Journal: International journal of cancer

Article Title: Selective pharmacological inhibitors of HDAC6 reveal biochemical activity but functional tolerance in cancer models.

doi: 10.1002/ijc.32169

Figure Lengend Snippet: Figure 1. Activity and selectivity of selected HDACi. (a) Chemical structure and literature data concerning enzymatic IC50 values against HDAC1 and 6, for the studied HDACi. Selectivity index is calculated by dividing the HDAC1 IC50 value by the HDAC6 IC50. (b) Immunoblotting results for acetylated α-tubulin, α-tubulin, acetylated histone 3 and histone 3 of SK-OV-3-Luc ovarian cancer cells treated with the HDACi at the indicated concentrations for 1 h. For quantification, see Supporting Information Figure 1A. Samples separated by horizontal lines were run on different gels. (c) Immunoblotting results for acetylated α-tubulin and α-tubulin of SK-OV-3-Luc cells treated with Tubathian A (8A) and Tubastatin A (Tub A) at a concentration of 1 μM during 1 h, followed by removal of the treatment and the addition of fresh medium for 1 h (wash out). (d) Immunoblotting results for acetylated α-tubulin and α-tubulin of SK-OV-3-Luc cells treated with Tubathian A (8A, 1 μM) during the indicated time range. In (b), (c) and (d) GAPDH was used as a loading control.

Article Snippet: HDAC inhibitors Tubastatin A (Tub A), Tubacin (Tub), Ricolinostat (ACY) and Vorinostat (SAHA) were purchased from Selleckchem (Munich, Germany).

Techniques: Activity Assay, Western Blot, Concentration Assay, Control

Journal: International journal of cancer

Article Title: Selective pharmacological inhibitors of HDAC6 reveal biochemical activity but functional tolerance in cancer models.

doi: 10.1002/ijc.32169

Figure Lengend Snippet: Figure 2. In vitro effects of HDACi on the proliferation and migration of SK-OV-3-Luc ovarian cancer cells. (a) Real-time analysis of cell confluency of treated SK-OV-3-Luc cells using IncuCyte technology (left panel). Microscopic pictures were taken every 2 h and confluency was determined using IncuCyte software (indicated by yellow mask, right panel). (b) Relative concentration of extracellular glutamine upon treatment of SK-OV-3-Luc cells with the indicated conditions for 48 h (NMR measurement). The concentrations were corrected for cell number. Median is shown. (c) Real-time monitoring of SK-OV-3-Luc trans-well migration by measuring electrical impedance (using xCELLigence technology) during 24 h. (d) Real-time monitoring of treated SK-OV-3-Luc cells scratch migration using IncuCyte Technology (left panel). Relative wound density was analyzed using IncuCyte software (scratch wound is indicated by yellow mask, right panel). In (a), (c) and (d) a representative example of three biological replicates is presented. Mean + SEM of six (a and d) or two (c) technical replicates is shown. Statistical analysis can be found in Supporting Information Figure 2. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: HDAC inhibitors Tubastatin A (Tub A), Tubacin (Tub), Ricolinostat (ACY) and Vorinostat (SAHA) were purchased from Selleckchem (Munich, Germany).

Techniques: In Vitro, Migration, Software, Concentration Assay

Journal: International journal of cancer

Article Title: Selective pharmacological inhibitors of HDAC6 reveal biochemical activity but functional tolerance in cancer models.

doi: 10.1002/ijc.32169

Figure Lengend Snippet: Figure 3. Effect of HDACi on parental and HDAC6 knockout (HAP1-KO) HAP1 cells. (a) Immunoblotting results for HDAC6, acetylated α-tubulin and α-tubulin of HAP1 and HAP1-KO cells. (b) Immunoblotting results for acetylated α-tubulin, α-tubulin, acetylated histone 3 and histone 3 of HAP1-KO cells treated for 1 h with the indicated conditions of HDACi. For quantification, see Supporting Information Figure 3A. Samples separated by horizontal lines were run on different gels. (c) Real-time analysis of HAP1-KO cell confluency using IncuCyte technology. A representative example of three biological replicates is presented. Mean + SEM of six technical replicates is shown. Statistical analysis can be found in Supporting Information Figure 3C. (d) Relative mitochondrial metabolic activity of HAP1 and HAP1-KO cells treated for 72 h with the indicated conditions. Mean + SEM is shown (N = 3). In (a) and (b) GAPDH was used as a loading control.

Article Snippet: HDAC inhibitors Tubastatin A (Tub A), Tubacin (Tub), Ricolinostat (ACY) and Vorinostat (SAHA) were purchased from Selleckchem (Munich, Germany).

Techniques: Knock-Out, Western Blot, Activity Assay, Control

Journal: International journal of cancer

Article Title: Selective pharmacological inhibitors of HDAC6 reveal biochemical activity but functional tolerance in cancer models.

doi: 10.1002/ijc.32169

Figure Lengend Snippet: Figure 5. Impact of HDAC6i Tubathian A (8A) on breast cancer progression in vivo. (a) Relative weight of the primary tumour in BALB/c mice six weeks after orthotopic injection of 4 T1-Luc murine breast cancer cells in the mammary fat pad. Mice were treated intraperitoneally with vehicle or HDAC6 inhibitor 8A (5 or 20 mg/kg) on a daily basis. Median is shown. (b) Quantification of ex vivo bioluminescence of the primary tumours 6 weeks after inoculation in mice treated daily as indicated. Median is shown. Representative ex vivo bioluminescence monitoring of primary tumours for each condition are presented. (c) Percentage of mice having lung metastasis. Total number is indicated. (d) Quantification of ex vivo bioluminescence in lungs of DMSO or 8A treated mice with metastasis. Median is shown. Representative ex vivo bioluminescence monitoring of metastasis for each condition are presented. (e) Immunoblotting results for acetylated α-tubulin, α-tubulin, acetylated histone 3 and histone 3 of primary tumours of mice treated with vehicle or HDAC inhibitor 8A (5 or 20 mg/kg), 1 h after final treatment. GAPDH was used as a loading control. (f) Quantification of acetylated α-tubulin/GAPDH (left panel) and acetylated histone 3/GAPDH (right panel) of primary tumour tissue in mice treated as indicated. The median is shown. (g) H&E staining of the primary tumour and lung of mice treated as indicated. Metastasis are encircled by a dotted line. Representative images of each treatment group are shown. CC: cancer cells; IC: immune cells; MT: muscle tissue. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: HDAC inhibitors Tubastatin A (Tub A), Tubacin (Tub), Ricolinostat (ACY) and Vorinostat (SAHA) were purchased from Selleckchem (Munich, Germany).

Techniques: In Vivo, Injection, Ex Vivo, Western Blot, Control, Staining

Journal: Nature Communications

Article Title: Capturing disease severity in LIS1-lissencephaly reveals proteostasis dysregulation in patient-derived forebrain organoids

doi: 10.1038/s41467-025-64980-0

Figure Lengend Snippet: A Bar graphs of –log₁₀-transformed p -values from GO molecular function enrichment for actin binding, actin filament binding, tubulin binding, and cytoskeleton structural constituent; nominal significance p = 0.05; p -value adjusted with Bonferroni correction; two-sided Hypergeometric test. B Representative day 20 organoids stained for acetylated α-tubulin (Ac-TUB); yellow dashed lines, apical/basal VZ regions. C Quantification of Ac-TUB strand density at day 20: n = 11 control (4 cell lines), 10 mild, 12 moderate, 18 severe (2 cell lines each), across four differentiations. VZ loops: 41, 54, 49, 80, respectively; two-sided Wilcoxon test. D Day 20 organoids stained for N-cadherin (N-CAD); yellow dashed lines, VZ edges and N-cadherin diameter expansion. E Quantification of apical N-CAD signal at day 20: VZ structures n = 32 control, 39 mild, 42 moderate, 43 severe for a total of 6, 8, 7, and 9 differentiations, respectively; 2 cell lines each; two-sided Kruskal–Wallis with post hoc Wilcoxon correction. F GO enrichment for cadherin binding; nominal significance p = 0.05; p -value adjusted with Bonferroni correction; two-sided Hypergeometric test. G Heatmap of log₂FC for WNT pathway genes in cycling progenitors by severity grade versus control (* P < 0.05, ** P < 0.01, *** P < 0.001); two-sided Wilcoxon test. H WNT-GFP reporter organoids at day 20; yellow dashed lines, VZ boundaries. I Mean value of WNT-GFP intensity per VZ: n = 20 control (2 cell lines), 20 mild (2 cell lines), 10 moderate (one cell line), 10 severe (one cell line); 3 organoids per condition from two differentiations per cell line; two-sided Kruskal–Wallis with post hoc Wilcoxon correction. J Quantification of cell division plane orientation: n = 13 control, 12 mild, 12 moderate, 15 severe. VZ loops: 59, 56, 43, 57; cells: 206, 359, 172, 249, respectively; Chi-square test. K Examples of division planes in control and severe organoids; yellow dashed lines, VZ edges; white dashed lines, division plane orientation. Scale bars: B left 200 μm, B right, H 50 μm; D , K 20 μm; All boxplots show median, 25th/75th percentiles, and 1.5 interquartile range whiskers. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were diluted according to the manufacturer’s instructions and incubated overnight at 4 °C with the following dilutions: Ac-TUB (1:500, Cell Signaling, cat. no. 5335S), AFP (1:200, Sino Biologicals, cat.no.

Techniques: Transformation Assay, Binding Assay, Staining, Control

Journal: EMBO Reports

Article Title: Human cerebral organoids reveal progenitor pathology in EML1‐linked cortical malformation

doi: 10.15252/embr.202154027

Figure Lengend Snippet: A Immunocytochemical analyses of iPS cells reveal that cells stain positive for the pluripotency‐associated markers Tra1‐60, Tra‐1‐81, and SSEA3. Following 4 weeks of in vitro differentiation and immunocytochemical analyses, derivatives of all three germ layers such as ß‐III‐tubulin+ (ß‐III‐Tub, ectoderm), alpha‐1‐fetoprotein+ (AFP, endoderm), and smooth muscle actin+ cells (SMA, mesoderm) could be detected. B High‐resolution SNP karyotyping reveals integrity of the karyotype. Note: (A) and (B) depict representative data of EML1 patient 1–derived iPS cells. (C), EML1 patient 1 and EML1 patient 2 (D)‐specific mutations were confirmed by sequencing. C EML1 patient 1 and EML1 patient 2. D Specific mutations were confirmed by sequencing. E Genetic validation of selected EML1 ‐heKO clones. PCR primers are designed to recognize the EML1 wild‐type allele or the integration of the puromycin cassette. Note: Detection of the integration of the puromycin cassette simultaneously to detection of WT allele indicates heterozygous EML1‐ heKO. F qRT–PCR shows reduced expression levels of EML1 in EML1‐ heKO‐derived iPS cells confirming the heterozygous EML1‐ heKO. Three biological replicates, data in graphs are represented as means ± SD. Significance based on two‐way ANOVA and Tukey’s multiple‐comparison test, *** P = 0.0001. Data information: For statistical analyses, see the source data for this figure. Scale bars, (A) 100 μm, SMA and AFP 25 μm. Source data are available online for this figure.

Article Snippet: Primary antibodies and concentrations were as follows: Acetylated α‐tubulin (AC‐TUB) (Cell Signaling Technology, 1:1,000) and ß‐Actin (Cell Signaling Technology, 1:15,000).

Techniques: Staining, In Vitro, Derivative Assay, Sequencing, Biomarker Discovery, Clone Assay, Quantitative RT-PCR, Expressing, Comparison

Journal: EMBO Reports

Article Title: Human cerebral organoids reveal progenitor pathology in EML1‐linked cortical malformation

doi: 10.15252/embr.202154027

Figure Lengend Snippet: Day 33 ± 2 control‐ and EML1 patient‐derived cerebral organoids stained for ß‐III‐tubulin and DAPI. VZ and CP areas are marked by white brackets, ectopic neural rosettes by red bracket, and heterotopic neurons by red arrows. Day 33 ± 2 control‐ and EML1 patient‐derived cerebral organoid stained for the adherens junction marker N‐cadherin (NCAD) expressed at the VZ surface and within the ectopic neural rosettes. Day 20 ± 2 cerebral organoids derived from controls, patients, and EML1 ‐heKOs stained for the neuronal marker MAPT and the adherens junction marker NCAD, counterstained with DAPI. Ectopic neural rosettes and neuronal heterotopia are highlighted with dotted yellow lines. Quantification of VZ areas with ectopic neural rosettes (three batches, three organoids analyzed per batch, significance based on Kruskal–Wallis test, P = 0.0001, Dunn’s post hoc test for multiple comparisons performed to define statistical differences between genotypes, and single data points presented are colored by batch). Quantification of heterotopic, disorganized, or organized cortical areas (three batches, three organoids per batch, significance based on Kruskal–Wallis test, P = 0.0001 for “organized” and “heterotopic”; no significant difference for “disorganized,” Dunn’s post hoc test performed for multiple comparisons to define statistical differences between genotypes). Data information: * marks statistical significance in relation to Control 1, # in relation to Control 2. P ‐values: ***/###< 0.001. VZ, ventricular zone; CP, cortical plate. Data in graphs are represented as means ± SD. Scale bars: (A) 100 µm; (B) 100 µm, enlarged 25 µm; and (C) 50 µm. Source data are available online for this figure.

Article Snippet: Primary antibodies and concentrations were as follows: Acetylated α‐tubulin (AC‐TUB) (Cell Signaling Technology, 1:1,000) and ß‐Actin (Cell Signaling Technology, 1:15,000).

Techniques: Control, Derivative Assay, Staining, Marker

Journal: EMBO Reports

Article Title: Human cerebral organoids reveal progenitor pathology in EML1‐linked cortical malformation

doi: 10.15252/embr.202154027

Figure Lengend Snippet: Immunocytochemical analyses of day 20 ± 2 control‐ and EML1 ‐deficient cerebral organoids stained for p‐Vim (mitotic cells) and TPX2 (labeling the mitotic spindle). Dotted lines indicate the VZ surface and RG‐cell division mode, squares indicate areas enlarged below. PAX6‐positive cortical progenitors derived from control, EML1 ‐heKO, and EML1 ‐heKO treated with EpoD exhibit ARL13B‐positive primary cilia. Immunoblot for acetylated tubulin in control and EML1 ‐deficient cortical progenitors under control‐ (−) or EpoD‐treated (+) condition. Quantification of relative protein signal intensities of the immunoblot shown in C based on n = 3 biological replicates of independent experiments. Signals were normalized to actin. Data information: Bar graphs show mean with SEM, significance based on two‐way ANOVA with Bonferroni’s multiple‐comparison test, ** P = 0.0047. For individual P ‐values, see the source data for this figure. Scale bars: (A) 50 μm, enlarged 10 µm; (B) 5 μm.

Article Snippet: Primary antibodies and concentrations were as follows: Acetylated α‐tubulin (AC‐TUB) (Cell Signaling Technology, 1:1,000) and ß‐Actin (Cell Signaling Technology, 1:15,000).

Techniques: Control, Staining, Labeling, Derivative Assay, Western Blot, Comparison

Journal: Bioactive Materials

Article Title: Spatiotemporally engineered microneedle for microenvironment remodeling propels mucosal regeneration after tracheal mucosal injury

doi: 10.1016/j.bioactmat.2026.01.026

Figure Lengend Snippet: Gel-AgNA/MgGA MN promote mucosal regeneration. (a) HE and Safranin O staining of rabbit tracheal samples harvested at Day 10 post-operation after treated with Gel, Gel-AgNA, Gel-MgGA, and Gel-AgNA/MgGA MN. (b) IF staining of CK14 (marker of basal cells, red) and AC-Tub (marker of cilia cell, green). (c) IF staining of ZO-1 (marker of tight junctions, orange). (d, e) Quantitative analysis of regenerated epithelial coverage and thickness (n = 9). (f) Masson and Sirius Red staining for collagen evaluation after various treatments (n = 5). Quantitative analysis of collagen volume fraction (g) and fiber orientation (h) . The pentagram indicates luminal side of trachea.

Article Snippet: Immunofluorescence staining of CK14 (Abcam, ab181595), AC-Tub (Proteintech, 66200-1-Ig), ZO-1 (Proteintech, 21773-1-AP), and Immunohistochemical (IHC) staining for CD31 (Servicebio, S1002) were conducted to reveal the conditions of mucosal regeneration, according to previous literature [ ].

Techniques: Staining, Marker

Journal: Journal of neurovirology

Article Title: HIV-associated neurodegeneration: exploitation of the neuronal cytoskeleton

doi: 10.1007/s13365-019-00737-y

Figure Lengend Snippet: a) Primary rat cortical neurons (7 days in vitro) were fixed, stained for acetylated tubulin (red), and imaged using a STEDYCON microscope (Abberior Instruments). Yellow, a coloring construct from the imaging system, indicates the enrichment of acetylated tubulin. b) Enlarged image of box in (a) showing that acetylated tubulin is enriched in the axon when compared to the rest of the cell. Scale bar: 5nm.

Article Snippet: Thus, PTMs on fully formed MTs modify many aspects of neuronal function. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1. caption a7 caption a8 Acetylated tubulin is enriched in the axon. a) Primary rat cortical neurons (7 days in vitro ) were fixed, stained for acetylated tubulin (red), and imaged using a STEDYCON microscope (Abberior Instruments).

Techniques: In Vitro, Staining, Microscopy, Construct, Imaging

Journal: Oncotarget

Article Title: Low concentrations of vorinostat decrease EB1 expression in GBM cells and affect microtubule dynamics, cell survival and migration

doi: 10.18632/oncotarget.27892

Figure Lengend Snippet: Analysis of tubulin, EB proteins, and PTM (post translational modification) level expression by Western blot on U87-MG cells treated for 24 h with vorinostat at various concentration in parallel with Histone H3 acetylation. ( A ) α tubulin, acetylated tubulin, tubulin ΔY, tubulin EEY, EB1 EEY and EB1 proteins level expression, 0 to 2.5 μM vorinostat. Ratios (%) acetylated tubulin/GAPDH, EB1 EEY/GAPDH and EB1/GAPDH, from at least three independent experiments are presented under the blots. ( B ) α tubulin, acetylated tubulin, EB1, EB2, EB3 and histone H3 proteins level expression, 0 to 10 μM vorinostat. Ratios (%) EB1/GAPDH, EB2/GAPDH and EB3/GAPDH and ratios (%) EB1/tubulin, EB2/tubulin and EB3/tubulin, from at least three independent experiments are presented under the blots. ( C ) Tubulin EEY and tubulin ΔY proteins level expression, 0 to 20 μM vorinostat. ( D ) α tubulin, acetylated tubulin and EB1 proteins level expression, 0 to 5 μM vorinostat, U87-MG sh0 et U87-MG shEB1. Ratios (%) EB1/GAPDH, from at least three independent experiments are presented under the blots.

Article Snippet: Indirect immunofluorescence was performed with the anti-EB3 antibody (EPR11421(B), Abcam), anti-acetylated tubulin antibody (Merck millipore), anti-detyrosinated tubulin antibody (Abcam) and anti-mouse antibody Alexa 568 nm (Molecular Probes); and FITC-coupled anti-α-tubulin antibody (clone DM1A; Sigma-Aldrich).

Techniques: Modification, Expressing, Western Blot, Concentration Assay

Journal: Oncotarget

Article Title: Low concentrations of vorinostat decrease EB1 expression in GBM cells and affect microtubule dynamics, cell survival and migration

doi: 10.18632/oncotarget.27892

Figure Lengend Snippet: ( A ) Immunofluorescence staining of α-tubulin (green) and endogenous EB3 (red) in U87-MG cells with 24 hours 5 μM vorinostat and control (left panel) and vorinostat effect on EB3 comet longer and area, **** p < 0.0001, vs control, Student’s t -test, (right panel), bar = 5 μm. ( B ) Analysis of EB3 protein level expression by Western blot of U87-MG cells transfected by EB3-GFP treated after 24 h of treatment with vorinostat 2 μM. ( C ) Representative image from time-lapse videomicroscopy of U87-MG cells transfected with EB3-GFP cells (top), representative image of EB3 comet trajectories in cells obtained from ICY ® software (gradient of colors according time, bottom) and variation of comet area under vorinostat, bar = 10 μm (right panel). ( D ) Parameters of EB3-GFP dynamics. All values are expressed as mean ± SEM of comets tracks analyzed (n.s.: non-significant., **** p ≤ 0.0001 each condition vs control, Student’s t -test).

Article Snippet: Indirect immunofluorescence was performed with the anti-EB3 antibody (EPR11421(B), Abcam), anti-acetylated tubulin antibody (Merck millipore), anti-detyrosinated tubulin antibody (Abcam) and anti-mouse antibody Alexa 568 nm (Molecular Probes); and FITC-coupled anti-α-tubulin antibody (clone DM1A; Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Transfection, Software

Journal: Oncotarget

Article Title: Low concentrations of vorinostat decrease EB1 expression in GBM cells and affect microtubule dynamics, cell survival and migration

doi: 10.18632/oncotarget.27892

Figure Lengend Snippet: ( A ) Analysis of tyrosinated tubulin, EB1, GFP-EB1 tyrosinated and detyrosinated level expression by Western blot of U87-MG cells transfected by GFP-EB1-detyr and GFP-EB1-tyr, treated after 24 h of treatment with vorinostat 5 μM. ( B ) Analysis of acetylated tubulin and EB1 protein level expression by Western blot of U87-MG cells, treated after 24 h of treatment with vorinostat 2 μM and/or MG132; 2 μM.

Article Snippet: Indirect immunofluorescence was performed with the anti-EB3 antibody (EPR11421(B), Abcam), anti-acetylated tubulin antibody (Merck millipore), anti-detyrosinated tubulin antibody (Abcam) and anti-mouse antibody Alexa 568 nm (Molecular Probes); and FITC-coupled anti-α-tubulin antibody (clone DM1A; Sigma-Aldrich).

Techniques: Expressing, Western Blot, Transfection