Journal: Autophagy

Article Title: Inhibition of WNT-CTNNB1 signaling upregulates SQSTM1 and sensitizes glioblastoma cells to autophagy blockers

doi: 10.1080/15548627.2017.1423439

Figure Lengend Snippet: WNT-CTNNB1 signaling transcriptionally upregulates SQSTM1 in GBM. (A) GBM cell lines were treated with WNT3A (100 ng/ml) and FH535 (10 µM) for 24 h. SQSTM1 mRNA levels were analyzed by qPCR and data presented as fold induction vs. control normalized to GAPDH. Values are mean ± s.e.m of triplicate measurements of 3 independent experiments. (B) SQSTM1 immunoblotting analysis in A172, U251-MG and U87-MG GBM cell lines and C17 and C65 primary GBM cultures treated with FH535 (10 µM, 24 h). ACTB was used as a loading control. (C) TCF4 and SQSTM1 mRNA levels were examined by qPCR in control (scrambled shRNA; Scr) or TCF4-silenced (shRNA TCF4) U251-MG and U87-MG cell lines. (D) SQSTM1 protein levels in control cells (Scr) or TCF4-silenced GBM cell lines. (E and F) A172, U251-MG and U87-MG cells were transfected with CTNNB1 or control (GAPDH) siRNAs (E) or with WT and S37Y CTNNB1 plasmids (F) and SQSTM1 and CTNNB1 protein levels were analyzed after 48 h. *p<0.05, **p<0.01 and ***p<0.001

Article Snippet: GBM cell lines A172, U251-MG and U87-MG were obtained from CLS Cell Lines Service (300108, 300385 and 300367, respectively).

Techniques: Western Blot, shRNA, Transfection

Journal: Autophagy

Article Title: Inhibition of WNT-CTNNB1 signaling upregulates SQSTM1 and sensitizes glioblastoma cells to autophagy blockers

doi: 10.1080/15548627.2017.1423439

Figure Lengend Snippet: Inhibition of WNT-CTNNB1 signaling increases the autophagic flux in GBM. (A) Total cell lysates from A172, U251-MG and U87-MG cells untreated or incubated with FH535 (10 µM, 24 h) were immunoblotted for MAP1LC3A/B and ubiquitinated (Ub) proteins. (B and C) U251-MG and U87-MG GBM cell lines and C17 and C65 primary GBM cultures (B) were treated with FH535 as above (B) or with 50 ng/ml DKK1 (C) for 24 h plus Baf (5 nM) for the last 2 h. Cell lysates were immunoblotted for MAP1LC3A/B. Values indicate the fold increase of MAP1LC3A/B-II vs. untreated control cells. ACTB was used as a loading control. (D) Representative images from U87-MG cells transfected with the ptfLC3 plasmid and treated with the indicated drugs. Pictures correspond to merge of red and green channels. Cells with red and green colocalizing (yellow) fluorescent MAP1LC3A/B puncta show autophagosomes, whereas red-only fluorescent MAP1LC3A/B dots indicate autolysosomes. Bar: 50 µm. The numbers of green and red puncta/cell were calculated and shown as a bar graph (note that whereas in CQ there is a balance between red and green puncta, the other treatments show a marked increase of red dots). Data are mean ± s.e.m of at least 40 different cells from 10 different fields from 3 independent experiments. *** p<0.001.

Article Snippet: GBM cell lines A172, U251-MG and U87-MG were obtained from CLS Cell Lines Service (300108, 300385 and 300367, respectively).

Techniques: Inhibition, Incubation, Transfection, Plasmid Preparation

Journal: Autophagy

Article Title: Inhibition of WNT-CTNNB1 signaling upregulates SQSTM1 and sensitizes glioblastoma cells to autophagy blockers

doi: 10.1080/15548627.2017.1423439

Figure Lengend Snippet: Inhibition of TCF diminishes MTOR signaling and promotes TFEB nuclear translocation. (A) U251-MG and U87-MG cells were treated with 10 or 20 µM FH535 or 5 µM rapamycin for 24 h. Western blots for phosphorylated RPS6KB (Thr389), EIF4E (Ser209), total RPS6KB, total EIF4E and ATF4 levels were analyzed as MTOR targets. Phosphorylated AKT (Ser473) and MAPK (Tyr202/204) were analyzed and compared to total AKT and MAPK. ACTB was used as a loading control. Plots represent the quantification of phosphorylated RPS6KB, phosphorylated EIF4E and ATF4 levels normalized vs. ACTB (shown as percent of the control; *p <0.05 and *** p<0.001; n≥3). (B) Immunoblot for TFEB from U251-MG, U87-MG and A172 untreated cells, cells treated with FH535 or deprived of serum for 24 h. The TFEB upper band corresponds to the phosphorylated/inactive TFEB form, whereas the lower band corresponds to dephosphorylated and active TFEB, as indicated by the deprivation condition. Small panels show A172 cell lysates treated with or without AP, demonstrating the TFEB band shift towards the lower band in the presence of the enzyme. ACTB was used as a loading control. The plot represents the quantification of the dephosphorylated:phosphorylated TFEB ratio (lower:upper band) (*p <0.05 and **p<0.01; n≥3). (C) TFEB immunostaining (green) overlapped with Hoechst staining (blue) in U251-MG and U87-MG cell lines and C65 primary GBM cells, after treatment with FH535 or serum deprivation and compared to control cells. Note that in all conditions except controls (where TFEB immunostaining appears perinuclear; arrows), TFEB immunostaining is predominantly nuclear and colocalizes with Hoechst. Bar: 50 µm. Single-channel TFEB pictures can also be found Figure S4. (D) Quantification of nuclear TFEB immunostaining in U251-MG, U87-MG and C65 GBM cells treated as indicated. *** p<0.001.

Article Snippet: GBM cell lines A172, U251-MG and U87-MG were obtained from CLS Cell Lines Service (300108, 300385 and 300367, respectively).

Techniques: Inhibition, Translocation Assay, Western Blot, Electrophoretic Mobility Shift Assay, Immunostaining, Staining

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Xanthone-1,2,4-triazine and Acridone-1,2,4-triazine Conjugates: Synthesis and Anticancer Activity.

doi: 10.3390/ph16030403

Figure Lengend Snippet: Figure 3. Comparison of the proportion of apoptotic (Annexin V+/PI-) and other dead cells (PI+) in glioblastoma A-172 (A) and human embryonic kidney HEK-293 (B) cultures, mean ± CI-95%.

Article Snippet: Cell Culture The studies were carried out on cultured cells of human glioblastoma (A-172, ATCC CRL 1620) [29], human breast cancer (Hs578T, ATCC HTB-126) [30] and human embryonic kidney 293 cells (HEK-293, ATCC CRL 1573) [31] obtained from the shared research facility “Vertebrate cell culture collection” (Institute of Cytology, RAS, Russia).

Techniques: Comparison

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Xanthone-1,2,4-triazine and Acridone-1,2,4-triazine Conjugates: Synthesis and Anticancer Activity.

doi: 10.3390/ph16030403

Figure Lengend Snippet: Figure 4. Fluorescence staining of human glioblastoma A-172 cells with Annexin V-FITC and PI dyes under the influence of the test substances 7a and 7e compared to cisplatin (6 µM concentration) and intact cells. Cells in early apoptosis stained green (Annexin V-FITC), cells in late apoptosis and necrosis stained red (PI), 200× magnification.

Article Snippet: Cell Culture The studies were carried out on cultured cells of human glioblastoma (A-172, ATCC CRL 1620) [29], human breast cancer (Hs578T, ATCC HTB-126) [30] and human embryonic kidney 293 cells (HEK-293, ATCC CRL 1573) [31] obtained from the shared research facility “Vertebrate cell culture collection” (Institute of Cytology, RAS, Russia).

Techniques: Fluorescence, Staining, Concentration Assay

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Xanthone-1,2,4-triazine and Acridone-1,2,4-triazine Conjugates: Synthesis and Anticancer Activity.

doi: 10.3390/ph16030403

Figure Lengend Snippet: Figure 7. Assessment of DNA synthesis intensity in human glioblastoma cell cultures of the A-172 line under the influence of compounds 7a and 7e. Blue color stains the nuclei of all cells using DAPI dye, and red nuclei of cells with active DNA synthesis (EdU), at 100× magnification.

Article Snippet: Cell Culture The studies were carried out on cultured cells of human glioblastoma (A-172, ATCC CRL 1620) [29], human breast cancer (Hs578T, ATCC HTB-126) [30] and human embryonic kidney 293 cells (HEK-293, ATCC CRL 1573) [31] obtained from the shared research facility “Vertebrate cell culture collection” (Institute of Cytology, RAS, Russia).

Techniques: DNA Synthesis

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: Inhibition of mTor reduces A172 sphere formation and the expression of NSC/progenitor markers. A172 cells were cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence or the presence of rapamycin for 3 days. The numbers of generated primary spheres were counted (A). The primary spheres were dissociated and cultured with EGF and bFGF in the absence of rapamycin for 3 days. The numbers of secondary spheres were counted (B). (C–E) Cells were cultured as described in (A). The expression of cd133 or gapdh mRNA was analyzed by RT-PCR (C). Cell lysates were subjected to immunoblot analysis with the indicated antibodies (D and E). (F–K) Cells were transfected with the indicated siRNA. Then, cells were reseeded in the stem/progenitor cell culture medium containing EGF and bFGF for assays to count primary sphere numbers (G and H), RT-PCR (J), and immunoblot (F and K) analysis after 3 days. The primary spheres were dissociated and cultured with EGF and bFGF for 3 days. The numbers of secondary spheres were counted (I). Results in (A), (B), (H), and (I) are presented as the mean ± SD of 3 independent experiments. *P < .05.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Inhibition, Expressing, Cell Culture, Generated, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: Inhibition of PI3K reduces A172 sphere formation and the expression of NSC/progenitor markers. (A) A172 cells were cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence or the presence of LY294002 for 3 days. The numbers of generated primary spheres were counted. (B) The primary A172 spheres were dissociated and cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence of LY294002 for 3 days. The numbers of secondary spheres were counted. (C–E) Cells were cultured as described in (A). Cell lysates were subjected to immunoblot analysis with the indicated antibodies (D and E). The expression of cd133 or gapdh mRNA was analyzed by RT-PCR (C). Results in (A) and (B) are presented as the mean ± SD of 3 independent experiments. *P < .05.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Inhibition, Expressing, Cell Culture, Generated, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: Effect of combination treatment with rapamycin and LY294002 on A172 cells. (A–E, G, and H) A172 cells were cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence or the presence of rapamycin (Rap) and/or LY294002 (LY) for 3 days. Cell lysates were subjected to immunoblot analysis with the indicated antibodies (A–C and H). The expression of cd133 or gapdh mRNA was analyzed by RT-PCR (G). Cells were cultured as described above and then observed under a phase-contrast microscope (magnification ×40) (D). The numbers of generated primary spheres were counted (E). The primary A172 spheres were dissociated and cultured with EGF and bFGF in the absence of the indicated inhibitors for 3 days. The numbers of secondary spheres were counted (F). Results in (E) and (F) are presented as the mean ± SD of 3 independent experiments.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Cell Culture, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Microscopy, Generated

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: Combination treatment with rapamycin and LY294002 inhibits self-renewal and elicits differentiation of A172 CSLCs. (A and B) A172 CSLCs were cultured in the stem/progenitor cell culture medium with EGF and bFGF for 14 days before inhibitor treatment. Then, A172 CSLCs were treated with the indicated inhibitors for 3 days, and observed under a phase-contrast microscope (magnification ×100). Arrows show neurite-like morphology (A). The numbers of spheres were counted (B) (rapamycin [Rap, 50 nM] and/or LY294002 [LY, 10 µM]). (C) Cells were cultured as described in (A) and plated on ornithine-coated coverslips with 10% serum for 6 h to facilitate cell adhesion to the dish. Cells were then immunolabeled with the indicated antibodies. (D) Results of quantitative analysis of 3 independent experiments for (C) are shown (Rap: rapamycin, 50 nM and/or LY: LY294002, 10 µM). Results in (B) are presented as the mean ± SD of 3 independent experiments. *P < .05. Data in (D) are the mean ± SD of values obtained from at least 200 cells in each of 3 independent experiments. *P < .05.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Cell Culture, Microscopy, Immunolabeling

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: NVP-BEZ235 inhibits A172 sphere formation and reduces the expression of NSC/progenitor markers. (A–C, E, and F) A172 cells were cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence or the presence of NVP-BEZ235 (BEZ) for 3 days. Cell lysates were subjected to immunoblot analysis with the indicated antibodies (A and F). The expression of cd133 or gapdh mRNA was analyzed by RT-PCR (E). Cells were cultured as described above and then observed under a phase-contrast microscope (magnification ×40) (B). The numbers of generated primary spheres were counted (C). The primary A172 spheres were dissociated and cultured with EGF and bFGF in the absence of the indicated inhibitors for 3 days. The numbers of secondary spheres were counted (D). Results in (C) and (D) are presented as the mean ± SD of 3 independent experiments.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Expressing, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Microscopy, Generated

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: NVP-BEZ235 inhibits self-renewal and elicits differentiation of A172 CSLCs. (A) A172 CSLCs were cultured in the stem/progenitor cell culture medium with EGF and bFGF for 14 days before NVP-BEZ235 (BEZ) treatment. Then, A172 CSLCs were treated with BEZ for 3 days and observed under a phase-contrast microscope (left, magnification ×100). The numbers of generated primary spheres were counted (right). (B and C) Cells were cultured as described above and were plated on ornithine-coated coverslips with 10% serum for 6 h to facilitate cell adhesion to the dish. Cells were then immunolabeled with the indicated antibodies. Results in (A) are presented as the mean ± SD of 3 independent experiments. *P < .05.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Cell Culture, Microscopy, Generated, Immunolabeling

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: NVP-BEZ235–treated cells have reduced capacity of tumor formation in vivo. Monolayer-cultured A172 CSLCs were treated with NVP-BEZ235 (BEZ, 1 µM) for 3 days, and the levels of indicated proteins were analyzed by immunoblotting (A and B). (B) The numbers of generated spheres were counted (right upper). Representative images of spheres are shown (right lower). Results in the graph are presented as the mean ± SD of 3 independent experiments. (C) Monolayer-cultured A172 CSLCs (left) and SJ28P3 cells (right) treated with vehicle (DMSO) or NVP-BEZ235 for 3 days in vitro (1 × 105) were injected subcutaneously into BALB/c− nu/nu mice (5 mice per group), and the size of subcutaneous tumors was measured at the indicated time points. (D) Monolayer-cultured A172 CSLCs (left) and SJ28P3 cells (right) treated with vehicle (DMSO) or NVP-BEZ235 for 3 days in vitro (1 × 104) were injected intracranially into BALB/c− nu/nu mice (5 mice per group), and the survival of mice was evaluated by the Kaplan–Meier analysis (log-rank test: *P = .048 and **P = .049; upper). Alternatively, mice were sacrificed at 30 days after the intracranial injection, and the tissue sections were stained with hematoxylin and eosin (lower).

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: In Vivo, Cell Culture, Western Blot, Generated, In Vitro, Injection, Staining

Journal: Oncotarget

Article Title: Type 5 phosphodiesterase regulates glioblastoma multiforme aggressiveness and clinical outcome

doi: 10.18632/oncotarget.14656

Figure Lengend Snippet: A . Wound healing assay and B . Histogram showing the mean percentage ±SD of closure of sh-scrT98G or sh1 or sh2-PDE5 cells monolayers in the presence or absence of 1 μM sildenafil or 100 μM 8br-cGMP. C . Migration assay on matrigel cushions through transwell filters of sh-scr or sh1-PDE5 T98G cells in the presence or absence of 1 μM sildenafil or 100 μM 8br-cGMP. Scale bars= 50 μm. D . Histogram showing the relative migration capacity (fold increase ± SD) of cells that passed through the filter in the different treatments compared to control cells. E . Migration assay on matrigel cushions through transwell filters of mock- or PDE5-overexpressing U87MG (upper panels) or GBM line 83 cells (lower panels). Scale bar= 20 μm. F . Histogram showing the mean percentage ± SD of cells that passed through the filter. G . Histogram of the mean percentage ±SD of secreted MMP-2 in gelatin zymography (bottom image) in sh-scr or sh1-PDE5 T98G and H . in mock- or PDE5-overexpressing U87MG cells and I . in sh-scr T98G stimulated with CNP, GSNO or sildenafil at the indicated concentrations. Data have been obtained from three independent experiments. (*p < 0.05; **p < 0.001).

Article Snippet: The GBM cell lines (T98G, U87MG, A172, U251, LN18) used in this study were grown in DMEM, (Euroclone; Milan, Italy) with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Wound Healing Assay, Migration, Control, Zymography

Journal: Oncotarget

Article Title: Type 5 phosphodiesterase regulates glioblastoma multiforme aggressiveness and clinical outcome

doi: 10.18632/oncotarget.14656

Figure Lengend Snippet: A . PKG1 low and B . PKG1 high GBM samples, as revealed by immuno-histochemistry. Black arrows point to PKG1 expression within the vessel walls. White arrow points to tumor cells. Scale bar=20μm. C . Expression levels of PKGII, PKG1α and PKG1β and total PKG1 in sh-scr and sh1-PDE5 T98G cells and in wtT98G, U87MG, A172, LN18, 83 GBM lines. D . pVASP levels in sh-scr or sh1-PDE5 T98G cells before and after Y-27632, 8br-cGMP (50 μM) treatments. Representative image out of 4 independent experiments. E . Phosphorylation levels of MYPT1 (pSer695) in sh-PDE5 or sh1-scr T98G upon the indicated treatments [8br-cGMP, 50 μM; Y-27632 (RI), 10 μM); KT5823 (PKGi), 2 μM]. F . Densitometric analysis of western blots from three independent experiments is shown. Bars represent the mean ±SD. G . sh-scr and sh1-PDE5 T98G invasion assays on matrigel cushions in the presence of Y-27632 or 8br-cGMP or both. H . Histogram showing the relative migration capacity (fold increase ± SD) of cells that reached the bottom of the matrigel cushion in the different treatments compared to control cells. (**p < 0.001).

Article Snippet: The GBM cell lines (T98G, U87MG, A172, U251, LN18) used in this study were grown in DMEM, (Euroclone; Milan, Italy) with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Immunohistochemistry, Expressing, Phospho-proteomics, Western Blot, Migration, Control

Journal: Oncotarget

Article Title: Type 5 phosphodiesterase regulates glioblastoma multiforme aggressiveness and clinical outcome

doi: 10.18632/oncotarget.14656

Figure Lengend Snippet: A . sh-scr (PDE5+) or sh1-PDE5 (PDE5-) T98G cells were treated with increasing X-ray doses from 2 to 4 Gy and probed with anti-PAR and anti-PARP-1 antibodies. B . sh-scr or sh1-PDE5 T98G cells were exposed to X-rays at 4 Gy in the presence or absence (+/-) of the PARP1 inhibitor olaparib (1 μM). Total protein PARylation levels were assayed after 24 h. C . Schematic representation of cGMP signaling pathway promoting migration and DNA repair in GBM cells.

Article Snippet: The GBM cell lines (T98G, U87MG, A172, U251, LN18) used in this study were grown in DMEM, (Euroclone; Milan, Italy) with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Migration