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