Journal: Journal of Pharmaceutical Analysis

Article Title: Targeting SH3GL1 for prognosis and immune response in breast cancer

doi: 10.1016/j.jpha.2025.101377

Figure Lengend Snippet: SH3-domain containing GRB2-like 1 ( SH3GL1 ) is generally highly expressed in breast cancer (BRCA) and is closely related to a variety of immune cells. (A) Hematoxylin and eosin (H&E) staining diagram of BRCA and immunohistochemistry (IHC) staining diagram of breast markers, including pan-cytokeratin, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2). (B) Western blot analysis of the protein expression levels of SH3GL1 in tissues of BRCA and adjacent normal tissue (Adj). ∗∗∗ P < 0.001, in comparison with Adj. (C) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) to detect the mRNA expression level of SH3GL1 in BRCA and Adj in clinical samples, ∗∗∗ P < 0.001, in comparison with Adj. (D) Flow cytometry for the proportion of natural killer (NK) cells, T cells, B cells, regulatory T cells (Treg), M0, M1, dendritic cell (DC) and mast cell in the tissues of BRCA and Adj, ∗∗∗ P < 0.001, in comparison with Adj. (E) RT-qPCR to detect the expression level of SH3GL1 in commonly used BRCA cell lines. Among them, MCF-10A was normal breast epithelial cell, BT474: three Yang; SKBR3: HER2 high expression; MCF-7: ER + ; MDA-MB-453: ER-HER2 + ; MDA-MB-231: Three Yin. ∗ indicates comparison with MCF-10A. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (F, G) Western blot analysis of the protein expression level of SH3GL1 in commonly used BRCA cell lines. In comparison with MCF-10A, ∗ P < 0.01, ∗∗ P < 0.01, ∗∗∗ P < 0.001. The expression values for different cell types were not ordered according to any specific rank. M0/M1: macrophage subtypes. TNBC: triple-negative breast cancer; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. BCRA-1 and BCRA-2 represent breast cancer samples obtained from two independent patients.

Article Snippet: In this study, the BRCA cell lines BT474 (human breast ductal carcinoma cell line, BFN60700229), SKBR3 (human BRCA cell line, BFN60700185), MCF-7 (human BRCA cell line, BFN607200603), MDA-MB-231 (human BRCA cell line, BFN608008564), and MDA-MB-453 (human BRCA cell line, BFN60700116), as well as the normal breast cell line MCF-10A (human breast epithelial cells, BFN60805246) were all purchased from BLUEFBIO (Shenzhen, Guangdong, China) and authenticated by the German DSMZ Leibniz Research Institute in November 2017 and June 2018 using a nano-composite PCR-based (short tandem repeat) STR profile.

Techniques: Staining, Immunohistochemistry, Western Blot, Expressing, Comparison, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Flow Cytometry

Journal: Journal of Pharmaceutical Analysis

Article Title: Targeting SH3GL1 for prognosis and immune response in breast cancer

doi: 10.1016/j.jpha.2025.101377

Figure Lengend Snippet: SH3-domain containing GRB2-like 1 ( SH3GL1 ) in breast cancer (BRCA) epithelial cells promotes their interaction with immune cells. (A) The mRNA expression level of SH3GL1 in normal organoids (Normal-Orgs) and BRCA organoids (BRCA-Orgs) was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). ∗∗ P < 0.01, in comparison with Normal-Orgs. (B) Western blot analysis of SH3GL1 protein expression in Normal-Orgs and BRCA-Orgs. ∗∗ P < 0.01, in comparison with Normal-Orgs. (C) RT-qPCR to detect the mRNA expression level of SH3GL1 in BRCA-Orgs and BT474 before and after co-culture. (D) Western blot analysis of SH3GL1 protein levels in BRCA-Orgs and BT474 before and after co-culture. (E) RT-qPCR to detect mRNA expression of tumor susceptibility marker CD44 in BRCA-Orgs and BT474 before and after co-culture. (F) Representative pictures of proliferating marker Ki67 in BRCA-Orgs before and after co-culture detected by IHC. (G) Statistics of the maximum passage times of BRCA-Orgs before and after co-culture. (H) Statistics on the number of BRCA-Orgs formed from single cells to organoids before and after co-culture. (I) Representative pictures of 5-ethynyl-2′-deoxyuridine (EdU) staining of BT474 cells before and after co-culture. The right side is the relative statistical map of EdU positive cells. (J) Representative images of scratch migration experiment of BT474 cells before and after co-culture. Statistical map of relative scratch closure area on the right. (K) RT-qPCR to detect the mRNA expression of SH3GL1 before and after co-culture of BRCA-Orgs and MDA-MB-231 after knockdown of SH3GL1 . (L,M) Western blot analysis of SH3GL1 protein expression in BRCA-Orgs and MDA-MB-231 cells before and after co-culture after SH3GL1 knockdown (L), and the statistical chart (M). (N) Representative pictures of proliferation marker Ki67 before and after co-culture detected by IHC after BCRA-Orgs SH3GL1 knockdown, and statistical graph. (O) Representative pictures of EdU staining of MDA-MB-231 cells before and after co-culture after SH3GL1 knockdown (red is EdU positive cells), and the relative statistical map of EdU positive cells. (P) Representative images of scratch migration experiment of MDA-MB-231 cells before and after co-culture after SH3GL1 knockdown, and statistical map of relative scratch closure area. In the D–P plots, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (Q) Representative picture of transwell migration induced PBMC (T cells) to BRCA-Orgs, Where fluorescence represents T cells, and statistical graph on the right. In the Q plot, ∗∗∗ P < 0.001, in comparison with Normal-Orgs or shNC. IHC: immunohistochemistry; CD44: tumor susceptibility marker; transwell: migration assay system; PBMC: peripheral blood mononuclear cells.

Article Snippet: In this study, the BRCA cell lines BT474 (human breast ductal carcinoma cell line, BFN60700229), SKBR3 (human BRCA cell line, BFN60700185), MCF-7 (human BRCA cell line, BFN607200603), MDA-MB-231 (human BRCA cell line, BFN608008564), and MDA-MB-453 (human BRCA cell line, BFN60700116), as well as the normal breast cell line MCF-10A (human breast epithelial cells, BFN60805246) were all purchased from BLUEFBIO (Shenzhen, Guangdong, China) and authenticated by the German DSMZ Leibniz Research Institute in November 2017 and June 2018 using a nano-composite PCR-based (short tandem repeat) STR profile.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison, Western Blot, Co-Culture Assay, Marker, Staining, Migration, Knockdown, Fluorescence, Immunohistochemistry, Transwell Migration Assay

Journal: Cancer Management and Research

Article Title: Elevated mRNA Level of Y-Box Binding Protein 1 Indicates Unfavorable Prognosis Correlated with Macrophage Infiltration and T Cell Exhaustion in Luminal Breast Cancer

doi: 10.2147/cmar.s311650

Figure Lengend Snippet: Figure 3 The correlation between YBX1 expression and clinicopathological parameters in BRCA. Notes: (A) The clustering of clinicopathological parameters according to the YBX1 mRNA level using cBioPeortal database. (B) Immunohistochemical staining of YB1 in BRCA samples with different molecular subtypes. (C) The different protein expression of YB1 in human breast epithelial cells and aseries of BRCA cell lines. *P<0.05 and ***P<0.001 vs control group. Abbreviations: BRCA, breast cancer; cBioPortal, the cBio Cancer Genomics Portal database; ns, not significant; TNBC, triple-negative breast cancer; YBX1, Y-box binding protein 1.

Article Snippet: Cell Lines and Antibodies Human breast epithelial MCF-10A cells and human BRCA cells (MCF-7, BT-474, SK-BR-3, MDA-MB-231) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China), and human BRCA cells namely HCC1428, HCC1569 and HCC1143 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Binding Assay

Journal: Cancer Management and Research

Article Title: Elevated mRNA Level of Y-Box Binding Protein 1 Indicates Unfavorable Prognosis Correlated with Macrophage Infiltration and T Cell Exhaustion in Luminal Breast Cancer

doi: 10.2147/cmar.s311650

Figure Lengend Snippet: Figure 7 Effect of YBX1 knockdown on the cytotoxicity of macrophages and CD8+ T cells in co-culture system of BRCA cells and immunocytes. Notes: (A) YBX1 was effectively knocked down by siRNAs in MCF-7, BT-474, and MDA-MB-231 cells. (B) BRCA cells with or without knockdown of YBX1 were cultured alone or co-cultured with peritoneal macrophages, and cell viability was evaluated. For luminal BRCA cells, namely MCF-7 and BT-474, the decrease of cell viability after YBX1 knockdown were more dramatic in co-cultures compared with the single culture system. However, no difference was observed for MDA-MB-231 TNBC cells. (C) IL-2 production by activated CD8+ Tcells co-cultured with BRCA cells transfected with YBX1 siRNA or scrambled negative control siRNA. YBX1 knockdown significantly increased IL-2 secretion in co-cultures of CD8+ Tcells with MCF-7 or BT-474 cells but not in the co-culture with MDA-MB-231 cells. (D) IFN-γ production by activated CD8 + Tcells co-cultured with BRCA cells transfected with YBX1 or scrambled negative control siRNA. YBX1 knockdown significantly increased IFN-γ secretion in the co- cultures of CD8+ Tcells and MCF-7 or BT-474 cells but not in the co-culture with MDA-MB-231 cells. (E) The relative survival of BRCA cells represented by the release of LDH in co-cultures of CD8+ Tcells and BRCA cells with or without YBX1 knockdown. YBX1 depletion markedly reduced the survival of MCF-7 and BT-474 cells but not MDA-MB-231 cells. **P<0.01, ***P<0.001 compared with corresponding negative control groups. Abbreviations: BRCA, breast cancer; IFN-γ, interferon-γ; IL-2, interleukin-2; LDH, lactate dehydrogenase; NC, negative control; YBX1, Y-box binding protein 1.

Article Snippet: Cell Lines and Antibodies Human breast epithelial MCF-10A cells and human BRCA cells (MCF-7, BT-474, SK-BR-3, MDA-MB-231) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China), and human BRCA cells namely HCC1428, HCC1569 and HCC1143 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Co-Culture Assay, Cell Culture, Transfection, Negative Control, Binding Assay

Journal: Oncotarget

Article Title: Polyphenol-rich extract of Pimenta dioica berries (Allspice) kills breast cancer cells by autophagy and delays growth of triple negative breast cancer in athymic mice

doi:

Figure Lengend Snippet: A. Dose-dependent decrease in viability of BrCa cells exposed to AAE for 72h. MTT assay was used to determine percent of viable cells. Each graph was drawn from data pooled from three independent experiments containing optical densities of three triplicate culture wells treated with AAE. Points shown are Mean ± Standard Error (SE). CM=complete medium B. Exposure time-dependent decrease in viability of BrCa cells with AAE. Culture wells received indicated amount of AAE for indicated duration. MTT assay was used to determine viability at the end of 48h. B (i): Viability of MCF7 cells; B (ii): Viability of MB231 cells. C. Normal (non-tumorigenic) breast epithelial cells are resistant to cytotoxicity by AAE. Cytotoxicity of AAE on proliferating and quiescent normal breast epithelial cell cultures (MCF10A). MCF10A cells cultured for 24h with or without growth factor containing basal medium [MEBM ± Growth Factors Mix (Single-Quots, Lonza Inc., Allendale, NJ)] were exposed to AAE for 72h and surviving MCF10A cells were estimated using MTT assay . D. Cytotoxicity of AAE on serum-starved and non-starved Human Lung Fibroblasts (HLFs). About 5 × 10 3 HLFs/well/48well clustures were cultured in in CM or RPMI + 0.1%BSA for starvation. Cells were then treated with AAE for 72h and viability quantified by MTT assay. E. AAE reduces clonogenic potential of BrCa cells when treated at low density. BrCa cells plated at low density (0.5 – 1 × 10 3 cells/well/2ml) in 6-well plates were incubated with several concentrations of AAE for 24h. Culture wells were then changed to normal culture medium without AAE and surviving cells were allowed to form colonies for 7-10 days. Data shown in Panel A-E are mean ± SEM, n ≥ 3.

Article Snippet: All human BrCa cell lines (MCF7, MDA-MB231, SKBr3, BT474, and T47D) and non-malignant breast epithelial cell lines MCF-10A and MCF-12A were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: MTT Assay, Cell Culture, Incubation

Journal: Oncotarget

Article Title: Polyphenol-rich extract of Pimenta dioica berries (Allspice) kills breast cancer cells by autophagy and delays growth of triple negative breast cancer in athymic mice

doi:

Figure Lengend Snippet: A & B. Lack of cell death caspase (Caspase3/7) activation by AAE in T47D and MB321 cells. T47D and MB231 cell cultures in 96-well clusters were treated with 50-150 μg/ml AAE for 24h. Duplicate wells are also treated with 1μM staurosporine, a known inducer of caspase 3/7 in these cells. Caspase 3/7 activity was measured by Caspase-Glo 3/7 assay systems (Promega, Madison, WI). Data shown are Mean ± SEM, n = 6. C. Lack of cleaved-PARP in BrCa cells treated with AAE for 24h. Cells (1.0 × 10 4 cells/well) seeded in 12-well plates were treated with indicated amount of AAE for 24h. Cleaved PARP levels were detected using western blots of cell lysates from indicated cultures. D & E. Apoptosis determined by TUNEL assay of 24h AAE-treated MCF7 ( D ) and MB231 ( E ) BrCa cells. Green (FITC) TUNEL-positive cells and DAPI images (630X 600x). Results from a typical experiment is shown. Similar results were obtained from two more repeats. UT: untreated, ST cells: 1 μM staurosporine.

Article Snippet: All human BrCa cell lines (MCF7, MDA-MB231, SKBr3, BT474, and T47D) and non-malignant breast epithelial cell lines MCF-10A and MCF-12A were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Activation Assay, Activity Assay, Caspase-Glo Assay, Western Blot, TUNEL Assay

Journal: Oncotarget

Article Title: Polyphenol-rich extract of Pimenta dioica berries (Allspice) kills breast cancer cells by autophagy and delays growth of triple negative breast cancer in athymic mice

doi:

Figure Lengend Snippet: A. Morphological features of autophagy in AAE treated MB231 cells as seen under phase-contrast microscopy. Magnification: 100X. Cells became vacuolated after AAE or rapamycin treatment for 24h. B. AAE induced autophagy in BrCa cells. Increased levels of LC3B protein in BrCa cells treated with AAE. C.(i). Immunofluorescence detection of LC3B puncta induction in AAE treated BrCa cells. Untreated cells are shown in (a) and AAE treated cells are shown in (b: 100 μg/ml) and (c: 150 μg/ml). Micrograph magnification: 630X. C. (ii): Quantification of number of puncta/cell in treated and control cultures. There was a significant (** p < 0.05) increase in LC3B puncta in treated cells as compared to that of control. D. Increased levels of autolysosomes in AAE treated-cells as visualized by red-fluorescence (mCherry) and quenching of GFP in acidic compartment of lysosomes resulting from fusion of autophagosome and lysosome. Confocal microscopy was used to obtain multicolor micrographs (Magnification: 630X); E. AAE induced autophagic flux in BrCa cells. BrCa cells were incubated with lysosomal inhibitors pepstatin A and E64d (5 μg/ml and 10 μg/ml each, for MCF-7 cells and 0.5 μg/ml for MB231 cells) and/or AAE (100 μg/ml) for 24h. Cell lysates were analyzed by immunoblotting for LC3BII (LC3B) levels, to distinguish between autophagy induction and inhibition of autophagosome turnover.

Article Snippet: All human BrCa cell lines (MCF7, MDA-MB231, SKBr3, BT474, and T47D) and non-malignant breast epithelial cell lines MCF-10A and MCF-12A were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Microscopy, Immunofluorescence, Control, Fluorescence, Confocal Microscopy, Incubation, Western Blot, Inhibition

Journal: Oncotarget

Article Title: Polyphenol-rich extract of Pimenta dioica berries (Allspice) kills breast cancer cells by autophagy and delays growth of triple negative breast cancer in athymic mice

doi:

Figure Lengend Snippet: A. & B. Immunoblotting of gene silencing by siRNA of ATG7 and ATG5 in MCF7 and MD231 cells. C. & D. : Percent of viable cells following various transfections with or without exposure to AAE. Transfected cells were harvested 48h after siRNA transfections, half the cell suspension used for protein estimation and immunoblotting. The other half of the harvested cells were incubated with 100 μg/ml AAE for 24h. Percent of live cells (viability) was determined after counting live and dead cells using trypan blue staining. Cell Viability was significantly rescued by autophagy inhibition of ATG7 and ATG5 compared to the background siRNA control group. E. Increased cytotoxicity in BrCa treated with both AAE and an autophagy inducer rapamycin. BrCa cells were treated either with AAE (25-75μg/ml) or rapamycin (10-50 nM) alone or with both drugs of various concentrations for another 72h. Cell viability was measured by MTT assay. Cell Viability decreased significantly in both treated MCF7 cells (* P < 0.05) and M231 cells (* P < 0.05) compared to AAE/rapamycin treated cells respectively. F. AAE and rapamycin synergistically induce cytotoxicity. The combination Index plot was generated using Compusyn (ComboSyn, Inc., Paramus, NJ, USA). Blue round circles indicate the data points in the drug combination treatment.

Article Snippet: All human BrCa cell lines (MCF7, MDA-MB231, SKBr3, BT474, and T47D) and non-malignant breast epithelial cell lines MCF-10A and MCF-12A were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Western Blot, Transfection, Suspension, Incubation, Staining, Inhibition, Control, MTT Assay, Generated

Journal: Oncotarget

Article Title: Polyphenol-rich extract of Pimenta dioica berries (Allspice) kills breast cancer cells by autophagy and delays growth of triple negative breast cancer in athymic mice

doi:

Figure Lengend Snippet: BrCa cell cultures were treated with AAE for 24 h, cells were harvested and cell extracts were analyzed by immunoblotting. The levels of total Akt and mTOR, phospho-Akt, phospho-mTOR, total S6 ribosomal protein and 4E-BP1, phospho-S6 and phospho-4E-BP1, total ULK1 and phospho-ULK1, as well as ATG13 were detected by immunoblotting. Positive control used for panel A is AKT inhibitor, SH-5 at 20 μM; for panel B is mTOR inhibitor rapamycin at 25 nM. Levels of β-actin was used as a loading control. Since protein lysates from same experiment were analyzed for detecting signaling proteins, a single β-actin blot is shown at the bottom. All blots were repeated using new samples twice, with comparable results.

Article Snippet: All human BrCa cell lines (MCF7, MDA-MB231, SKBr3, BT474, and T47D) and non-malignant breast epithelial cell lines MCF-10A and MCF-12A were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Western Blot, Positive Control, Control

Journal: Annals of Translational Medicine

Article Title: The expression landscape of FOXP3 and its prognostic value in breast cancer

doi: 10.21037/atm-22-3080

Figure Lengend Snippet: FOXP3 expression in cell lines. (A) FOXP3 mRNA expression data in various cancer cell lines from the HPA database; (B) FOXP3 protein located in the nucleoplasm of MCF-7 cells from the HPA database (IF, ×400); (C) FOXP3 mRNA expression in MCF-7, MDA-MB-231, MDA-MB-453, BT-474, and SK-BR-3 BRCA cell lines. PBMC was regarded as the positive control; (D) FOXP3 protein expression decreased in MCF-7 transfected with FOXP3 siRNA by western blot; (E) FOXP3 mRNA expression decreased in MCF-7 transfected with FOXP3 siRNA by qRT-PCR; (F) FOXP3 knockdown enhanced the ability of MCF-7 cell migration evaluated by wound healing assay (microscope, ×100); (G) FOXP3 knockdown enhanced the invasion ability of MCF-7 cell in transwell chamber stained with crystal violet (microscope, ×200). **, P<0.01, ***, P<0.001. ns, no significance; qRT-PCR, quantitative real-time PCR; HPA, human protein atlas; IF, immunofluorescence; PBMC, peripheral blood mononuclear cell.

Article Snippet: In addition, we purchased five BRCA cell lines (MCF-7, MDA-MB-231, MDA-MB-453, BT-474, and SK-BR-3) from the American Type Culture Collection (ATCC) bank and cultured these cells in 10% FBS + RPMI1640 in our laboratory.

Techniques: Expressing, Positive Control, Transfection, Western Blot, Quantitative RT-PCR, Knockdown, Migration, Wound Healing Assay, Microscopy, Staining, Real-time Polymerase Chain Reaction, Immunofluorescence