ipa3  (Millipore)

Journal: Biomolecules

Article Title: Inhibition of the RAC/PAK Signaling Axis Enhances the Potency of MAPK Cascade Inhibitors Against Uveal Melanoma

doi: 10.3390/biom15101425

Figure Lengend Snippet: GNAQ- and GNA11-mutant uveal melanoma cells are sensitive to PAK inhibitors. ( A ). The indicated concentrations of a pan-PAK inhibitor PF3758309 were added to the cultures of 92.1, Mel202 and MP41 uveal melanoma cells. Five days later, cell numbers were compared using the methylene blue staining and extraction method. The values for each cell line were normalized to those in the respective control cultures. Error bars denote standard deviations of quadruplicates. ( B ). 92.1, Mel202 and MP41 uveal melanoma cells were treated with a group I PAK inhibitor IPA3 and the results were analyzed as in A. Half-maximal inhibitory concentrations (IC50) with corresponding 95% confidence intervals (95% CI) are shown.

Article Snippet: IPA3 was purchased from Selleckchem (S7093) and Cayman Chemical (4759).

Techniques: Mutagenesis, Staining, Extraction, Control

Journal: Biomolecules

Article Title: Inhibition of the RAC/PAK Signaling Axis Enhances the Potency of MAPK Cascade Inhibitors Against Uveal Melanoma

doi: 10.3390/biom15101425

Figure Lengend Snippet: MEK inhibitors and PAK inhibitors synergistically suppress uveal melanoma cells. ( A ). 92.1 cells treated with MEKi selumetinib (30 nM), PAKi PF3758309 (4 nM), or the corresponding vehicle (DMSO) control were fixed and visualized by methylene blue staining. ( B – G ). Bliss synergy scores for various MEKi/PAKi combinations in uveal melanoma cell lines. ( B ). 92.1 treated with MEKi selumetinib and PAKi PF3758309. ( C ). 92.1 treated with MEKi selumetinib and PAKi IPA3. ( D ). 92.1 treated with MEKi binimetinib and PAKi IPA3. ( E ). 92.1 treated with MEKi mirdametinib and PAKi IPA3. ( F ). MP41 treated with MEKi binimetinib and PAKi IPA3. ( G ). MP41 treated with MEKi selumetinib and PAKi IPA3. All cultures were analyzed 5 days after the addition of the indicated compounds. Relative survival in ( B – G ) was measured using methylene blue staining and extraction method. Each condition was tested in quadruplicate. SynergyFinder was used to calculate Bliss synergy scores with p -values for each experiment. “SEL”—selumetinib, “BIN”—binimetinib, “MIR”—mirdametinib, “PF”—PF3758309, “IPA”—IPA3.

Article Snippet: IPA3 was purchased from Selleckchem (S7093) and Cayman Chemical (4759).

Techniques: Control, Staining, Extraction

Journal: Biomolecules

Article Title: Inhibition of the RAC/PAK Signaling Axis Enhances the Potency of MAPK Cascade Inhibitors Against Uveal Melanoma

doi: 10.3390/biom15101425

Figure Lengend Snippet: Inhibitors of PAK and IMPDH augment MEK inhibitor selumetinib in suppressing the signaling of the MAPK cascade. ( A ). Lysates of 92.1 cells treated for 72 h with selumetinib (30 nM; “SEL”), PF3758309 (4 nM; “PF”) or their combination (“Combo”) were compared to those of untreated control cultures (“UT”; exposed to the vehicle only) by probing with the antibodies for phosphorylated ERK (top), total ERK (middle) and β-actin (bottom). ( B ). Lysates of 92.1 cells treated for 72 h with selumetinib (30 nM; “SEL”), IPA3 (4 µM), or their combination (“Combo”) were analyzed as in A. ( C ). Lysates of 92.1 cells treated for 72 h with selumetinib (30 nM; “SEL”), ribavirin (10 µM; “RBV”) or their combination (“Combo”) were analyzed as in A. ( D ). Three independent experiments performed as in A were analyzed quantitatively, and the intensity of the pERK band was normalized to that of β-actin in each sample. The resulting values from each experiment were further normalized to those in parallel untreated controls. The bars represent averages and the error bars—standard deviations of the three experiments. ( E ). Three independent experiments were performed as in B and analyzed as in D. ( F ). Three independent experiments were performed as in C and analyzed as in D. “*” denotes p < 0.05. “**” denotes p < 0.01. The original Western Blotting images are in the .

Article Snippet: IPA3 was purchased from Selleckchem (S7093) and Cayman Chemical (4759).

Techniques: Control, Western Blot

Journal: BMC Biology

Article Title: Vimentin-mediated buffering of internal integrin β1 pool increases survival of cells from anoikis

doi: 10.1186/s12915-024-01942-w

Figure Lengend Snippet: Effects of inhibiting vimentin S38 phosphorylation on metastasis. A HeLa cells were suspended for 2 h in the presence of varying concentrations of IPA3 as indicated, and Ser38 phosphorylation of vimentin was measured as in Fig. A. B HeLa cells suspended for 2 h in the presence or absence of 40 μM IPA3 were analyzed for surface integrin β1 by using flow cytometry. C The mean fluorescence intensities of integrin β1 staining in each condition were normalized against the untreated control and presented as a one-dimensional scatter plot. *, p < 0.05 (paired T test, n = 3). D HeLa and HeLa_VIMKO cells suspended for 24 h in the presence or absence of 40 μM IPA3 were analyzed by flow cytometry as in Fig. A. E Percentages of PI-positive cells in ( D ) are shown. ***, p < 0.0001, n.s.: not significant (one-way ANOVA followed by Tukey multiple-comparison test, n = 3). F Schematic of an in vivo experiment using BLAB/c mice. 1 × 10. cells of 4T1 were injected into the tail vein, and 5 mg/kg IPA3 or DMSO were treated through intraperitoneal injection every day for 14 days. G Representative pictures of lungs from mice treated with control vehicle (top) or IPA3 (bottom) are shown. H The number of metastatic nodules in the lungs in each group was counted and presented as a graph. ***, p < 0.0001 (unpaired T test, n = 5 for each group)

Article Snippet: 1.5 × 10 5 cells suspended on a poly-HEMA-coated surface for 0 to 24 h in the presence or absence of 20 ~ 40 μM RGDS peptides (RnD system, 3498–10), 10 ~ 40 μM IPA3 (Sigma, I2285), or 10 μM withaferin A (APExBio, B7199).

Techniques: Flow Cytometry, Fluorescence, Staining, Control, Comparison, In Vivo, Injection