Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: The 8701-BC cells express a soluble factor that is responsible of HAS2 induction in NHDF. ( A ) Quantitative RT-PCR analysis of HAS2 gene in NHDF and NHDF cultured in 8701-BC 48 h CM. The results are expressed as fold change with respect to NHDF cultured in RPMI1640 and each bar represents mean ± S.D. of two independent experiments. * p < 0.05. ( B ) SDS PAGE after Blue Coomassie staining of different amounts in term of total proteins (2.5, 5, 10 μg respectively) of 8701-BC 48 h CM. Bands at MW of approximately 55 kDa were excised and analyzed by MALDI-TOF as reported in Material and Methods. ( C ) Quantitative RT-PCR analysis of the c10orf118 (Q7z3E2) gene expression level in different breast cancer cell lines (MCF-7, MDA-MB-231 and 8701-BC), expressed as fold change with respect to NHDF. Bars represent mean ± S.D. of triplicate samples. * p < 0.05, *** p < 0.001.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Quantitative RT-PCR, Cell Culture, SDS Page, Staining, Gene Expression

Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: C10orf118 is found both in cell lysate and secreted media of different breast cancer cell lines. ( A ) Schematic view of the different isoforms of c10orf118 protein as reported by ensembl.org database, reporting the amino acid position and the molecular weight. Grey box depicts the region recognized by the antibody HPA018019 (Sigma Aldrich) and schematic view of the c10orf118 human recombinant peptide bearing the first 211 amino acids and a GST tag at N-terminal position. ( B ) Left panel, immunoblot of c10orf118 (full size, arrow) and tubulin in 30 μg of total proteins of cell lysate from NHDF, MCF-7 and MDA-MB-231 cells, respectively; right panel, immunoprecipitation and immunoblot of c10orf118 (full size, arrow) in 25 μL CM from culture of MDA-MB-231 and MCF-7.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Molecular Weight, Recombinant, Western Blot, Immunoprecipitation

Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: Endogenous c10orf118 localizes in the Golgi apparatus in MCF-7 cells. ( A ) Confocal microscopy images of MCF-7 cells stained for c10orf118 (green) and Golgin-97 (red), a Golgi apparatus marker. ( B ) Confocal microscopy images of MCF-7 cells stained for c10orf118 (green) and calnexin (red), a specific endoplasmic reticulum marker. ( C ) Line-scan of the staining performed in the section marked with a green line in the merged view and relative quantification. Overlap of the two signals is apparent. ( D ) Line-scan of the staining in the section marked with a green line in the merged view and relative quantification. ( E ) Confocal microscopy images of MCF-7 cells nucleofected with 30 nmol of scrambled siRNA and siRNA against c10orf118 for 48 h and stained with antibody against c10orf118 (green) or Golgin-97 (red). White bars, 20 µm.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Confocal Microscopy, Staining, Marker, Quantitative Proteomics

Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: Role of C10orf118 in MCF-7 cell function. ( A ) MTT assay of MCF-7 cells nucleofected with 30 pmol of a siRNA against c10orf118 or a scrambled siRNA. Data are expressed as percentage of control ± S.D. of three independent experiments. ( B ) Wound healing assay performed in MCF-7 cells nucleofected with 30 pmol of siRNA against c10orf118 or scrambled control. The graph represents the percentage of wound closure ± S.D after 24 h from the initial scratch of four independent experiments. Images were analysed using the software tool TScratch. ( C ) Western blot of 30 µg of MCF-7 cells lysates nucleofected for 48 h with an overexpressing vector for c10orf118 (pCMW-AC-c10orf118-GFP) or the relative empty vector (pCMW-AC-GFP) and treated with 5 µM MG-132 for 24 h. M = protein marker. The images report a representative immunoblot for c10orf118 and GAPDH and ( D ) the relative quantification. The analysis was performed measuring the optical density of the bands and values are expressed as percentage variation of the control ± S.D. of four independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Cell Function Assay, MTT Assay, Control, Wound Healing Assay, Software, Western Blot, Plasmid Preparation, Marker, Quantitative Proteomics

Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: c10orf118 silencing influences HA-related genes expression in MCF-7. Quantitative RT-PCR analyses of MCF-7 cells nucleofected for 48 h with 30 pmol of c10orf118 siRNA or a scrambled siRNA. The graphs represent the mRNA levels of ( A ) c10orf118, ( B ) HAS2, ( C ) the long non-coding RNA HAS2-AS1 and ( D ) the HA receptor CD44. Data are represented as mean ± S.D. of six independent experiments. ** p < 0.01, and *** p < 0.001.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: Specificity of c10orf118 effect on HAS2 synthesis by NHDF. ( A ) Quantitative RT-PCR analysis of HAS2 expression by NHDF co-cultured with MCF-7 alone or in the presence of an anti-c10orf118 polyclonal antibody or an anti-α-actin antibody as negative control. Each bar represents mean ± S.D. of triplicates. * p < 0.05. ( B ) Quantitative RT-PCR analysis of HAS2 expression by NHDF in the presence of increasing concentrations of a recombinant c10orf118 peptide (Hr_Q71-211). Bars represent mean ± S.D. of three replicates. * p < 0.05.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Negative Control, Recombinant

Journal: Cancers

Article Title: The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk

doi: 10.3390/cancers13051105

Figure Lengend Snippet: C10orf118 is highly expressed in breast tumour tissues that express ER and its level of expression is correlated with higher overall survival. ( A ) Quantitative analysis of c10orf118 expression normalized to actin as obtained from immunoblot of cancer patient specimens. ( B ) Kaplan-Meier survival plots obtained by http://kmplot.com/analysis/ (accessed date 20 January 2021) to evaluate the overall survival (OS), relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients in the database expressing high (red line) and low (black line) levels of the c10orf118 mRNA.

Article Snippet: The following human Taqman gene expression assays were used: HAS2 (Hs00193435_m1), HAS3 (Hs00193436_m1), HAS2-AS1 (Hs03309447_m1), CD44 (Hs01075861_m1), C10orf118 (Hs00215984_m1) and β-actin (Hs99999903_m1) as a housekeeping gene.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Identification of a P62-TIF-IA axis that drives nucleolar fusion and the senescence associated secretory phenotype

doi: 10.1101/2023.12.05.570133

Figure Lengend Snippet: (A to D) TIF-IA accumulation parallels nucleolar fusion and precedes NF-κB activation in oncogene-induced senescence (OIS). IMR90 ER:Ras cells were treated with 4-hydroxytamoxifen (4-OHT) for the times specified to induce oncogenic Ras expression. IMR90 ER:Stop cells serve as a control and retain proliferative capacity with 4 -OHT. (A) Immunomicrographs showing nucleolar (areas devoid of DAPI staining) accumulation of TIF-IA in ER:Ras cells 72hs post 4-OHT exposure. (B, C) Levels and localisation of TIF-IA were monitored in ER:Stop and ER:Ras cells exposed to 4-OHT in time course studies using (B) Western blot analysis, performed on whole cell extracts. See Fig S1C for quantification. N=3 (C) Immunocytochemistry. See Fig S1D for representative images of full time course. FIJI™ software was used to quantify nuclear TIF-IA intensity and nucleolar area (areas devoid of DAPI staining). At least five fields, minimum of 150 cells, were analysed per experiment. N=5. (D) qRT-PCR was used to monitor expression of early NF-κB target gene, NFKB1A (IκB), in ER:Stop and ER:Ras cells treated with 4-OHT for the times given. (E to I) Nucleolar accumulation parallels nucleolar fusion and precedes transcription of SASP factors in therapy-induced senescence (TIS). HTC116 cells were treated with etoposide (100uM) for the times specified in hours (h). (E and F) Immunocytochemistry was used to determine TIF-IA levels and localisation. (E) Representative immunomicrographs. (F) Cell Profiler™ was used to quantify the intensity of cytoplasmic and nuclear TIF-1A and the ratio for each cell determined. Five fields of view (minimum 80 cells) per treatment were quantified for each time point (n=3). (G) Representative phase-contrast images of live HCT116 cells treated with 100μM Etoposide for the times indicated ( x40 magnification). Nucleolar fusion is evident. (H) Nucleolar area was determined from phase contrast images using FIJI™ in at least 500 nuclei/treatment. (n=2). (I) Expression of the indicated NF-κB target genes was quantified using qRT-PCR. The bars represent the mean ± the standard error (n=3). (J) TIF-IA and N-Ras immunohistochemistry were performed on liver sections from mice culled 6 days after hydrodynamic delivery of Nras G12V/D38A ( n =5) or Nras G12V ( n =5) transposons. TIF-IA accumulation is observed in oncogenic Nras G12V expressing, but not control Nras G12V/D38A expressing, hepatocytes (left violin plot), while N-ras expression is similar (right violin plot). Top panel: representative images at 10X (scale bar 100uM) and 40X (scale bar 20uM) magnification. Ten regions of interest (ROIs) were randomly selected from each slide and the intensity of TIF-1A and N-Ras staining quantified using QuPath™ software (v0.2.3). The left violin plot shows the percentage of TIF-1A positive cells for each ROI, whereas the right plot exhibits NRas staining. There were 5 mice analysed from each group. Statistical significance throughout was determined using one-way ANOVA, with Tukey’s multiple comparison, Mann Whitney U test or Student’s t-Test, as appropriate. Ns-non-significant.

Article Snippet: A549 WT and A549-ATG5 -/- (ΔATG5) cells were a kind gift from Professor Simon Wilkinson (Institute of Genetics & Cancer, University of Edinburgh), whereas WT IMR90 fibroblasts and HCT116 colon cancer cells were obtained from the ATCC.

Techniques: Activation Assay, Expressing, Control, Staining, Western Blot, Immunocytochemistry, Software, Quantitative RT-PCR, Immunohistochemistry, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Identification of a P62-TIF-IA axis that drives nucleolar fusion and the senescence associated secretory phenotype

doi: 10.1101/2023.12.05.570133

Figure Lengend Snippet: A to F IMR90 ER:Stop or ER:Ras fibroblasts were pre-treated with two independent TIF-IA siRNA (SiTIF-1A#1 & SiTIF-1A#2) or a non-sense sequence (SiControl) prior to addition of 4-OHT for various times. (A) Schematic showing the treatment schedule for siRNA and 4-OHT delivery. (B) BrDU incorporation was used to assess cell proliferation. BrDU positive cells were stained by immunocytochemistry and quantified manually by fluorescent microscopy. At least 100 cells from at least 10 fields of view were analysed for each treatment group (n=2). (C) The percentage of cells with senescence associated heterochromatin foci (SAHF) (large, bright DAPI foci) was determined by microscopic analysis of the DAPI channel 120h after 4-OHT exposure, with a minimum of 200 cells counted manually per treatment for each replicate (n=2). (D) Anti-C23 (nucleolar marker) immunocytochemistry. Top panel: representative images from 4-OHT treated cells. Bottom panel: quantification of nucleolar area (measured as areas devoid of DAPI using FIJI™ software) for at least 50 cells per condition (n=2). (E) qRT-PCR demonstrating TIF-IA depletion inhibits Ras-induced transcription of SASP factors. Induction in ER:Ras siControl cells was compared to that in ER:Stop SiControl cells. Expression in ER:Ras, TIF-IAsiRNA treated cells is given as a percentage of the induction in SiControl cells for each gene and expressed as the mean ± SEM (n=3). (F) IMR90 cells were treated with 4-OHT for 8 days and the effect of TIF-1A knockdown on senescence was determined. Top:Typical fields of view after treatment. Below: the percentage of β-Galactosidase positive cells was quantified in 10 fields of view per replicate by light microscopy (n=3). (G to I) HCT116 cells were transfected with control or two independent TIF-IA siRNAs (#siTIF-IA 1, #siTIF-IA 2) prior to treatment with 100uM Etoposide for 48h (G and H) or 72h (I). Depletion of TIF-1A mRNA was assessed by qRT-PCR (H). (G) Live images were acquired as in and nucleolar area determined using FIJI™ software. At least 125 nucleoli per treatment were assessed from 3 individual replicates. (H) Expression of TIF-IA, IκB (NF-KB1A) and the SASP factors, IL-1α and IL-8 was quantified by qRT-PCR. Bars represent the relative expression compared to etoposide siControl +/-SE (n=3). (G) β-galactosidase assays were performed on etoposide treated cells. The percentage of senescent cells (indicated by blue stain) was determined by light microscopy. Ten fields of view, at least 50 cells/field, were counted for each experimental condition. N=3. (J) IMR90 ER:Ras cells were transfected with pEGFP-C1 or pEGFP-TIF-IA, treated for 5 days with DMSO or 4OHT then immunocytochemistry performed for the nucleolar marker, C23 (inset shows example immunomicrograph). Nucleolar size was quantified using FIJI™ software and area of C23 stain. 33-100 cells were imaged per replicate (n=3). (K) HCT116 cells were transfected with eGFP-C1 or eGFP-TIF-IA. qRT-PCR was used to determine the levels of the given NF-κB target genes 72h later. GFP expression (relative to GAPDH) was used to normalise for transfection efficiency. Statistical significance throughout was determined using either one-way ANOVA, with Tukey’s multiple comparison, Mann Whitney or Student’s t-Test, dependent on normality.

Article Snippet: A549 WT and A549-ATG5 -/- (ΔATG5) cells were a kind gift from Professor Simon Wilkinson (Institute of Genetics & Cancer, University of Edinburgh), whereas WT IMR90 fibroblasts and HCT116 colon cancer cells were obtained from the ATCC.

Techniques: Sequencing, BrdU Incorporation Assay, Staining, Immunocytochemistry, Microscopy, Marker, Software, Quantitative RT-PCR, Expressing, Knockdown, Light Microscopy, Transfection, Control, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Identification of a P62-TIF-IA axis that drives nucleolar fusion and the senescence associated secretory phenotype

doi: 10.1101/2023.12.05.570133

Figure Lengend Snippet: (A) TIF-IA accumulation is associated with reduced 47S transcription in IMR90 cells. qRT-PCR was used to monitor 47S transcription in ER:Stop and ER:Ras cells treated with 4OHT for the times given. Each point represents a technical replicate (n=3) (B) Depleting TIF-IA alone reduced 47S transcription but has no effect on transcription of SASP factors. siRNA was used to deplete TIF-IA in ER:STOP cells as in then qRT-PCR used to monitor expression of the given NF-κB target genes 144h later. Each point represents a technical replicate (n=5) (C) qRT-PCR was used to monitor 47S transcription in IMR90 (n=3) and HCT116 (n=4) cells treated with the given concentrations of etoposide for 48h. Right: Representative immunomicrographs showing nucleolar accumulation of TIF-IA. Statistical significance throughout was determined using a students T test.

Article Snippet: A549 WT and A549-ATG5 -/- (ΔATG5) cells were a kind gift from Professor Simon Wilkinson (Institute of Genetics & Cancer, University of Edinburgh), whereas WT IMR90 fibroblasts and HCT116 colon cancer cells were obtained from the ATCC.

Techniques: Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Identification of a P62-TIF-IA axis that drives nucleolar fusion and the senescence associated secretory phenotype

doi: 10.1101/2023.12.05.570133

Figure Lengend Snippet: (A) The levels of TIF-1A transcription were investigated by qRT-PCR in IMR90 ER:Stop and ER:Ras cells exposed to 4-OHT for the times indicated in hours (n=3). (B) HCT116 cells were treated with DMSO (control) or etoposide for 8h. Whole cell lysates were immunoprecipitated with IgG or TIF-IA antibody then precipitated proteins analysed by quantitative mass spectrometry. Volcano plot shows a comparison of IgG v TIF-IA for DMSO treated cells. Blue dot=non-significant. Red dot=significant change (fold change (FC) >2, P<0.05). (C) Immmunomicrograph showing p62-TIF-IA co-localisation in cytoplasmic foci in IMR90 cells. (D) Top: Immunomicrograph of IMR90 cells treated with the lysosome inhibitor, bafilomycin A (BafA1) or DMSO carrier. Bottom: Cell profiler was used to quantify cytoplasmic and nuclear TIF-IA intensity. A minimum of 50 cells were analysed/treatment for each biological repeat. N=2. (E) Representative immunomicrographs (63X) showing level and localisation of TIF-IA and p62 in wild type (WT) A549 cells and those in which the autophagy gene, atg5, has been deleted (ΔATG5). White arrow indicates co-localised foci in WT cells. N=5. (F) Immunoblot performed on whole cell extracts from WT and Δatg5 cells. n=5 (G) Immunoblot showing levels of TIF-IA and p62 in whole cell lysates following siRNA depletion of p62. n=2. (H) TIF-IA was immunoprecipitated (IP) from WT or Δatg5 A549 cells then recovered proteins analysed by immunoblot (IB) for p62. Stripped gels were re-probed for TIF-IA. Input levels of protein are shown. Rabbit IgG acts as a control. n=3 (I) P62 binds to a high molecular weight (HMW) form of TIF-IA in a manner dependent on the dimerization and ubiquitin binding domains. Wild type and Δatg5 A549 cells were transfected with plasmids expressing GFP-P62 WT, GFP-p62 ΔPB1 (deleted for the PB1 domain required for dimerization) or GFP-p62ΔUBA (deleted for the ubiquitin binding domain). GFP-tagged proteins were immuoprecipitated (IP) from whole-cell lysates using GFP-TRAP beads. Precipitated proteins were subjected to immunoblotting (IB) for TIF-IA and GFP. Input levels of TIF-IA are shown. N=2. Actin is used as a loading control throughout.

Article Snippet: A549 WT and A549-ATG5 -/- (ΔATG5) cells were a kind gift from Professor Simon Wilkinson (Institute of Genetics & Cancer, University of Edinburgh), whereas WT IMR90 fibroblasts and HCT116 colon cancer cells were obtained from the ATCC.

Techniques: Quantitative RT-PCR, Control, Immunoprecipitation, Mass Spectrometry, Comparison, Western Blot, High Molecular Weight, Ubiquitin Proteomics, Binding Assay, Transfection, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: The PI3K inhibitor pictilisib and the multikinase inhibitors pazopanib and sorafenib have an impact on Rac1 level and migration of medulloblastoma in vitro

doi: 10.1111/jcmm.17604

Figure Lengend Snippet: Medulloblastoma cells of different subgroups reveal distinct actin cytoskeletal features, nuclear shape, and differential distribution of Rac1. (A) Actin skeleton of Daoy and MEB‐Med‐8A cells was stained with Alexa Fluor‐555 coupled phalloidin 24 h after seeding and imaged by fluorescence microscopy (40x objective). Grey scale images were inverted to show phalloidin‐staining (actin) in black and merged with RGB images showing nuclei (DAPI counterstain) in red. Dashed white lines outline nuclear shapes displaying a characteristic deformation of the nucleus in MEB‐Med‐8A cells. Actin cytoskeleton was arranged in a prominent circular structure underlying the cellular cortex in Daoy cells or in distinct perinuclear accumulations in MEB‐Med‐8A cells (black arrowheads, detail view) situated in the characteristic nuclear indentation. Pictures are representative of three independent experiments. Scales indicate 50 μm. (B) Microfilaments (actin, red), nucleus (DAPI, blue) and Rac1 (green) were stained in medulloblastoma cell lines and Z‐stacks were prepared by means of confocal laser‐scanning microscopy with a 40x water immersion objective (NA 1.15). The extended focus pictures show subcellular distribution of Rac1 with respect to actin filaments. Rac1 is seen in the cytoplasm in both cell lines and additionally localizes to the cortical network at the plasma membrane in Daoy cells, while in MEB‐Med‐8A cells Rac1 is located to the microfilament accumulation in close vicinity to the nucleus but does not seem to be present at high concentrations at the cell boundary. Scales indicate 20 μm. (C) Daoy and MEB‐Med‐8A cells were stained for Rac1 (grayscale) and DAPI (red) and imaged by confocal laser‐scanning microscopy as described in (B). Extended focus pictures show the subcellular distribution of Rac1 in the two different cell lines with respect to the nucleus and cytoplasm. Detailed views (inserts) in grayscale and 16‐colour lookup tables (LUT) illustrate that the major proportion of Rac1 is in a ring like structure around the nucleus and thinned out towards the cellular border. In MEB‐Med‐8A cells the maximum signal of Rac1 was captured in an aggregation equivalent to the actin accumulation and situated close to the nucleus or within the nuclear indentation characteristic of MM8A. Scales indicate 50 μm. (D) Subcellular fractions of medulloblastoma cells were analysed by immunoblot to assess the presence of Rac1 in the cytoplasmatic and nuclear compartment of both cell lines. Either fraction shows positive signals for Rac1 upon detection using a Rac1 specific antibody (BD Biosciences). The expected molecular weight (21 kDa) is confirmed by the molecular weight marker shown in the middle lane (kDa). Results are representative of three independent experiments.

Article Snippet: The medulloblastoma cell line Daoy (HTB 186) was obtained from ATCC and the medulloblastoma cell line MEB‐Med‐8A was kindly provided by Prof. T. Pietsch, University of Bonn.

Techniques: Staining, Fluorescence, Microscopy, Confocal Laser Scanning Microscopy, Clinical Proteomics, Membrane, Western Blot, Molecular Weight, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: The PI3K inhibitor pictilisib and the multikinase inhibitors pazopanib and sorafenib have an impact on Rac1 level and migration of medulloblastoma in vitro

doi: 10.1111/jcmm.17604

Figure Lengend Snippet: PI3K‐ and multikinase inhibition alter the actin cytoskeleton and interfere with migration of Daoy cells. (A) Daoy cells were seeded and Pictilisib (GDC‐0941, 1 μM), Pazopanib (15 μM), and Sorafenib (10 μM) were applied for 24 h. Cells were stained with Alexa Fluor‐555 coupled phalloidin to visualize the actin cytoskeleton (grayscale, inverted) and DAPI as nuclear counterstain (red). Images were captured using a fluorescence microscope (40x objective) and processed in ImageJ. Data are representative of two independent experiments. Scales indicate 25 μm. (B) A confluently grown monolayer of Daoy cells was scratch wounded, debris was gently washed off and fresh medium including kinase inhibitors was applied. After 24 h the actin cytoskeleton was stained with Alexa Fluor‐555 coupled phalloidin (grayscale, inverted) and DAPI as nuclear counterstain (red). Fluorescence microscopy was performed using a 10x objective. Two images of representative staining from two independent experiments are presented except for Sorafenib. In the latter we focused on showing a larger scratch border to better present elongated cells. Scales indicate 50 μm. (C) Scratches were performed as described above and treated with kinase inhibitors. After incubation for 0, 6, 12, 24, and 30 h non‐stained living cells were imaged using an inverted microscope in phase contrast taking care to image exactly the same scratch region. The kinetic of the scratch closure was analysed using NIS imaging software by measuring the size of the area of the cell free zone in each distinct scratch region after the indicated time interval. Results are shown in comparison to the untreated cells. All values below an asterisk show significantly lower scratch closure compared with the DMSO control group (* p < 0.05). The data represent the mean ± SD of two independent experiments. (D and E) Daoy cells were seeded in 6‐well plates and inhibitors were applied. The fraction of PI‐positive dead cells (D) and number of viable cells (E) was assessed after 6, 12 and 24 h by flow cytometry. All values below an asterisk show significantly changed cell survival compared with the DMSO control group (* p < 0.05). The data shown represent the mean ± SD of three independent experiments. (F) Daoy cells were treated with inhibitors for 24 h and cell lysates were prepared followed by SDS‐PAGE and immunoblot to assess total protein levels of (p‐)Akt and (p‐)Erk with GAPDH as loading control. The same GAPDH bands are shown in Figure since they were part to the same experiment. The data shown are representative of two independent experiments.

Article Snippet: The medulloblastoma cell line Daoy (HTB 186) was obtained from ATCC and the medulloblastoma cell line MEB‐Med‐8A was kindly provided by Prof. T. Pietsch, University of Bonn.

Techniques: Inhibition, Migration, Staining, Fluorescence, Microscopy, Incubation, Inverted Microscopy, Imaging, Software, Comparison, Control, Flow Cytometry, SDS Page, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: The PI3K inhibitor pictilisib and the multikinase inhibitors pazopanib and sorafenib have an impact on Rac1 level and migration of medulloblastoma in vitro

doi: 10.1111/jcmm.17604

Figure Lengend Snippet: Levels of activated GTP‐bound Rac1 are reduced after PI3K blockage and inhibition of tyrosine kinases. (A and B). Confluently grown monolayers of Daoy or MEB‐Med‐8A cells were scratch wounded and treated with Pictilisib (GDC‐0941), Pazopanib or Sorafenib. After 12 h (MEB‐Med‐8A) or 24 h (Daoy) cells were fixed and stained with a Rac1 antibody to assess relative subcellular distribution of Rac1 at the scratch border. Scratch areas were imaged by confocal laser scanning microscopy using a 40x water immersion objective. Z‐stacks were performed to analyse Rac1 signals from top to bottom of the cells in 1024 × 1024 resolution using Galvano mode for the 488 laserline. Extended focus pictures were prepared using ImageJ software and show Rac1 distribution (grayscale) in the cells at the scratch border. Pictures presented are representative for two individual experiments. Scales indicate 50 μm. (B and D) Lysates of drug treated MEB‐Med‐8A (12 h) or Daoy (24 h) cells were prepared and a pulldown of GTP‐bound Rac1 was performed with a PAK PBD‐1 fusion protein followed by immunoblot analysis of Rac1. From the same lysates SDS‐PAGE followed by immunoblot to Rac1 were prepared to analyse the level of total cytoplasmatic Rac1. Densitometry analysis was performed using ImageJ 1.53c software. Signals of GTP‐bound Rac1 and total Rac1 levels were determined, and levels were compared with untreated control cells. Levels of total Rac1 were normalized to GAPDH as a loading control. The same GAPDH bands are shown in Figures and since the same blotted membranes were used in both experimental parts to determine expression levels of the respective proteins in relation to GAPDH of the same membrane. The data shown are representative of two independent experiments for the pulldown and four independent experiments for total cytoplasmatic Rac1 levels.

Article Snippet: The medulloblastoma cell line Daoy (HTB 186) was obtained from ATCC and the medulloblastoma cell line MEB‐Med‐8A was kindly provided by Prof. T. Pietsch, University of Bonn.

Techniques: Inhibition, Staining, Confocal Laser Scanning Microscopy, Software, Western Blot, SDS Page, Control, Expressing, Membrane