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