protein translocation  (Seca)

Journal: International Journal of Molecular Sciences

Article Title: Identification of ANT2 as a Druggable Target for Endocrine-Resistant ERα-Positive Breast Cancer

doi: 10.3390/ijms27083704

Figure Lengend Snippet: POH-binding protein ANT2 affects ER expression and growth of ER-positive BC cells. ( A ) Silver staining of POH-binding proteins. Purified POH-binding proteins from whole-cell extracts of MCF7 cells incubated with POH-immobilized FG beads were detected and identified using mass spectrometry. ( B ) Confirmation of the interaction of POH with RPS5 and ANT2. Bound RPS5 and ANT2 were detected by Western blotting with anti-RPS5 and anti-ANT2 antibodies, respectively. ( C , D ) Effects of RPS5 or ANT2 depletion on ERα expression. ERα expression was analyzed by Western blotting in MCF7 cells treated with negative control siRNA (siNeg) or siRNAs targeting different sequences (#1 and #2) of RPS5 ( C ) and ANT2 ( D ) gene for 48 h. β-Actin was used as a loading control. ( E ) Confirmation of the direct binding of POH with the recombinant ANT2 protein. Purified recombinant FLAG-ANT2 was incubated with POH-immobilized FG beads, and bound FLAG-ANT2 was detected by Western blotting with the anti-FLAG antibody. ( F ) The most stable docking pose of POH with ANT2 evaluated by the MD simulation and the scoring function of Smina. ( G ) Interaction network between ANT2 amino acid residues and POH. Interaction analysis was performed using ProLIF. For visual clarity, the original ProLIF-generated figure was modified. HB, hydrogen bond; VdW, van der Waals contact. ( H ) Effects of POH and bongkrekic acid (BKA) on intracellular ATP levels in ER-positive BC cells. MCF7 cells were treated with 1 mM POH, 100 µM BKA, or DMSO for 2 h and 6 h. Intracellular ATP levels were measured using the CellTiter-Glo ® 2.0 Cell Viability Assay. Points, means ( n = 5); bars, SD. ns, not significant; ** p < 0.01; **** p < 0.0001. ( I ) Effects of ANT2 depletion on colony formation of ER-positive BC cells. MCF7 cells were treated with siNeg or siANT2 (#1 and #2) for 16 days. Colonies were fixed and stained with crystal violet. Representative images of stained colonies are shown (upper panel). Colony formation rates are shown in the graph (lower panel). Bars, means ( n = 3). ** p < 0.01.

Article Snippet: Similarly, the recombinant FLAG-tagged ANT2 protein (TP308949; OriGene, Rockville, MD, USA) was incubated with empty or POH-immobilized beads for 4 h at 4 °C.

Techniques: Binding Assay, Expressing, Silver Staining, Purification, Incubation, Mass Spectrometry, Western Blot, Negative Control, Control, Recombinant, Generated, Modification, Viability Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Identification of ANT2 as a Druggable Target for Endocrine-Resistant ERα-Positive Breast Cancer

doi: 10.3390/ijms27083704

Figure Lengend Snippet: ANT2 expression is correlated with clinical aggressiveness in ERα-positive BC. ( A ) The comparison of SLC25A5 mRNA expression between BC and normal tissue using UALCAN ( http://ualcan.path.uab.edu (accessed on 14 April 2025)). **** p < 0.0001. ( B ) The comparison of ANT2 protein expression between BC and normal tissue using UALCAN. **** p < 0.0001. ( C ) Correlation analysis between the expressions of SLC25A5 and MKI67 in the TCGA BC data using GEPIA ( http://gepia.cancer-pku.cn/ (accessed on 25 June 2025)). **** p < 0.0001. ( D ) The comparison of SLC25A5 expression between Luminal A and B subtypes based on the TCGA dataset using cBioPortal ( https://www.cbioportal.org/ (accessed on 9 July 2025)). **** p < 0.0001. ( E ) Prognosis analysis of ER-positive BC patients treated with endocrine therapy. Patients were stratified by SLC25A5 expression, and recurrence-free survival (RFS) was analyzed using Kaplan–Meier Plotter ( https://kmplot.com/ (accessed on 30 June 2025)).

Article Snippet: Similarly, the recombinant FLAG-tagged ANT2 protein (TP308949; OriGene, Rockville, MD, USA) was incubated with empty or POH-immobilized beads for 4 h at 4 °C.

Techniques: Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Identification of ANT2 as a Druggable Target for Endocrine-Resistant ERα-Positive Breast Cancer

doi: 10.3390/ijms27083704

Figure Lengend Snippet: The fatty acid elongation pathway is involved in Fulvestrant-resistant cells. ( A ) The top 20 pathways of enrichment analysis of upregulated genes in 182R-1 cells compared with MCF7 cells based on RNA-seq data. ( B ) The comparison of fatty acid elongation-related DEGs between MCF7 and 182R-1 cells based on RNA-seq data. ( C ) The top 14 pathways of enrichment analysis of downregulated genes upon ANT2 depletion by siANT2 (#2) in 182R-1 cells based on RNA-seq data. ( D ) The comparison of fatty acid elongation-related DEGs with or without ANT2 depletion by siANT2 (#2) in 182R-1 cells based on RNA-seq data. ( E , F ) Prognosis analysis of ER-positive BC patients treated with endocrine therapy. Patients were stratified by ELOVL7 ( E ) and ELOVL6 ( F ) expression, and recurrence-free survival (RFS) was analyzed using the Kaplan–Meier Plotter ( https://kmplot.com/ (accessed on 30 June 2025)). ( G , H ) Correlation analysis between the expressions of SLC25A5 and ELOVL7 ( G ) or ELOVL6 ( H ) in the TCGA BC data using GEPIA ( http://gepia.cancer-pku.cn/ (accessed on 25 June 2025)). **** p < 0.0001. ( I , J ) Correlation analyses between the expressions of MKI67 and ELOVL7 ( I ) or ELOVL6 ( J ) in the TCGA BC data using GEPIA. **** p < 0.0001. ( K , L ) The comparison of ELOVL7 ( K ) and ELOVL6 ( L ) expression between Luminal A and B subtypes based on the TCGA dataset using cBioPortal ( https://www.cbioportal.org/ (accessed on 9 July 2025)). ** p < 0.01, **** p < 0.0001. ( M , N ) ROC curve analysis of ELOVL7 ( M ) and ELOVL6 ( N ) expression in responder and non-responder groups to endocrine therapy in ER-positive BC using the ROC Plotter ( https://rocplot.com/ (accessed on 28 May 2025)).

Article Snippet: Similarly, the recombinant FLAG-tagged ANT2 protein (TP308949; OriGene, Rockville, MD, USA) was incubated with empty or POH-immobilized beads for 4 h at 4 °C.

Techniques: RNA Sequencing, Comparison, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Identification of ANT2 as a Druggable Target for Endocrine-Resistant ERα-Positive Breast Cancer

doi: 10.3390/ijms27083704

Figure Lengend Snippet: Targeting ANT2 leads to lipid droplet accumulation in Fulvestrant-resistant cells. ( A – D ) Effects of ANT2 depletion and POH treatment on lipid droplet accumulation in Fulvestrant-resistant cells. 182R-1 cells were treated with negative control siRNA (siNeg) or siANT2 (#1 and #2) for 72 h ( A , B ) and 500 µM POH for 72 h ( C , D ). Cells were fixed and stained with ShoyakuGreen (green, lipid droplets) and Hoechst 33342 (blue, cell nuclei). Representative images of stained cells are shown ( A , C ). Scale bars, 5 µm. Lipid droplet area per cell is shown in the graph ( B , D ). **** p < 0.0001. ( E ) Effects of ANT2 depletion on colony formation of Fulvestrant-resistant cells. 182R-1 cells were treated with siNeg or siANT2 (#1 and #2) for 16 days. Colonies were fixed and stained with crystal violet. Representative images of stained colonies are shown (upper panel). Colony formation rates are shown in the graph (lower panel). Columns, means ( n = 3); bars, SD. ** p < 0.01.

Article Snippet: Similarly, the recombinant FLAG-tagged ANT2 protein (TP308949; OriGene, Rockville, MD, USA) was incubated with empty or POH-immobilized beads for 4 h at 4 °C.

Techniques: Negative Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Identification of ANT2 as a Druggable Target for Endocrine-Resistant ERα-Positive Breast Cancer

doi: 10.3390/ijms27083704

Figure Lengend Snippet: In silico drug screening identified candidate repurposed ligands for ANT2. ( A ) Workflow of in silico screening for ANT2 ligands (see Materials and Methods section for details). ( B , C ) The most stable docking pose of Venetoclax ( B ) and Nystatin A1 ( C ) with ANT2 evaluated by the MD simulation and the scoring function of Smina. ( D , E ) Interaction networks between amino acid residues and Venetoclax ( D ) or Nystatin A1 ( E ). The interaction analyses were performed using ProLIF. For visual clarity, the figures output by ProLIF were modified. HB, hydrogen bond; VdW, van der Waals contact.

Article Snippet: Similarly, the recombinant FLAG-tagged ANT2 protein (TP308949; OriGene, Rockville, MD, USA) was incubated with empty or POH-immobilized beads for 4 h at 4 °C.

Techniques: In Silico, Drug discovery, Modification

Journal: International Journal of Molecular Sciences

Article Title: Identification of ANT2 as a Druggable Target for Endocrine-Resistant ERα-Positive Breast Cancer

doi: 10.3390/ijms27083704

Figure Lengend Snippet: Venetoclax and Nystatin exhibit tumor-suppressive effects against ER-positive BC, including endocrine-resistant cells. ( A , B ) Analysis of cell growth of ER-positive BC cells after the treatment of ANT2 ligands. MCF7 cells were treated with Venetoclax ( A ) and Nystatin ( B ) at the indicated concentrations for 120 h. Cell viability was measured with a Cell Counting Kit-8 assay. The data obtained with dimethyl sulfoxide (DMSO) were taken as 100%. Points, means ( n = 4); bars, SD. * p < 0.05; **** p < 0.0001, significantly different from the DMSO-treated control. ( C , D ) Analysis of the expression of ERα protein after the dose-dependent treatment of ANT2 ligands. ERα expression was analyzed by Western blotting in MCF7 cells treated with Venetoclax ( C ) and Nystatin ( D ) at the indicated concentrations for 24 h. β-Actin was used as a loading control. ( E , F ) Analysis of cell growth of Fulvestrant-resistant cells after the treatment of ANT2 ligands. 181R-1 cells were treated with Venetoclax ( E ) and Nystatin ( F ) at the indicated concentrations for 120 h. Cell viability was measured with a Cell Counting Kit-8 assay. The data obtained with dimethyl sulfoxide (DMSO) were taken as 100%. Points, means ( n = 4); bars, SD. * p < 0.05; **** p < 0.0001, significantly different from the DMSO-treated control. ( G , H ) Effects of ANT2 ligand treatment on lipid droplet accumulation in Fulvestrant-resistant cells. 182R-1 cells were treated with 10 µM Venetoclax ( G ) and 100 µM Nystatin ( H ) for 72 h. Cells were fixed and stained with ShoyakuGreen (green, lipid droplets) and Hoechst 33342 (blue, cell nuclei). Representative images of stained cells are shown (upper panel). Scale bars, 5 µm. Lipid droplet area per cell is shown in the graph (lower panel). **** p < 0.0001. ( I ) Effects of ANT2 depletion on Venetoclax sensitivity in Fulvestrant-resistant cells. 182R-1 cells were transfected with siNeg or siANT2 (#1 and #2). Venetoclax was added on day 3 at the indicated concentrations, and cell viability was measured on day 8. Cell viability was measured using a Cell Counting Kit-8 assay. For each group (siNeg, siANT2 (#1), and siANT2 (#2)), the value at 0 µM Venetoclax was set as 100%. Points, means ( n = 4); bars, SD. * p < 0.05; ** p < 0.01, significantly different from the DMSO-treated control. ( J ) Schematic overview of endocrine resistance and its overcoming by ANT2 targeting in ER-positive BC.

Article Snippet: Similarly, the recombinant FLAG-tagged ANT2 protein (TP308949; OriGene, Rockville, MD, USA) was incubated with empty or POH-immobilized beads for 4 h at 4 °C.

Techniques: Cell Counting, Control, Expressing, Western Blot, Staining, Transfection