Journal: International Journal of Molecular Sciences

Article Title: Polysialic Acid Sustains the Hypoxia-Induced Migration and Undifferentiated State of Human Glioblastoma Cells

doi: 10.3390/ijms23179563

Figure Lengend Snippet: NCAM and PSA expression in different GBM cell cultures and hypoxic areas of GBM tissues. ( A ) Western blot qualitative analysis showing NCAM isoforms and PSA expression levels in a mouse glioma cell line (GL261) and four human GBM cell lines (U251, A172, U373, U87-MG) compared to normal neonatal rat astrocytes, included as positive control. ( B ) Real-time PCR analysis showing mRNA expression of polysialyltransferases II and IV (ST8SiaII and ST8SiaIV) in low grade (GL18-1 and GL18-3) and high grade (GL18-2, GL18-4, GL18-5, GL18-7, GL18-15) glioma patient-derived primary cultures compared to U87-MG and U251 GBM cell lines and a brain metastasis. ( C ) Immunohistochemical analysis showing the expression of NCAM, PSA and HIF-1α in formalin-fixed, paraffin embedded sections of a GBM tissue compared to a normal, non-tumoral brain.

Article Snippet: Proteins (20 μg) were resolved on 8% SDS-PAGE and immunoblotted with the following antibodies: anti-PSA (MBS488177, MyBioSource—dilution 1:1000) and anti-NCAM (AB-90239, Immunological Sciences—dilution 1:1000).

Techniques: Expressing, Western Blot, Positive Control, Real-time Polymerase Chain Reaction, Derivative Assay, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded

Journal: International Journal of Molecular Sciences

Article Title: Polysialic Acid Sustains the Hypoxia-Induced Migration and Undifferentiated State of Human Glioblastoma Cells

doi: 10.3390/ijms23179563

Figure Lengend Snippet: PolySTs, NCAM and PSA expression in U87-MG cells under chronic hypoxia. ( A ) Real-Time PCR analysis showing the time-course (0–72 h) of mRNA expression of polysialyltransferases II and IV (ST8SiaII and ST8SiaI(V) in U87-MG cells exposed to low oxygen concentrations. ( B ) Western blot analysis showing the time-course (0–72 h) of HIF-1α activation, NCAM and PSA expression in U87-MG cells under hypoxia. ( C ) Cytofluorimetric analysis showing the extracellular expression of NCAM (CD56) in U87-MG cells exposed for 72 h either to chronic hypoxia (red histogram) or normoxia (blue histogram). ( D ) Cytofluorimetric analysis showing the extracellular expression of PSA in U87-MG cells exposed for 72 h either to chronic hypoxia (light green histogram) or normoxia (green histogram). All results are expressed as the mean ± SD of three independent experiments. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Article Snippet: Proteins (20 μg) were resolved on 8% SDS-PAGE and immunoblotted with the following antibodies: anti-PSA (MBS488177, MyBioSource—dilution 1:1000) and anti-NCAM (AB-90239, Immunological Sciences—dilution 1:1000).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Polysialic Acid Sustains the Hypoxia-Induced Migration and Undifferentiated State of Human Glioblastoma Cells

doi: 10.3390/ijms23179563

Figure Lengend Snippet: Effects of F-NANA on the proliferation, chemoresistance to TMZ and levels of NCAM and PSA in U87-MG cells under hypoxia. ( A ) Western blot analysis showing the expression of NCAM and PSA in U87-MG cells grown either under normal or low oxygen concentrations, in presence or not of 100 μM F-NANA. ( B ) MTS assay showing the proliferation (expressed as absorbance at 492 nm) of U87-MG cells exposed for 72 h either to chronic hypoxia or normoxia, in presence or not of 100 μM F-NANA. ( C ) Viability evaluation in U87-MG cells grown either in normoxia or hypoxia and treated with 200 μM TMZ, in presence or not of 100 μM F-NANA. All results are expressed as the mean ± SD of three independent experiments. *: p < 0.05; ***: p < 0.001.

Article Snippet: Proteins (20 μg) were resolved on 8% SDS-PAGE and immunoblotted with the following antibodies: anti-PSA (MBS488177, MyBioSource—dilution 1:1000) and anti-NCAM (AB-90239, Immunological Sciences—dilution 1:1000).

Techniques: Western Blot, Expressing, MTS Assay

Journal: International Journal of Molecular Sciences

Article Title: Polysialic Acid Sustains the Hypoxia-Induced Migration and Undifferentiated State of Human Glioblastoma Cells

doi: 10.3390/ijms23179563

Figure Lengend Snippet: Effect of PSA inhibition on the migration of GBM cells under hypoxic conditions. ( A ) Transwell migration assay showing the number of migrated U87-MG cells under normal and low oxygen concentration, in presence or not of 100 μM F-NANA. ( B ) Western blot analysis showing the expression of NCAM and PSA in a patient-derived GBM primary culture (GL18-15) exposed to 72 h chronic hypoxia, in presence or not of 100 μM F-NANA. ( C ) Transwell migration assay showing the percentage versus control of migrated GL18-15 cells under normal and low oxygen concentration, in presence or not of 100 μM F-NANA. Results are presented as the mean ± SD of three independent experiments. **: p < 0.01; ***: p < 0.001.

Article Snippet: Proteins (20 μg) were resolved on 8% SDS-PAGE and immunoblotted with the following antibodies: anti-PSA (MBS488177, MyBioSource—dilution 1:1000) and anti-NCAM (AB-90239, Immunological Sciences—dilution 1:1000).

Techniques: Inhibition, Migration, Transwell Migration Assay, Concentration Assay, Western Blot, Expressing, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Polysialic Acid Sustains the Hypoxia-Induced Migration and Undifferentiated State of Human Glioblastoma Cells

doi: 10.3390/ijms23179563

Figure Lengend Snippet: PolySTs, NCAM and PSA expression during U87NS cell differentiation. ( A ) Phase-contrast micrographs showing floating U87NS differentiated in medium supplemented with 10% FBS for 5 days. Images were acquired at day 0, 1, 3 and 5. ( B ) Real-time PCR analysis of ST8SiaII and IV mRNAs expression in U87NS differentiated for 5 days in medium supplemented with 10% FBS. ( C ) Western blot analysis showing the levels of NCAM and PSA in U87NS differentiated for 5 days in medium supplemented with 10% FBS. ( D ) Western blot analysis of NCAM and PSA expression in U87NS differentiated for 3 days in medium supplemented with 10% FBS, in presence or not of 100 μM F-NANA. Results are presented as the mean ± SD of three independent experiments. 1d: day 1; 3dd: day 3; 5dd: day 5. **: p < 0.01; ***: p < 0.001.

Article Snippet: Proteins (20 μg) were resolved on 8% SDS-PAGE and immunoblotted with the following antibodies: anti-PSA (MBS488177, MyBioSource—dilution 1:1000) and anti-NCAM (AB-90239, Immunological Sciences—dilution 1:1000).

Techniques: Expressing, Cell Differentiation, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Polysialic Acid Sustains the Hypoxia-Induced Migration and Undifferentiated State of Human Glioblastoma Cells

doi: 10.3390/ijms23179563

Figure Lengend Snippet: F-NANA reduces the levels of PSA in brain tissues from wild-type mice. Representative cropped Western blottings and densitometric analysis of PSA ( A ) and NCAM ( B ) levels in brain tissues from vehicle- and F-NANA-treated wild-type mice at 7 weeks of age. Data are represented as mean ± SD, N = 5 for each group of mice. *, p < 0.05; (Unpaired t -test). Representative cropped Western blottings and densitometric analysis of SNA ( C ) and MAL II ( D ) levels in brain tissues from the vehicle- and F-NANA-treated wild-type mice at 7 weeks of age. Data are represented as mean ± SD, N = 5 for each group of mice. ( E ) Mouse body weight measured at the end of the treatment. Values are represented as mean ± SD. ( N = 5 for each group of mice). (Unpaired t -test). ( F ) Motor performance assessed by Rotarod test. Values are represented as mean ± SD. ( N = 5 for each group of mice). (Unpaired t -test).

Article Snippet: Proteins (20 μg) were resolved on 8% SDS-PAGE and immunoblotted with the following antibodies: anti-PSA (MBS488177, MyBioSource—dilution 1:1000) and anti-NCAM (AB-90239, Immunological Sciences—dilution 1:1000).

Techniques: Western Blot

Journal: Oncotarget

Article Title: Identification of novel genes that regulate androgen receptor signaling and growth of androgen-deprived prostate cancer cells

doi:

Figure Lengend Snippet: A. LNCaP cells were transfected with control or IGSF8-targeting siRNAs (5 nM) and cultured in hormone-free conditions for 6 days. Expression of AR and PSA was analyzed by immunoblotting. Quantification of AR signal intensity normalized to GAPDH signal is shown on the right, relative to AR level of untransfected LNCaP cells (mean of 3 independent experiments +/– SD). B. LNCaP were transfected as above, followed by fixation and immunostaining with DAPI and anti-AR antibodies. Images were taken and nuclear staining was quantified. The results represent average quantification of the AR to DAPI ratio of fluorescence intensity (30 nuclei per siRNA) +/– SD. C. LNCaP cells were transfected as in A and cultured in hormone-free conditions for 6 days in the presence of 10 μM of Enzalutamide or DMSO. Gene expression was analyzed by QPCR. The results represent mean of 3 independent experiments +/– SD.

Article Snippet: The following antibodies were used: rabbit anti AR (, H-280, sc-13062, Santa Cruz, CA), rabbit anti PSA (K92110R, BioDesign Saint-Priest, France) and mouse anti GAPDH (6C5, sc-32233, Santa Cruz, CA).

Techniques: Transfection, Control, Cell Culture, Expressing, Western Blot, Immunostaining, Staining, Fluorescence, Gene Expression