gsk 3α β inhibitor  (TargetMol)

Journal: iScience

Article Title: A developmental atlas of mouse vestibular-like inner ear organoids

doi: 10.1016/j.isci.2025.111817

Figure Lengend Snippet: Modulation of TGFβ, BMP, FGF, and WNT signaling results in the formation of inner ear sensory epithelia in vitro (A) Overview of the mouse embryonic stem cell (mESC)-derived inner ear organoid protocol. DIV, days in vitro ; ↓ , inhibition; ↑, activation; OEPD, otic epibranchial progenitor domain. (B) By DIV8, non-neural ectoderm gives rise to ECAD+/SOX2+/PAX8+ OEPD. (C) By DIV11, OEPD cells further differentiate to form ECAD+/SOX2+/PAX8+ otic vesicle structures. (D) By DIV14, otic vesicle structures mature into sensory epithelia, complete with SOX2+/JAG1+ supporting cells and MYO7A + hair cells. Scale = 50 μm, n ≥ 3.

Article Snippet: On DIV8, organoids were washed with 1X PBS and transferred to maturation media containing 1% matrigel and 3 μM CHIR99021 (a GSK-3α/β inhibitor, Selleckchem) to induce Wnt signaling.

Techniques: In Vitro, Derivative Assay, Inhibition, Activation Assay

Journal: iScience

Article Title: A developmental atlas of mouse vestibular-like inner ear organoids

doi: 10.1016/j.isci.2025.111817

Figure Lengend Snippet: scRNA-seq of DIV3, DIV4 and DIV8 inner ear organoids (A and B) scRNA-seq of DIV3 organoids reveals the transition of mESCs to ectoderm and neural ectoderm, as well as a small population of mesendoderm, as demonstrated by select marker gene expression. (C and D) scRNA-seq of DIV4 organoids reveals the further transition of ectoderm to non-neural ectoderm, as demonstrated by select marker gene expression. See also Figure S1 . (E) qPCR validation of differential gene expression between mESCs, DIV3, and DIV4 organoids, n = 4. p value ∗<0.05, ∗∗<0.01, ∗∗∗∗<0.0001, ns = not significant. Significance was assessed by two-way ANOVA followed by Tukey’s multiple comparisons test. Data are represented as mean ± SD. (F) Immunostaining for the pluripotency markers OCT4 and SOX2 reveals the specification of an outer layer of OCT4-/SOX2-/ECAD+ ectoderm lineage cells at DIV4. Scale, 50 μm, n ≥ 3. (G and H) scRNA-seq of DIV8 organoids shows the formation of Sox2+/Pax8+ otic-epibranchial progenitor domain cells (OEPD), in addition to mesenchyme, neural precursor cells (NPCs)/glia, neurons, and surface ectoderm (SE). (I and J) Validation of TWIST1/2+/ECAD-SOX2- mesenchymal cells and TUBB3+ neurons at DIV8. (K and L) EPCAM+/SOX2+/PAX8+ OEPD and EPCAM+/P63+ SE develop adjacently but do not overlap. Scale = 50μm, n ≥ 3. (M) While the overall level of Epcam expression does not change between DIV4 and DIV7, Pax8 and Trp63 are rapidly induced between DIV4 and DIV5. PPE, pre-placodal ectoderm; n = 3. p value ∗<0.05, ∗∗∗∗<0.0001. Significance was assessed by two-way ANOVA followed by Tukey’s multiple comparisons test. Data are represented as mean ± SD.

Article Snippet: On DIV8, organoids were washed with 1X PBS and transferred to maturation media containing 1% matrigel and 3 μM CHIR99021 (a GSK-3α/β inhibitor, Selleckchem) to induce Wnt signaling.

Techniques: Marker, Gene Expression, Biomarker Discovery, Immunostaining, Expressing

Journal: iScience

Article Title: A developmental atlas of mouse vestibular-like inner ear organoids

doi: 10.1016/j.isci.2025.111817

Figure Lengend Snippet:

Article Snippet: On DIV8, organoids were washed with 1X PBS and transferred to maturation media containing 1% matrigel and 3 μM CHIR99021 (a GSK-3α/β inhibitor, Selleckchem) to induce Wnt signaling.

Techniques: Generated, Recombinant, Software

Journal: BMC Cancer

Article Title: Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

doi: 10.1186/1471-2407-10-526

Figure Lengend Snippet: GSK-3 expression, phosphorylation and localization in MM cells . (A) WB for total GSK-3α and β (top panels), phosphorylated GSK-3α (on Ser 21) and GSK-3β (on Ser 9) (middle panels), and β-actin as loading control (bottom panels) in (from left to right): 4 PBMC samples of healthy subjects, non-malignant in vitro -generated PC (nPC), CD138 + purified PC from 9 MM patients, 4 MMCLs; (B) WB for total GSK-3α and β (top panels), phosphorylated GSK-3α (on Tyr 279) and GSK-3β (on Tyr 216) (middle panels) and β-actin as loading control (bottom panels), in three primary MM plasma cell samples (MM 7, 3, 1). (C-E): GSK-3 immunostaining and confocal microscopy analysis in MMCLs OPM-2, INA-6, U-266 and RPMI-8226 (C), three CD138+ primary malignant PC from patients (MM1, MM9, MM10) (D) and BMMC (E). In the merged images GSK-3 is detected by green fluorescence and nuclei are in blue. Scale bars = 10 μm. For all the images: 600× magnification, oil objective.

Article Snippet: GSK-3α/β inhibitors SB216763 and SB415286 were purchased from Sigma-Aldrich, Italy.

Techniques: Expressing, In Vitro, Generated, Purification, Immunostaining, Confocal Microscopy, Fluorescence

Journal: BMC Cancer

Article Title: Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

doi: 10.1186/1471-2407-10-526

Figure Lengend Snippet: Effects of GSK-3 inhibitors on MM cell proliferation . (A, B) Graphs showing representative in vitro kinase assays performed using human recombinant GSK-3β (hrGSK-3β) and total protein lysates (+ = 1 μg, ++ = 4 μg) of OPM-2 cells (A) and RPMI-8226 cells (B) that were cultured with the two specific GSK-3 inhibitors, SB216763 at 10 μM and SB415286 at 4 μM. The graphs show a remarkable inhibition of both hrGSK-3β protein (leftmost graphs) and endogenous GSK-3 kinase activity (rightmost graphs) (y axis: counts per minute). Data represent mean ± SD, n = 3. * indicates p < 0.05 by one-way ANOVA test. (C) Dose-response graphs of U-266 (left) and INA-6 cells (right) incubated for 48 hours with increasing concentrations of SB415286. Cell proliferation was assessed by 3 H-thymidine-incorporation assay. Data represent mean ± SD, n = 4, * indicates p < 0.05 by one-way ANOVA test.

Article Snippet: GSK-3α/β inhibitors SB216763 and SB415286 were purchased from Sigma-Aldrich, Italy.

Techniques: In Vitro, Recombinant, Cell Culture, Inhibition, Activity Assay, Incubation, Thymidine Incorporation Assay

Journal: BMC Cancer

Article Title: Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

doi: 10.1186/1471-2407-10-526

Figure Lengend Snippet: GSK-3 inhibition causes MM cell apoptosis and disruption of the mitochondrial membrane potential . (A) Histograms showing the percentage of surviving (annexin V negative) U-266, RPMI-8226 and INA-6 cells (left graph) and of normal PBMC control cells (right graph) after treatment without (black bars) or with (white bars) 4 μM SB415286 for 48 hours. Data represent mean ± SD, n = 5 (cell lines) or 4 (PBMC). * indicates p < 0.05 by Student's t-test. (B) Histograms showing FACS analysis of the amount of JC-1 monomer-containing, green fluorescent-INA-6 cells (left graph) or U-266 cells (right graph), treated with the same conditions as in A. Data are expressed as ratio over untreated cells. Data represent mean ± SEM, n = 5. * indicates p < 0.01 by Student's t-test. (C) Representative WB analysis of phospho Tyr 279 GSK-3α and phospho Tyr 216 GSK-3β, total GSK-3, PARP cleavage and Smac/DIABLO protein expression on cell lysates of RPMI-8226 cells grown with 5 μM or 10 μM SB216763 for 24 hours (top panels) and WB analysis of PARP protein in INA-6 cells grown with 4 μM SB415286 for 8 and 24 hours (bottom). β-Actin is used to check protein loading. (D) Representative WB analysis of nuclear (n) and cytosolic (c) proteins from U-266 cells untreated or treated with 5 μM SB216763, showing the levels of total GSK-3, Tyr phosphorylated GSK-3, β-catenin, total ERK 1/2, Thr/Tyr phosphorylated ERK 1/2, PARP and nucleophosmin and GAPDH as loading controls.

Article Snippet: GSK-3α/β inhibitors SB216763 and SB415286 were purchased from Sigma-Aldrich, Italy.

Techniques: Inhibition, Expressing

Journal: BMC Cancer

Article Title: Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

doi: 10.1186/1471-2407-10-526

Figure Lengend Snippet: Effects of GSK-3α and β knockdown by RNA interference on MM cell survival . (A) WB analysis of the levels of GSK-3α (left panels) or GSK-3β (right panels) upon nucleofection with: un = untransfected; scr = scrambled siRNAs oligos; GSK-3α or GSK-3β=GSK-3α or GSK-3β-directed siRNAs oligos. (B, left panels) Light microscopy microphotographs of U-266 cells transfected with scrambled (scr), GSK-3α or GSK-3β-directed siRNAs oligos. Scale bars = 10 μm. (B, middle plots) Representative dot plot graphs and FACS analysis of U-266 cells transfected with scrambled (scr), GSK-3α or GSK-3β-directed siRNAs oligos. (B, right panels) Histograms summarizing the results of FACS analysis of U-266 cells transfected with scrambled (scr) siRNAs oligos, GSK-3α (top) or GSK-3β (bottom)-specific siRNAs oligos,. In y-axis dead cells are cells displaying a low FSC/high SSC cyto-morphological profile. Data represent mean ± SEM, n = 6. * indicates p < 0.05 (Student's t test). (C) Representative WB analysis of the levels of unprocessed PARP after nucleofection of U-266 cells with: scrambled, GSK-3α or GSK-3β siRNAs oligos. (D) Light microscopy microphotographs and histograms summarizing the results of FACS analysis of U-266 cells transfected with scrambled (scr), GSK-3α or GSK-3β-directed siRNAs oligos and treated with 5 nM BZ. In y-axis dead cells are cells displaying a low FSC/high SSC cyto-morphological profile. Data represent mean ± SEM, n = 3. * indicates p < 0.05 (one-way ANOVA test). (E) Histogram plot summarizing the results of the FACS analysis of U-266 cells transfected with scrambled (scr) or GSK-3α/GSK-3β-directed siRNA oligos and treated with 5 nM BZ. In y-axis dead cells are cells displaying a low FSC/high SSC cyto-morphological profile. Data represent mean ± SEM, n = 3. * indicates p < 0.05 (one-way ANOVA). (F) WB analysis of GSK-3α, GSK-3β, AKT, Ser 473-phosphorylated AKT and MCL-1 protein levels in U-266 cells untransfected (lane 1), treated with 5 nM BZ (lane2), transfected with scrambled (scr, lane 3) or GSK-3α/β (lane 4), GSK-3α (lane 5) or GSK-3β (lane 6)-directed siRNAs oligos without (-, lanes 3-6) or with (+, lanes 7-10) exposure to 5 nM BZ for 18 hours.

Article Snippet: GSK-3α/β inhibitors SB216763 and SB415286 were purchased from Sigma-Aldrich, Italy.

Techniques: Light Microscopy, Transfection

Journal: BMC Cancer

Article Title: Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

doi: 10.1186/1471-2407-10-526

Figure Lengend Snippet: Bortezomib modulates GSK-3 intracellular localization and activation . (A) Immunofluorescence microscopy of INA-6 and U-266 cells untreated or treated with 5 nM BZ for 18 hours and stained for GSK-3 (green fluorescence) and the nuclei (DAPI, blue fluorescence) showing partial nuclear re-localization of GSK-3 upon BZ treatment. Scale bars = 10 μm. On the bottom, graph showing the percentage of MM cells scored as having cytosolic only GSK-3 in the untreated (black bars) or 5 nM BZ-treated (white bars) conditions. Data represent mean ± SD, n = 3. * indicates p < 0.05. (B) Representative WB analysis of phospho Ser 9 GSK-3β/Ser 21 GSK-3α and total GSK-3α/β in nuclear (n) and cytoplasmic (c) protein fractions from untreated (-) or 5 nM BZ-treated (18 hours) U-266 cells. β-Actin and nuclear PARP are used as markers for protein loading. (C) Representative WB analysis of phospho Tyr 279 GSK-3α/Tyr 216 GSK-3β and total GSK-3α/β in nuclear (n) and cytoplasmic (c) protein fractions from untreated (-) or 5 nM BZ-treated (+) (18 hours) U-266 cells, in the presence or absence of 5 μM or 10 μM SB216763. GAPDH and nucleophosmin were used as protein loading markers.

Article Snippet: GSK-3α/β inhibitors SB216763 and SB415286 were purchased from Sigma-Aldrich, Italy.

Techniques: Activation Assay, Immunofluorescence, Microscopy, Staining, Fluorescence