Journal: International Journal of Molecular Sciences

Article Title: Tamoxifen Activates Transcription Factor EB and Triggers Protective Autophagy in Breast Cancer Cells by Inducing Lysosomal Calcium Release: A Gateway to the Onset of Endocrine Resistance

doi: 10.3390/ijms25010458

Figure Lengend Snippet: Subcellular localization of TFEB in MCF7 and MCF7-TamR cells as detected using different techniques. The panels show representative confocal images of both MCF7 and MCF7-TamR cells in basal growth conditions, either stained with immunofluorescence ( a ) or following transient transfection with TFEB-GFP ( b ). ( c , d ) Fraction of cells displaying nuclear TFEB, as determined by immunofluorescence or transient transfection of TFEB-GFP, respectively. ( e ) Mean fluorescence intensity of nuclei without or with nuclear TFEB (blue or orange dots, respectively), from samples prepared with the indicated techniques. ( f ) Fraction of cells with nuclear TFEB as calculated in cells stably transfected with TFEB-GFP. IF, immunofluorescence; TFEB-GFP, cells transiently transfected with the TFEB-GFP chimera. Data represent the mean ± SD of three independent experiments. Statistical significance was assessed with Student’s t -test; ***: p < 0.001; §§§: p < 0.001 vs. the corresponding control nuclei. Scale bar: 20 µm.

Article Snippet: For transient transfection, the cells were seeded at 3 × 10 4 /cm 2 (MCF7 and MCF7-TamR) or 4 × 10 4 /cm 2 (MDA-MB-415, T47D and ZR-75-1) in 24-well tissue culture plates (Jet Biofil) and transfected 24 h later with a plasmid encoding human TFEB-GFP [a gift from Shawn Ferguson (Addgene plasmid #38119)] using the K2 Transfection Reagent (T060-0.75, Biontex, München, Germany).

Techniques: Staining, Immunofluorescence, Transfection, Fluorescence, Stable Transfection

Journal: International Journal of Molecular Sciences

Article Title: Tamoxifen Activates Transcription Factor EB and Triggers Protective Autophagy in Breast Cancer Cells by Inducing Lysosomal Calcium Release: A Gateway to the Onset of Endocrine Resistance

doi: 10.3390/ijms25010458

Figure Lengend Snippet: Effects of Tam washout and starvation on the subcellular localization of TFEB-GFP in MCF7-TamR cells. ( a ) Cells treated with 5 µM Tam as indicated in or after Tam removal (washout) either in the absence or presence of 10 ng/mL of leptomycin B (LMB). ( b ) Cells grown in the absence of Tam starved for 4 h in HBSS-glucose and subsequently refed for 2 h with complete growth medium in the absence or presence of 10 ng/mL of LMB. The bar charts below each set of images represent the percentage of cells with nuclear TFEB. Starv: starvation; Ref: refeeding; LMB: 10 ng/mL leptomycin B; Tam: 5 µM tamoxifen; WO: washout; w/o Tam: MCF7-TamR cells grown in the absence of Tam to avoid Tam-induced TFEB relocation events. Data represent the mean ± SD of three independent experiments. Statistical significance was assessed using ANOVA followed by Dunnett’s post hoc test; *: p < 0.05; ***: p < 0.001. Scale bar: 20 µm.

Article Snippet: For transient transfection, the cells were seeded at 3 × 10 4 /cm 2 (MCF7 and MCF7-TamR) or 4 × 10 4 /cm 2 (MDA-MB-415, T47D and ZR-75-1) in 24-well tissue culture plates (Jet Biofil) and transfected 24 h later with a plasmid encoding human TFEB-GFP [a gift from Shawn Ferguson (Addgene plasmid #38119)] using the K2 Transfection Reagent (T060-0.75, Biontex, München, Germany).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Tamoxifen Activates Transcription Factor EB and Triggers Protective Autophagy in Breast Cancer Cells by Inducing Lysosomal Calcium Release: A Gateway to the Onset of Endocrine Resistance

doi: 10.3390/ijms25010458

Figure Lengend Snippet: Effect of Tam on the subcellular localization of TFEB-GFP in parental and Tam-resistant cells. Representative images of subcellular localization of TFEB in parental ( a ) or Tam-resistant ( b ) MDA-MB-415, T47D and ZR-75-1 cells transiently transfected with a plasmid encoding TFEB-GFP, in normal growth condition (upper panels) or in the presence of 5 µM Tam for 24 h (lower panels). Tam: 5 µM Tam. Scale bar: 20 µm.

Article Snippet: For transient transfection, the cells were seeded at 3 × 10 4 /cm 2 (MCF7 and MCF7-TamR) or 4 × 10 4 /cm 2 (MDA-MB-415, T47D and ZR-75-1) in 24-well tissue culture plates (Jet Biofil) and transfected 24 h later with a plasmid encoding human TFEB-GFP [a gift from Shawn Ferguson (Addgene plasmid #38119)] using the K2 Transfection Reagent (T060-0.75, Biontex, München, Germany).

Techniques: Transfection, Plasmid Preparation