A protein-centric view of PLK1 solubility (thermal stability) in response to all treatments. Log 2 fold change is plotted on the y-axis. The points represent each of the 256 treatments that were performed. Orange points indicate compounds known to target PLK1. Blue points indicate other compounds that result in a significant decrease in solubility of PLK1. Green dashed lines mark a SD of 3.5 cutoff for PLK1. ( E and F ) K562 cells were treated with the indicated concentrations of palbociclib or BI 2536 for 15 minutes. Changes in protein thermal stability are represented as a log 2 fold change in soluble protein abundance for each treatment in reference to a DMSO-treated control. ( E ) PLK1 activity was determined using a western blot for p-TCTP (S46) and total TCTP ( F ). ( G ) Chemical structures of BI 2536, palbociclib and NVP-TAE-226. Common structural features with BI 2536 are highlighted in blue for Palbociclib and NVP-TAE-226. ( H ) Co-crystal structure of CDK6 bound to palbociclib (blue) with BI 2536 (gray) modeled into the active site. ( I ) Co-crystal structure of PLK1 bound to BI 2536 (gray) with palbociclib (blue) modeled into the active site. Figure 2—source data 1. This table contains cell-based PISA data for K562 cells treated with increasing concentrations of BI 2536 and palbocilicb. Figure 2—source data 2. This table contains cell-based PISA data for K562 cells treated with increasing concentrations of BI 2536 and NVP-TAE-226. Figure 2—source data 3. PDF file containing original western blots for , indicating the relevant bands and treaments. Figure 2—source data 4. Original files for western blot analysis displayed in . Figure 2—source data 5. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—source data 6. Original files for western blot analysis displayed in .

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Solubility, Quantitative Proteomics, Control, Activity Assay, Western Blot

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: ( A-B ) A protein-centric view of p38α ( A ) and PIK3CB ( B ) solubility (thermal stability) in response to all treatments. Log 2 fold change is plotted on the y-axis. The points represent each of the 256 treatments that were performed. Orange points indicate compounds known to target each respective protein. Blue points indicate other compounds that result in a significant thermal stabilization. Green dashed lines mark a SD of 3.5 cutoff for each treatment. ( C-D ) K562 cells were treated with the indicated concentrations of NVP-TAE-226 or BI 2536 for 15 min. Changes in protein thermal stability are represented as a log 2 fold change in soluble protein abundance for each treatment in reference to a DMSO-treated control ( C ). PLK1 activity was determined using a western blot for p-TCTP (S46) and total TCTP ( D ).

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Solubility, Quantitative Proteomics, Control, Activity Assay, Western Blot

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: ( A and B ) Plots depicting the mean log 2 fold change values for RB1 ( A ) and CRKL ( B ) in response to treatment with all compounds assayed using cell ( x )- and lysate ( y )-based PISA. Dashed lines indicate a log 2 fold change of +/-0.2. C. K562 cells were treated with each indicated compound at 10 µM for 15 min. Western blots were used to assess levels of p-CRKL (Y207) and total CRKL. D. A plot of the log2 fold change of CRKL in response to each indicated compound. ( E ) Plots highlighting the significant changes in protein solubility (thermal stability) following treatment with AZD-7762. The mean log 2 fold change of duplicate measurements is plotted on the x-axis and the mean nSD is plotted on the y-axis. Blue boxes contain proteins that exhibit an increase in solubility (increase in melting temperature) and orange boxes contain proteins that exhibit a decrease in solubility (decrease in melting temperature). Orange points represent the known target of AZD-7762 and the blue points represents tyrosine kinases that experience a significant change in soluble protein abundance (thermal stability). ( F ) K562 lysates were treated with the indicated concentrations of AZD-7762 for 15 min and assayed using PISA. Changes in protein thermal stability are represented as a log 2 fold change in soluble protein abundance for each treatment in reference to a DMSO-treated control. ( G ) K562 cells were treated with the indicated concentration of bafetinib or AZD-7762 for 15 min and assayed using phosphoproteomic profiling. Plots depict the relative abundance of several phosphorylation sites following treatment with the indicated compounds. * indicate significant changes, which were determined using a permutation-based FDR (FDR – 0.05, S0 – 0.1, N=4). Error bars represent the stabdard deviation. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 5—source data 2. Original files for western blot analysis displayed in . Figure 5—source data 3. This table contains lysate-based PISA data for K562 lysates treated with 100 nM, 1 µM, or 10 µM AZD-7762. Figure 5—source data 4. This table contains phosphoproteomic data for K562 cells treated with 1 µM AZD-7762, 10 µM AZD-7762, 1 µM bafetinib, or 10 µM bafetini. Figure 5—source data 5. This table contains proteomic data for K562 cells treated with 1 µM AZD-7762, 10 µM AZD-7762, 1 µM bafetinib, or 10 µM bafetinib.

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Western Blot, Solubility, Quantitative Proteomics, Control, Concentration Assay, Phospho-proteomics

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: ( A and B ) A protein-centric view of CRKL solubility (thermal stability) in K562 cells ( A ) and lysates ( B ) in response to all treatments. Log 2 fold change is plotted on the y-axis. The points represent each treatment that was performed in each screen. Green dashed lines mark a SD of 3.5 cutoff for each treatment. ( C ) Plots depicting the log 2 fold change values for CRK in response to treatment with all compounds assayed using cell- and lysate-based PISA. ( D ) Changes in protein solubility (thermal stability) for the indicated proteins in cell- and lysate-based PISA are represented as a log 2 fold change in soluble protein abundance for each treatment in reference to a DMSO-treated control.

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Solubility, Quantitative Proteomics, Control

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: ( A-C ) K562 lysates were treated with the indicated concentrations of AZD-7762 for 15 min and assayed using PISA. Changes in protein thermal stability are represented as a log 2 fold change in protein soluble abundance for each treatment in reference to a DMSO-treated control. Significant changes were determined using a permutation-based FDR (FDR – 0.05, S0 – 0.1).

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Control

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet: ( A-F ) K562 cells were treated with the indicated concentration of bafetinib or AZD-7762 for 15 min and assayed using phosphoproteomic profiling. ( A-D ) Data is displayed as a volcano plot. Significant changes were determined using a permutation-based FDR (FDR – 0.05, S0 – 0.1). ( E ) PCA plot of phosphoproteomic data. ( F ) Total number of significant changes that result from each treatment (left) or significant changes with a log2 fold change >1 or < –1 (right).

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Concentration Assay

Journal: eLife

Article Title: Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

doi: 10.7554/eLife.95595

Figure Lengend Snippet:

Article Snippet: Cell line ( H. sapiens ) , K562 , ATCC , CLL-243 , .

Techniques: Recombinant, Software

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization of Temozolomide-Treated and Non Temozolomide-Treated Glioblastoma Cells Released Extracellular Vesicles and Their Role in the Macrophage Response

doi: 10.3390/ijms21218353

Figure Lengend Snippet: The expression levels of epidermal growth factor receptor (EGFR), EGFRvIII, isocitrate dehydrogenase 1 (IDH1), podoplanin (PDPN), Hsp25, Hsp70, and Hsp90 in U-87 MG cells and in isolated lEVs and sEVs after 200 µM temozolomide (TMZ) for 48 h treatment evaluated by Western blotting analysis. The expression of EGFR, EGFRvIII, IDH1, and PDPN is normalized with the housekeeping protein (β-actin) and reported as relative expression respect to parental cells. The values for all experimental groups reported in the histograms represent the means ± SD ( n = 3) of three independent experiments; (*) p < 0.05 compared to respective origin cells. Representative images of proteins blots are reported.

Article Snippet: From the temozolomide (TMZ, Selleckchem.com, Munich, Germany) stock solution (100 mM in dimethyl sulfoxide, DMSO obtained by solubilizing 10mg of TMZ in 0.515 mL of DMSO), 50, 100, 200, and 400 μM TMZ were added to 15 × 10 4 /well cells for 24, 48, and 72 h, to assay the most effective conditions, the most sensitive cells and the effects on EVs release.

Techniques: Expressing, Isolation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization of Temozolomide-Treated and Non Temozolomide-Treated Glioblastoma Cells Released Extracellular Vesicles and Their Role in the Macrophage Response

doi: 10.3390/ijms21218353

Figure Lengend Snippet: Macrophage polarization markers after exposure to 200 µM temozolomide (TMZ) for 48 h treated or non-TMZ- treated U-87 MG cells derived lEVs, sEVs and free EVs CM for 24 h. The expression of CD86, CD163, STAT6 and iNOS is normalized with the housekeeping protein (β-actin) and reported as relative expression respect to control M0. The mRNA levels of TLR4 and NF-kB are normalized to GAPDH mRNA level and expressed as relative expression respect to M0. The values for all experimental groups reported in the histograms represent the means ± SD (n = 3) of three independent experiments; (*) p < 0.05 compared to control M0. Representative images of proteins blots are reported.

Article Snippet: From the temozolomide (TMZ, Selleckchem.com, Munich, Germany) stock solution (100 mM in dimethyl sulfoxide, DMSO obtained by solubilizing 10mg of TMZ in 0.515 mL of DMSO), 50, 100, 200, and 400 μM TMZ were added to 15 × 10 4 /well cells for 24, 48, and 72 h, to assay the most effective conditions, the most sensitive cells and the effects on EVs release.

Techniques: Derivative Assay, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization of Temozolomide-Treated and Non Temozolomide-Treated Glioblastoma Cells Released Extracellular Vesicles and Their Role in the Macrophage Response

doi: 10.3390/ijms21218353

Figure Lengend Snippet: Macrophage polarization markers after exposure to 200 µM temozolomide (TMZ) for 48 h treated or non TMZ- treated U-87 MG cells derived lEVs, sEVs and free EVs CM for 24 h. The mRNA levels of CCL17, IL-10, TNFα, IL-8, IFNα, IL-1β, IL-4, and IL-6 are normalized to GAPDH mRNA level and expressed as a relative expression with respect to M0. The values for all experimental groups reported in the histograms represent the means ± SD (n = 3) of three independent experiments; (*) p < 0.05 compared to the control M0.

Article Snippet: From the temozolomide (TMZ, Selleckchem.com, Munich, Germany) stock solution (100 mM in dimethyl sulfoxide, DMSO obtained by solubilizing 10mg of TMZ in 0.515 mL of DMSO), 50, 100, 200, and 400 μM TMZ were added to 15 × 10 4 /well cells for 24, 48, and 72 h, to assay the most effective conditions, the most sensitive cells and the effects on EVs release.

Techniques: Derivative Assay, Expressing, Control