Journal: International Journal of Molecular Sciences

Article Title: Combined Inhibition of Hedgehog and HDAC6: In Vitro and In Vivo Studies Reveal a New Role for Lysosomal Stress in Reducing Glioblastoma Cell Viability

doi: 10.3390/ijms24065771

Figure Lengend Snippet: Hh and HDAC6 inhibition impaired U87-MG cell viability. ( A ) MTT assay of U87-MG cells treated for 48 h with vehicle (CT), 8 μM of TubA (T8), 10 μM of cyclo (C10) and 8 μM of TubA + 10 μM cyclo (T8C10). ( B – E ) Representative images of the head region of 3 dpf zebrafish embryos xenotransplanted with labeled U87-MG cells pretreated with ( B ) CT, ( C ) T8, ( D ) C10, and ( E ) T8C10. ( F ) Quantification of the tumor area at t1 (24 h postinjection, hpi) normalized to the tumor area at t0 (immediately after U87 injection). ( G ) Western blot analyses and quantification of pERK and pAKT protein expression levels following 48 h treatments with or without T8 and C10, alone or in combination. Data are expressed as the percentage of the control ( A , G ) or mean ± standard deviation ( F ). CT—vehicle; TubA/T—tubastatin A; cyclo/C—cyclopamine. One-way ANOVA with Tukey post hoc correction. *** p < 0.001; ** p < 0.01; * p < 0.05; nonsignificant data are not shown. Scale bare indicates 100 μm.

Article Snippet: For cell treatments, tubastatin A (TubA; Merck KGaA, Darmstadt, Germany) and Bafilomycin-A1 (BafA1; MedChemExpress, Princeton, NJ, USA) were dissolved in dimethyl-sulfoxide (DMSO).

Techniques: Inhibition, MTT Assay, Labeling, Injection, Western Blot, Expressing, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Combined Inhibition of Hedgehog and HDAC6: In Vitro and In Vivo Studies Reveal a New Role for Lysosomal Stress in Reducing Glioblastoma Cell Viability

doi: 10.3390/ijms24065771

Figure Lengend Snippet: Autophagy was impaired following combination treatments with inhibitors of HDAC6 and Hh pathway in U87-MG cells and zebrafish embryos. ( A , B ) Western blot analysis and quantification of LC3 protein expression levels after treatments with 8 μM of TubA (T8), 10 μM of cyclo (C10), and 8 μM of TubA + 10 μM of cyclo (T8C10) in the presence (grey)/absence (black) of ( A ) 3-MA or ( B ) BafA1. ( C ) Western blot analysis and quantification of p62/SQSTM1 protein expression following 48 h treatments with or without T8 and C10, alone or in combination (T8C10). Data are expressed as the percentage of the control. ( D ) Schematic representation of the pharmacological treatments. ( E – H ) Confocal images of the trunk region of the Tg( CMV:eGFPmap1Lc3b ) zebrafish embryos at 2 dpf after treatment with ( E ) DMSO, ( F ) 25 μM of TubA (T25), ( G ) 5 μM of cyclo (C5), and ( H ) 25 μM of TubA + 5 μM cyclo (T25C5). ( I ) Count of Lc3b puncta (green puncta) in the trunk region of treated embryos. ( J – M ) Confocal images of the trunk region of the Tg( CMV:eGFPmap1Lc3b ) zebrafish embryos at 2 dpf stained with p62 antibody after treatment with ( J ) DMSO, ( K ) T25, ( L ) C5, and ( M ) T25C5. ( N ) Percentage of Lc3b/p62 puncta in the trunk region of treated embryos. Asterisks indicate the colocalization. Scale bar indicates 100 μm. CT—vehicle; TubA/T—tubastatin A; cyclo/C—cyclopamine. One-way ANOVA with Tukey post hoc test. Data are presented as mean ± standard deviation *** p < 0.001; ** p < 0.01; * p < 0.05.

Article Snippet: For cell treatments, tubastatin A (TubA; Merck KGaA, Darmstadt, Germany) and Bafilomycin-A1 (BafA1; MedChemExpress, Princeton, NJ, USA) were dissolved in dimethyl-sulfoxide (DMSO).

Techniques: Western Blot, Expressing, Control, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Combined Inhibition of Hedgehog and HDAC6: In Vitro and In Vivo Studies Reveal a New Role for Lysosomal Stress in Reducing Glioblastoma Cell Viability

doi: 10.3390/ijms24065771

Figure Lengend Snippet: Combined HDAC6 and Hh inhibition altered acidic compartments. ( A ) U87-MG cells treated with vehicle (CT), 8 μM of TubA (T8), 10 μM of cyclo (C10), and 8 μM of TubA +10 μM cyclo (T8C10) for 48 h and stained with acridine orange. The AO accumulation into acidic compartments is represented by red dots. Green fluorescence arose from AO binding to DNA and RNA ( B ). Quantitative analysis of red fluorescence was calculated as corrected total cell fluorescence (CTCF) ± standard deviation. ( C ) LysoTracker Red staining in U87-MG cells treated with or without T8, C10, or T8C10. ( D ) Western blot analysis of LAMP1 protein expression normalized against GAPDH. Data are expressed as the percentage of the CT. ( E ) Schematic description of the pharmacological treatments in zebrafish. ( F – I ) Representative images of the trunk region of zebrafish embryos treated with 25 μM of TubA (T25) or 5 μM of cyclo (C5), alone or in combination, and stained with LysoTracker Red. ( I ) Quantification of the LysoTracker Red intensity in the trunk region of treated embryos. Scale bar indicates 40 μm ( A ) and 100 μm ( B , E – H ). Data are presented as mean ± standard deviation. CT—vehicle; TubA/T—tubastatin A; cyclo/C—cyclopamine. One-way ANOVA with Tukey post hoc correction. *** p < 0.001; * p < 0.05; nonsignificant data are not shown.

Article Snippet: For cell treatments, tubastatin A (TubA; Merck KGaA, Darmstadt, Germany) and Bafilomycin-A1 (BafA1; MedChemExpress, Princeton, NJ, USA) were dissolved in dimethyl-sulfoxide (DMSO).

Techniques: Inhibition, Staining, Fluorescence, Binding Assay, Standard Deviation, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Combined Inhibition of Hedgehog and HDAC6: In Vitro and In Vivo Studies Reveal a New Role for Lysosomal Stress in Reducing Glioblastoma Cell Viability

doi: 10.3390/ijms24065771

Figure Lengend Snippet: Sphingolipid metabolism and distribution were impaired following Hh and HDAC6 inhibition. ( A ) Radioactivity distribution among different sphingolipids after [1- 3 H]sphingosine administration at the metabolic steady state in U87-MG cells untreated (controls, CT) or treated with 8 μM of TubA (T8), 10 μM of cyclo (C10), and 8 μM of TubA + 10 μM cyclo (T8C10). Data are presented as mean of nCi/mg protein ± standard deviation. ( B ) Radioactivity of the total lipid extract (TLE), ceramide (Cer), and sphingomyelin (SM) following 2 h of [ 3 H]SM administration in U87-MG cells CT or treated with T8, C10, or T8C10. Data are presented as mean of nCi/mg protein ± standard deviation. ( C ) Uptake and subcellular localization of fluorescent BODIPY-SM after treatment in U87-MG cells treated with or without T8C10 at 0 min, 5 min, 15 min, and 60 min. CT—vehicle; TubA/T—tubastatin A; cyclo/C—cyclopamine. One-way ANOVA with Tukey post hoc correction. *** p < 0.001; ** p < 0.01; * p < 0.05; nonsignificant data are not shown. Scale bar indicates 40 μm.

Article Snippet: For cell treatments, tubastatin A (TubA; Merck KGaA, Darmstadt, Germany) and Bafilomycin-A1 (BafA1; MedChemExpress, Princeton, NJ, USA) were dissolved in dimethyl-sulfoxide (DMSO).

Techniques: Inhibition, Radioactivity, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Combined Inhibition of Hedgehog and HDAC6: In Vitro and In Vivo Studies Reveal a New Role for Lysosomal Stress in Reducing Glioblastoma Cell Viability

doi: 10.3390/ijms24065771

Figure Lengend Snippet: LC3 and LAMP1 colocalization and BODIPY-SM distribution were impaired following Hh and HDAC6 inhibition. ( A ) Double immunostaining against LC3 and LAMP1 of U87-MG cells treated in the presence or absence of both 8 μM of TubA and 10 μM of cyclo. Asterisks show some LC3 puncta, indicating autophagosomes, that did not colocalize with the red ones, indicating lysosomes. ( B ) BODIPY-SM and LysoTracker Red DND-99 staining of U87-MG cells treated in the presence or absence of both 8 μM of TubA and 10 μM of cyclo. CT—control, TubA/T—tubastatin A; cyclo/C—cyclopamine. Scale bar indicates 40 μm.

Article Snippet: For cell treatments, tubastatin A (TubA; Merck KGaA, Darmstadt, Germany) and Bafilomycin-A1 (BafA1; MedChemExpress, Princeton, NJ, USA) were dissolved in dimethyl-sulfoxide (DMSO).

Techniques: Inhibition, Double Immunostaining, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Combined Inhibition of Hedgehog and HDAC6: In Vitro and In Vivo Studies Reveal a New Role for Lysosomal Stress in Reducing Glioblastoma Cell Viability

doi: 10.3390/ijms24065771

Figure Lengend Snippet: Mechanism of action of TubA and cyclo. The path of cyclo is indicated in blue while that of TubA is indicated in green. cyclo: cyclopamine; TubA: tubastatin A. SMO: smoothened; PTCH1: Ptached1; GLI: glioma-associated oncogene. SM: sphingomyelin, GSL: glycosphingolipid. Image generated with the BioRender.com tool.

Article Snippet: For cell treatments, tubastatin A (TubA; Merck KGaA, Darmstadt, Germany) and Bafilomycin-A1 (BafA1; MedChemExpress, Princeton, NJ, USA) were dissolved in dimethyl-sulfoxide (DMSO).

Techniques: Generated