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Journal: RSC Chemical Biology
Article Title: A stress-responsive p38 signaling axis in choanoflagellates
doi: 10.1039/d4cb00122b
Figure Lengend Snippet: High-throughput screening of a small molecule library revealed inhibitors of S. rosetta cell proliferation. (A) Treatment of S. rosetta cultures with 1255 different small molecules (see Table S1, ESI ) resulted in a distribution of cell counts, assessed by flow cytometry, at the 24-hour endpoint. S. rosetta cell counts were normalized to the average of DMSO controls within the same plate (dark grey). Compounds determined to significantly inhibit S. rosetta cell proliferation by flow cytometry (based on two-tailed p -value <0.05 calculated from z -score), fall below the dotted line and are indicated in red. Compounds that were not detected as significant inhibitors by flow cytometry but were identified by imaging (based on two-tailed p -value <0.05 calculated from z -score) are in blue. Compounds that were not significant inhibitors for either screen are indicated in light grey. Sorafenib (SO), a focus of this study, is labeled. (B) The range of normalized cell counts measured by flow cytometry for compounds that significantly inhibited S. rosetta cell proliferation. Compounds that were the focus of further study – genistein (GE), glesatinib (GL), PP121, masitinib (MA), sotrastaurin (SOT) – are labeled. (C) Comparison of normalized values of compounds that inhibited S. rosetta cell proliferation, assessed by flow cytometry and the corresponding normalized values determined by imaging. Compounds determined to significantly inhibit S. rosetta cell proliferation (based on two-tailed p -value <0.05 calculated from z -score) by flow cytometry fall below the dotted line on the y -axis and by imaging, to the left of the dotted line on the x -axis.
Article Snippet:
Techniques: High Throughput Screening Assay, Flow Cytometry, Two Tailed Test, Imaging, Labeling, Comparison
Journal: RSC Chemical Biology
Article Title: A stress-responsive p38 signaling axis in choanoflagellates
doi: 10.1039/d4cb00122b
Figure Lengend Snippet: Glesatinib and sorafenib, two multi-target tyrosine kinase inhibitors, disrupt S. rosetta cell proliferation and tyrosine phosphosignaling. (A) Treatment of S. rosetta cultures with 1 μM sorafenib and glesatinib led to a complete block of cell proliferation, while treatment with 1 μM masitinib or PP121 led to a partial reduction in cell proliferation relative to DMSO-treated cultures. Two biological replicates were conducted per treatment, and each point represents the mean of three measurements from each biological replicate. For timepoints at 40, 60, and 85 hours, cell densities of inhibitor-treated cultures were significantly different from vehicle (DMSO) ( p -value <0.01). Significance was determined by a two-way ANOVA multiple comparisons test. (B) S. rosetta cultures treated with 1 μM or 10 μM sorafenib, glesatinib, or PP121 for 24 hours had reduced normalized cell density, whereas masitinib only had reduced normalized cell density at 10 μM. Normalized cell densities were determined to be reduced if differences between treatments and vehicle (DMSO) were significant ( p -value <0.01) Significance was determined by determined by a two-way ANOVA multiple comparisons test. Movies show S. rosetta cells treated with 10 μM glesatinib that undergo cell lysis (Movie S1, ESI ) and sorafenib, that have cell body deformation (Movies S2 and S3, ESI ), in comparison to DMSO control (Movie S4, ESI ). (C) Western blot analysis of S. rosetta cultures treated with 1 μM sorafenib and glesatinib for 1 hour showed a decrease in tyrosine phosphorylation of proteins at ∼60 kDa, ∼45 kDa, and ∼35 kDa (indicated by arrows and detected with pY1000 anti-phosphotyrosine antibody) compared to vehicle (DMSO) control. Masitinib and PP121 did not reduce the phosphotyrosine signal.
Article Snippet:
Techniques: Blocking Assay, Lysis, Comparison, Control, Western Blot, Phospho-proteomics