rabbit  (Alomone Labs)

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: ASIC3 is expressed in almost all CSCs and can be activated by GMQ. ( a ) Immunofluorescence image of GBM CSCs MES 0315 in culture stained for ASIC3 (red) and for hoechst (blue). Arrowhead points to an ASIC3 negative cell. ( b ) Immunofluorescence image of GBM CSCs MES 0315 in culture stained for CD133/Prominin1 (green) and for hoechst (blue). ( c ) Immunofluorescence image of GBM CSCs MES 0315 in culture stained for Ki67 (green) and for hoechst (blue). ( d ) Histogram showing the percentage of positive cells for ASIC3, CD133 and Ki67 n ≥ 3 independent experiments. The graph was made with GraphPad Prism v9 ( e ) High magnification image of ASIC3 (left), hoechst (middle) and double staining (right), arrowheads point to a membrane enriched ASIC3 staining. ( f ) Representative traces of a GMQ-induced current in GBM-MES 0315 cells in the presence or absence of APETx2 (black and red traces respectively). ( g ) Box plot of membrane current density upon stimulation with GMQ alone (red) or in the presence of APETx2 (black). Plot displays the mean (+), median (internal horizontal line), first and third quartiles (upper and lower box edges) and standard deviation (whiskers) of data distribution (n = 11). Dots represent individual cells. Significant difference was assessed by Mann–Whitney non-parametric test (****p < 0.0001). Scale Bar = 20 µm for ( a–c ) and 10 µm for ( e ). All data were analysed by using the Prism 8 (GraphPad) software and traces were generated by Origin 2018 (Origin Lab.).

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Double Staining, Membrane, Standard Deviation, MANN-WHITNEY, Software, Generated

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: The ASIC3 isoform is enriched in glioblastoma CSCs. ( a ) Western blot analysis of ASIC3 and ASIC1a expression in human primary GBM-CSC lines from the three GBM subtypes ( PN proneural, MES mesenchymal, CL classical). ASIC3 (n ≥ 3) ( b ) and ASIC1a (n = 3) ( b ’) protein expression quantification normalized on actin levels in the different GBM-CSC lines. ( c ) Western Blot analysis of CD133/prominin1 and ASIC3 expression in a healthy human brain lysate, in the GBM-MES CSCs 0315 line and in a biopsy from the same patient used to generate the CSC line 0315. ( c ’) ASIC3 and CD133/prominin1 protein expression quantification normalized for actin levels, in healthy human brain, GBM MES 0315 line and MES 0315 patient’s biopsy (n = 4). Statistic was performed by a two-way Anova with Bonferroni’s post-test:(****) p < 0,0001. ( d ) Data from publically available Repository for Molecular BRAIN Neoplasia Data (TCGA datasets Affymetrix HT HG U133A) were used to determine the relative expression of ASIC3 mRNA (ACCN3) in four human glioma subtypes (Classical, Mesenchymal, Neuronal, and Proneuronal), as well as in healthy patients. All 454 glioma patients with available mRNA were examined. ( e ) Western Blot analysis of ASIC3 expression in GBM-CSCs MES 0315 growth in 3D and 2D culture conditions after 24hin culture. Original blots were horizontally cropped at the appropriate MW region in order to perform multiple staining on the same blot. In the figure, portions of the same blot are separated by white lines. Black lines are used to separate independent blots presented in the same figure. Original blots are shown in supplementary figure S1 and S4. Dots represent independent experiments. Measurements were taken from distinct samples. Graphs for ( b,b’,c’ ) were analyzed with GraphPad Prism 9, graph for ( d ) was obtained from https://www.betastasis.com/glioma/tcga_gbm/ .

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: Western Blot, Expressing, Staining

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: Generation of a virtual 3D model of the human ASIC3 channel. ( a ) AlphaFold model of human ASIC3 represented as cartoons colored by model quality (from low quality pLDDT = 50 in blue, to high quality pLDDT = 100 in red); inset: close up view of the extracellular domain of ASIC3, highlighting with spheres amino acids Glu78 and Glu421, known to be involved in GMQ binding . ( b ) Superposition of the two structural models (depicted as cartoon) selected after MD simulation using cluster analysis: 682 (68.2 ns) as blue and 812 (81.2 ns) as cyan and extent of the region explored during in silico docking analysis. ( c,d) Close-up views of the interaction between charged (yellow carbon atoms) and non-charged (orange carbons) GMQ with protein residues [sticks with carbon atoms colored in cyan (812) or blue (682)].

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: Binding Assay, In Silico

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: In silico docking analysis .

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: In Silico

Journal: Scientific Reports

Article Title: Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

doi: 10.1038/s41598-024-71623-9

Figure Lengend Snippet: ASIC3 downregulation and its pharmacological blocking reduces the effect of GMQ. ( a ) Blot analysis of ASIC3 expression in GBM CSCs MES 0315 cells after infection with lentiviruses carrying specific anti-ASIC3 shRNAs (shRNA1 and shRNA2). Original blots were horizontally cropped at the appropriate MW region in order to perform multiple staining on the same blot. Portions of the same blot are separated by white lines. ( a ’) ASIC3/Actin protein expression ratio of MES 0315 before and after infection with Lentiviruses carrying shRNA1 or shRNA2 (n = 7) and normalized on control samples. CTRL vs shRNA1: P = 0,0007; CTRL vs shRNA2 P = : 0,0007. Original blots are shown in supplementary figure S9a and S9b. ( b ) Effect of ASIC3 downregulation on GMQ-induced GBM CSCs growth. Plot represents the number of MES 0315 cells after 96 h of GMQ treatments (3 and 10 μM), normalized to untreated samples: control (n = 4); shRNA1 (n = 3); shRNA2 (n = 3). (****) P < 0,0001. ( c ) Plot represents the % of GBM CSCs MES 0315 after 96 h of 3 μM GMQ treatment, in the presence or absence of 1 μM APETx2 toxin, normalized to the untreated sample, (n = 3); Unt vs GMQ p = 0.0214; Untr vs APETX2 + GMQ p = 0.9058; APETX2 vs GMQ p = 0.0189; GMQ vs APETX2 + GMQ p = 0.0029. Unpaired T-test for ( a ’), two-way Anova with Bonferroni’s post-test for ( b ) and One-way Anova with Bonferroni’s post-test for ( c ). Dots represent independent experiments. All graphs were analysed with GraphPad Prism v9.

Article Snippet: The coverslips were incubated with either an anti-human ASIC3 (1:500, Cat: ASC-018; Alomone Labs, Jerusalem, Israel), an anti-human CD133 (1:800, Cat: D4 W4N; Cell Signaling Technology, Denvers, MA, USA) or an anti-human Ki67 (1:250, Cat: ab16667, Abcam, Cambridge, UK) primary antibody overnight at 4 °C.

Techniques: Blocking Assay, Expressing, Infection, Staining, Control