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Procell Inc mouse breast cancer cell lines emt6

2-DG induces death of <t>EMT6</t> and 4T1 cells. (A1, A2) CCK-8 assays showed dose-dependent cytotoxicity (n = 6). Data were mean ± SD * P < 0.05, ** P < 0.01, ^ P < 0.05, ^^ P < 0.01. (B1, B2) Crystal violet staining revealed concentration-dependent reduction in clonogenicity (n = 3, ** P < 0.01). (C1, C2) Light microscopy images displayed morphological changes, with red arrows indicating cellular swelling, membrane rupture, and gas bubble extrusion (n = 3). (D) CCK-8 assays showed no significant cell viability changes in HC11 cells across 2-DG concentrations at 24 h and 48 h (n = 6, Data were mean ± SD).

Mouse Breast Cancer Cell Lines Emt6, supplied by Procell Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Administration of 2-deoxy-D-glucose induces pyroptosis in murine breast cancer cells via cAMP/PKA/HK2 to impair tumor survival"

Article Title: Administration of 2-deoxy-D-glucose induces pyroptosis in murine breast cancer cells via cAMP/PKA/HK2 to impair tumor survival

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2025.1724476

2-DG induces death of EMT6 and 4T1 cells. (A1, A2) CCK-8 assays showed dose-dependent cytotoxicity (n = 6). Data were mean ± SD * P < 0.05, ** P < 0.01, ^ P < 0.05, ^^ P < 0.01. (B1, B2) Crystal violet staining revealed concentration-dependent reduction in clonogenicity (n = 3, ** P < 0.01). (C1, C2) Light microscopy images displayed morphological changes, with red arrows indicating cellular swelling, membrane rupture, and gas bubble extrusion (n = 3). (D) CCK-8 assays showed no significant cell viability changes in HC11 cells across 2-DG concentrations at 24 h and 48 h (n = 6, Data were mean ± SD).

Figure Legend Snippet: 2-DG induces death of EMT6 and 4T1 cells. (A1, A2) CCK-8 assays showed dose-dependent cytotoxicity (n = 6). Data were mean ± SD * P < 0.05, ** P < 0.01, ^ P < 0.05, ^^ P < 0.01. (B1, B2) Crystal violet staining revealed concentration-dependent reduction in clonogenicity (n = 3, ** P < 0.01). (C1, C2) Light microscopy images displayed morphological changes, with red arrows indicating cellular swelling, membrane rupture, and gas bubble extrusion (n = 3). (D) CCK-8 assays showed no significant cell viability changes in HC11 cells across 2-DG concentrations at 24 h and 48 h (n = 6, Data were mean ± SD).

Techniques Used: CCK-8 Assay, Staining, Concentration Assay, Light Microscopy, Membrane

2-DG induces pyroptosis in EMT6 and 4T1 cells. (A1, A2) CCK-8 assays demonstrated that Z-VAD-FMK, but not Nec-1, abrogated 2-DG-induced cytotoxicity (n = 6, ** P < 0.01). (B1, B2) Immunoblotting assays showed no significant upregulation of necroptosis markers (n = 3, P > 0.05). (C1, C2) Immunoblot analyses revealed activation of pyroptosis mediators (n = 3, ** P < 0.01). (D1, D2) Quantification of LDH release and IL-1β/IL-18 secretion confirmed pyroptotic features (n = 3, ** P < 0.01).

Figure Legend Snippet: 2-DG induces pyroptosis in EMT6 and 4T1 cells. (A1, A2) CCK-8 assays demonstrated that Z-VAD-FMK, but not Nec-1, abrogated 2-DG-induced cytotoxicity (n = 6, ** P < 0.01). (B1, B2) Immunoblotting assays showed no significant upregulation of necroptosis markers (n = 3, P > 0.05). (C1, C2) Immunoblot analyses revealed activation of pyroptosis mediators (n = 3, ** P < 0.01). (D1, D2) Quantification of LDH release and IL-1β/IL-18 secretion confirmed pyroptotic features (n = 3, ** P < 0.01).

Techniques Used: CCK-8 Assay, Western Blot, Activation Assay

GSDME is the executor of 2-DG-induced pyroptosis. (A1, A2) Immunofluorescence staining (n = 3) showed diffuse GSDME localization in 2-DG-treated EMT6 and 4T1 cells. (B1, B2) Immunoblot assays (n = 3) confirmed effective GSDME silencing by si GSDME, ** P < 0.01. (C1, C2) Light microscopy images (n = 3) revealed fewer pyroptotic cells in 2-DG + si GSDME groups compared to 2-DG alone. (D1, D2-F1, F2) Quantification assays (n = 3) showed reduced secretion of IL-1β, IL-18, and LDH in 2-DG + si GSDME groups versus 2-DG alone. Data were mean ± SD, ** P < 0.01.

Figure Legend Snippet: GSDME is the executor of 2-DG-induced pyroptosis. (A1, A2) Immunofluorescence staining (n = 3) showed diffuse GSDME localization in 2-DG-treated EMT6 and 4T1 cells. (B1, B2) Immunoblot assays (n = 3) confirmed effective GSDME silencing by si GSDME, ** P < 0.01. (C1, C2) Light microscopy images (n = 3) revealed fewer pyroptotic cells in 2-DG + si GSDME groups compared to 2-DG alone. (D1, D2-F1, F2) Quantification assays (n = 3) showed reduced secretion of IL-1β, IL-18, and LDH in 2-DG + si GSDME groups versus 2-DG alone. Data were mean ± SD, ** P < 0.01.

Techniques Used: Immunofluorescence, Staining, Western Blot, Light Microscopy

Caspase-3 mediates 2-DG-induced GSDME activation as an upstream factor. (A1, A2) Immunoblot analyses (n = 3) showed 2-DG induces GSDME cleavage in EMT6/4T1 cells, while Z-VAD-FMK (pan-caspase inhibitor) attenuates these changes. Quantification (normalized to β-actin): mean ± SD; ** P < 0.01, ^^ P < 0.01. (B1, B2) Quantification assays (n = 3) showed Z-VAD-FMK suppressed 2-DG-induced IL-1β release in both cell lines. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (C1, C2) Quantification assays (n = 3) showed Z-VAD-FMK suppressed 2-DG-induced IL-18 release in both cell lines. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (D1, D2) LDH release assays (n = 3) showed Z-VAD-FMK pretreatment reduced 2-DG-induced cytotoxicity in EMT6/4T1 cells. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (E1, E2) Immunoblot analyses (n = 3) showed 2-DG upregulated cleaved caspase-3 (17 kDa, activated), while Z-DEVD-FMK (caspase-3 inhibitor) reduced this activation. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (F1, F2) Immunoblot analyses (n = 3) showed Z-DEVD-FMK modulated 2-DG-mediated expression of full-length GSDME (55 kDa) and GSDME-NT (35 kDa). Data were mean ± SD, ** P < 0.01, ^^ P < 0.01.

Figure Legend Snippet: Caspase-3 mediates 2-DG-induced GSDME activation as an upstream factor. (A1, A2) Immunoblot analyses (n = 3) showed 2-DG induces GSDME cleavage in EMT6/4T1 cells, while Z-VAD-FMK (pan-caspase inhibitor) attenuates these changes. Quantification (normalized to β-actin): mean ± SD; ** P < 0.01, ^^ P < 0.01. (B1, B2) Quantification assays (n = 3) showed Z-VAD-FMK suppressed 2-DG-induced IL-1β release in both cell lines. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (C1, C2) Quantification assays (n = 3) showed Z-VAD-FMK suppressed 2-DG-induced IL-18 release in both cell lines. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (D1, D2) LDH release assays (n = 3) showed Z-VAD-FMK pretreatment reduced 2-DG-induced cytotoxicity in EMT6/4T1 cells. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (E1, E2) Immunoblot analyses (n = 3) showed 2-DG upregulated cleaved caspase-3 (17 kDa, activated), while Z-DEVD-FMK (caspase-3 inhibitor) reduced this activation. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (F1, F2) Immunoblot analyses (n = 3) showed Z-DEVD-FMK modulated 2-DG-mediated expression of full-length GSDME (55 kDa) and GSDME-NT (35 kDa). Data were mean ± SD, ** P < 0.01, ^^ P < 0.01.

Techniques Used: Activation Assay, Western Blot, Expressing

2-DG inhibits HK2 and activates the cAMP/PKA pathway to cause pyroptosis in EMT6 and 4T1 cells. (A) Top enriched pathways, such as the cAMP signaling system, were found by comparing genes linked to breast cancer (GeneCards) and 2-DG targets (SwissTargetPrediction), which were displayed as a bar plot of -log10(P-values). (B) Western blot analyses (n = 3) show that 2-DG upregulated phosphorylated CREB (p-CREB) and PKA (p-PKA), consistent with pathway activation. Quantification (normalized to β-actin) was presented as mean ± SD, ** P < 0.01. (C) Western blot analyses (n = 3) demonstrating 2-DG downregulated HK2. Quantification (normalized to β-actin) was presented as mean ± SD, ** P < 0.01. (D) Western blot (n = 3) showing H-89 attenuated 2-DG-induced HK2 downregulation and PKA phosphorylation changes. Quantification (β-actin-normalized, mean ± SD, ** P < 0.01). (E) Co-immunoprecipitation (Co-IP) tests (n = 3) verify that HK2 and Caspase-3 interact physically. Protein expression was confirmed by input controls, whereas IgG acted as a negative control. Pyroptosis signals may be modulated by this relationship. (F) Western blot experiments (n = 3) showing how 2-DG and Insulin affect the amounts of Caspase-3, Cleaved-caspase-3, GSDME, and GSDME-NT protein expression. An effective loading control was β-actin. Quantification of relative protein expression is shown in the bar graph. ** P < 0.01. Insulin was used at a concentration of 100 nM to treat EMT6 and 4T1 cells for 48 h, aiming to activate HK2 expression.

Figure Legend Snippet: 2-DG inhibits HK2 and activates the cAMP/PKA pathway to cause pyroptosis in EMT6 and 4T1 cells. (A) Top enriched pathways, such as the cAMP signaling system, were found by comparing genes linked to breast cancer (GeneCards) and 2-DG targets (SwissTargetPrediction), which were displayed as a bar plot of -log10(P-values). (B) Western blot analyses (n = 3) show that 2-DG upregulated phosphorylated CREB (p-CREB) and PKA (p-PKA), consistent with pathway activation. Quantification (normalized to β-actin) was presented as mean ± SD, ** P < 0.01. (C) Western blot analyses (n = 3) demonstrating 2-DG downregulated HK2. Quantification (normalized to β-actin) was presented as mean ± SD, ** P < 0.01. (D) Western blot (n = 3) showing H-89 attenuated 2-DG-induced HK2 downregulation and PKA phosphorylation changes. Quantification (β-actin-normalized, mean ± SD, ** P < 0.01). (E) Co-immunoprecipitation (Co-IP) tests (n = 3) verify that HK2 and Caspase-3 interact physically. Protein expression was confirmed by input controls, whereas IgG acted as a negative control. Pyroptosis signals may be modulated by this relationship. (F) Western blot experiments (n = 3) showing how 2-DG and Insulin affect the amounts of Caspase-3, Cleaved-caspase-3, GSDME, and GSDME-NT protein expression. An effective loading control was β-actin. Quantification of relative protein expression is shown in the bar graph. ** P < 0.01. Insulin was used at a concentration of 100 nM to treat EMT6 and 4T1 cells for 48 h, aiming to activate HK2 expression.

Techniques Used: Western Blot, Activation Assay, Phospho-proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Negative Control, Control, Concentration Assay

Image Search Results

Genome-wide CRISPR-Cas9 screen identifies genetic regulators of CD47 expression in murine cancer cell lines. (A) Schematic of the FACS-based CRISPR screening pipeline. Mouse cancer cells (B16F10A, MC38, and EMT6) were transduced with the mTKO genome-wide CRISPR-Cas9 library, followed by puromycin selection and passaging for 5 days to ensure stable sgRNA integration. Cells were stained with fluorophore-conjugated anti-CD47 antibodies and sorted by FACS into the top 30% (CD47 high ) and bottom 30% (CD47 low ) populations. Genomic DNA was extracted, and sgRNA abundance was determined by NGS and analyzed using DrugZ to calculate NormZ scores. (B) Ranked NormZ scores of sgRNAs in B16F10A, MC38, and EMT6 cells. Negative NormZ scores indicate positive regulators of CD47 (gene knockouts reduce CD47 expression), while positive NormZ scores indicate negative regulators (gene knockouts increase CD47 expression). CD47 itself ranked as the top positive regulator in all three cell lines, validating the screen’s robustness. (C) GO enrichment analysis of significant negative regulators of CD47 expression (|NormZ| > 3) in B16F10A and MC38 cells. Negative regulators were significantly enriched in pathways related to mitochondrial signaling, including oxidative phosphorylation, mitochondrial translation, and respiratory chain complex assembly.

Journal: Frontiers in Immunology

Article Title: FACS-based genome-wide CRISPR screening platform identifies modulators of CD47

doi: 10.3389/fimmu.2025.1684539

Figure Lengend Snippet: Genome-wide CRISPR-Cas9 screen identifies genetic regulators of CD47 expression in murine cancer cell lines. (A) Schematic of the FACS-based CRISPR screening pipeline. Mouse cancer cells (B16F10A, MC38, and EMT6) were transduced with the mTKO genome-wide CRISPR-Cas9 library, followed by puromycin selection and passaging for 5 days to ensure stable sgRNA integration. Cells were stained with fluorophore-conjugated anti-CD47 antibodies and sorted by FACS into the top 30% (CD47 high ) and bottom 30% (CD47 low ) populations. Genomic DNA was extracted, and sgRNA abundance was determined by NGS and analyzed using DrugZ to calculate NormZ scores. (B) Ranked NormZ scores of sgRNAs in B16F10A, MC38, and EMT6 cells. Negative NormZ scores indicate positive regulators of CD47 (gene knockouts reduce CD47 expression), while positive NormZ scores indicate negative regulators (gene knockouts increase CD47 expression). CD47 itself ranked as the top positive regulator in all three cell lines, validating the screen’s robustness. (C) GO enrichment analysis of significant negative regulators of CD47 expression (|NormZ| > 3) in B16F10A and MC38 cells. Negative regulators were significantly enriched in pathways related to mitochondrial signaling, including oxidative phosphorylation, mitochondrial translation, and respiratory chain complex assembly.

Article Snippet: Mouse melanoma cell line B16F10A, mouse colon cancer cell line MC38, mouse breast cancer cell line EMT6, two mouse macrophage cells J774A-1, RAW264.7 are purchased from the American Type Culture Collection (ATCC).

Techniques: Genome Wide, CRISPR, Expressing, Transduction, Selection, Passaging, Staining, Phospho-proteomics

DNAJC13 is a conserved positive regulator of CD47 expression identified across multiple cancer cell lines. (A) Venn diagram of significant positive regulators of CD47 expression identified in the genome-wide CRISPR screen. CD47 and DNAJC13 were the only two genes consistently identified as significant positive regulators (|NormZ| > 3) across all three cell lines (B16F10A, MC38, and EMT6). (B) Correlation of DNAJC13 and CD47 expression in human cancers. Scatter plots generated from TCGA datasets via GEPIA2 show a positive correlation between DNAJC13 and CD47 expression in breast cancer (BRCA), colon adenocarcinoma (COAD), and skin cutaneous melanoma (SKCM). Pearson correlation coefficients (r) and p-values are indicated. (C) Western blot validation of DNAJC13 regulation of CD47 expression. CRISPR-Cas9–mediated DNAJC13 knockout (three independent sgRNAs: DNAJC13#1, #2, #3) markedly reduced CD47 protein levels compared with vector control in B16F10A, MC38, and EMT6 cells. GAPDH was used as a loading control. (D) Flow cytometry analysis of surface CD47 expression. Representative FACS histograms show reduced surface CD47 expression in DNAJC13 KO cells (green, yellow, and orange peaks; three independent sgRNAs) compared with vector controls (red) in B16F10A, MC38, and EMT6 cells. Isotype control is shown in blue. Quantification of Mean fluorescence intensity (MFI) is shown on the right.

Journal: Frontiers in Immunology

Article Title: FACS-based genome-wide CRISPR screening platform identifies modulators of CD47

doi: 10.3389/fimmu.2025.1684539

Figure Lengend Snippet: DNAJC13 is a conserved positive regulator of CD47 expression identified across multiple cancer cell lines. (A) Venn diagram of significant positive regulators of CD47 expression identified in the genome-wide CRISPR screen. CD47 and DNAJC13 were the only two genes consistently identified as significant positive regulators (|NormZ| > 3) across all three cell lines (B16F10A, MC38, and EMT6). (B) Correlation of DNAJC13 and CD47 expression in human cancers. Scatter plots generated from TCGA datasets via GEPIA2 show a positive correlation between DNAJC13 and CD47 expression in breast cancer (BRCA), colon adenocarcinoma (COAD), and skin cutaneous melanoma (SKCM). Pearson correlation coefficients (r) and p-values are indicated. (C) Western blot validation of DNAJC13 regulation of CD47 expression. CRISPR-Cas9–mediated DNAJC13 knockout (three independent sgRNAs: DNAJC13#1, #2, #3) markedly reduced CD47 protein levels compared with vector control in B16F10A, MC38, and EMT6 cells. GAPDH was used as a loading control. (D) Flow cytometry analysis of surface CD47 expression. Representative FACS histograms show reduced surface CD47 expression in DNAJC13 KO cells (green, yellow, and orange peaks; three independent sgRNAs) compared with vector controls (red) in B16F10A, MC38, and EMT6 cells. Isotype control is shown in blue. Quantification of Mean fluorescence intensity (MFI) is shown on the right.

Techniques: Expressing, Genome Wide, CRISPR, Generated, Western Blot, Biomarker Discovery, Knock-Out, Plasmid Preparation, Control, Flow Cytometry, Fluorescence

DNAJC13 negatively correlates with macrophage infiltration and regulates macrophage-mediated phagocytosis. (A) Correlation between gene expression and macrophage infiltration in human cancers. Scatter plots generated using TIMER2 show negative correlations between CD47 expression (top row) or DNAJC13 expression (bottom row) and macrophage infiltration levels in breast cancer (BRCA), colon adenocarcinoma (COAD), and skin cutaneous melanoma (SKCM). Spearman’s correlation coefficients (Rho) and p-values are indicated. (B, C) DNAJC13 loss enhances macrophage phagocytosis in vitro . Flow cytometry analysis of macrophage-mediated phagocytosis in co-culture assays. DNAJC13 knockout (KO) or CD47 KO cancer cells (B16F10A, MC38, EMT6) were co-cultured with (B) RAW 264.7 or (C) J774 macrophages for the indicated time. Representative FACS plots show increased phagocytosis (higher engulfment rate) in DNAJC13-KO and CD47-KO groups compared with vector controls.

Journal: Frontiers in Immunology

Article Title: FACS-based genome-wide CRISPR screening platform identifies modulators of CD47

doi: 10.3389/fimmu.2025.1684539

Figure Lengend Snippet: DNAJC13 negatively correlates with macrophage infiltration and regulates macrophage-mediated phagocytosis. (A) Correlation between gene expression and macrophage infiltration in human cancers. Scatter plots generated using TIMER2 show negative correlations between CD47 expression (top row) or DNAJC13 expression (bottom row) and macrophage infiltration levels in breast cancer (BRCA), colon adenocarcinoma (COAD), and skin cutaneous melanoma (SKCM). Spearman’s correlation coefficients (Rho) and p-values are indicated. (B, C) DNAJC13 loss enhances macrophage phagocytosis in vitro . Flow cytometry analysis of macrophage-mediated phagocytosis in co-culture assays. DNAJC13 knockout (KO) or CD47 KO cancer cells (B16F10A, MC38, EMT6) were co-cultured with (B) RAW 264.7 or (C) J774 macrophages for the indicated time. Representative FACS plots show increased phagocytosis (higher engulfment rate) in DNAJC13-KO and CD47-KO groups compared with vector controls.

Techniques: Gene Expression, Generated, Expressing, In Vitro, Flow Cytometry, Co-Culture Assay, Knock-Out, Cell Culture, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Administration of 2-deoxy-D-glucose induces pyroptosis in murine breast cancer cells via cAMP/PKA/HK2 to impair tumor survival

doi: 10.3389/fimmu.2025.1724476

Figure Lengend Snippet: 2-DG induces death of EMT6 and 4T1 cells. (A1, A2) CCK-8 assays showed dose-dependent cytotoxicity (n = 6). Data were mean ± SD * P < 0.05, ** P < 0.01, ^ P < 0.05, ^^ P < 0.01. (B1, B2) Crystal violet staining revealed concentration-dependent reduction in clonogenicity (n = 3, ** P < 0.01). (C1, C2) Light microscopy images displayed morphological changes, with red arrows indicating cellular swelling, membrane rupture, and gas bubble extrusion (n = 3). (D) CCK-8 assays showed no significant cell viability changes in HC11 cells across 2-DG concentrations at 24 h and 48 h (n = 6, Data were mean ± SD).

Article Snippet: Procell Life Science & Technology Co., Ltd. (Wuhan, China) provided the mouse breast cancer cell lines EMT6 and 4T1, as well as the non-cancerous murine mammary epithelial cell line HC11.

Techniques: CCK-8 Assay, Staining, Concentration Assay, Light Microscopy, Membrane

Journal: Frontiers in Immunology

Article Title: Administration of 2-deoxy-D-glucose induces pyroptosis in murine breast cancer cells via cAMP/PKA/HK2 to impair tumor survival

doi: 10.3389/fimmu.2025.1724476

Figure Lengend Snippet: 2-DG induces pyroptosis in EMT6 and 4T1 cells. (A1, A2) CCK-8 assays demonstrated that Z-VAD-FMK, but not Nec-1, abrogated 2-DG-induced cytotoxicity (n = 6, ** P < 0.01). (B1, B2) Immunoblotting assays showed no significant upregulation of necroptosis markers (n = 3, P > 0.05). (C1, C2) Immunoblot analyses revealed activation of pyroptosis mediators (n = 3, ** P < 0.01). (D1, D2) Quantification of LDH release and IL-1β/IL-18 secretion confirmed pyroptotic features (n = 3, ** P < 0.01).

Techniques: CCK-8 Assay, Western Blot, Activation Assay

Journal: Frontiers in Immunology

Article Title: Administration of 2-deoxy-D-glucose induces pyroptosis in murine breast cancer cells via cAMP/PKA/HK2 to impair tumor survival

doi: 10.3389/fimmu.2025.1724476

Figure Lengend Snippet: GSDME is the executor of 2-DG-induced pyroptosis. (A1, A2) Immunofluorescence staining (n = 3) showed diffuse GSDME localization in 2-DG-treated EMT6 and 4T1 cells. (B1, B2) Immunoblot assays (n = 3) confirmed effective GSDME silencing by si GSDME, ** P < 0.01. (C1, C2) Light microscopy images (n = 3) revealed fewer pyroptotic cells in 2-DG + si GSDME groups compared to 2-DG alone. (D1, D2-F1, F2) Quantification assays (n = 3) showed reduced secretion of IL-1β, IL-18, and LDH in 2-DG + si GSDME groups versus 2-DG alone. Data were mean ± SD, ** P < 0.01.

Techniques: Immunofluorescence, Staining, Western Blot, Light Microscopy

Journal: Frontiers in Immunology

Article Title: Administration of 2-deoxy-D-glucose induces pyroptosis in murine breast cancer cells via cAMP/PKA/HK2 to impair tumor survival

doi: 10.3389/fimmu.2025.1724476

Figure Lengend Snippet: Caspase-3 mediates 2-DG-induced GSDME activation as an upstream factor. (A1, A2) Immunoblot analyses (n = 3) showed 2-DG induces GSDME cleavage in EMT6/4T1 cells, while Z-VAD-FMK (pan-caspase inhibitor) attenuates these changes. Quantification (normalized to β-actin): mean ± SD; ** P < 0.01, ^^ P < 0.01. (B1, B2) Quantification assays (n = 3) showed Z-VAD-FMK suppressed 2-DG-induced IL-1β release in both cell lines. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (C1, C2) Quantification assays (n = 3) showed Z-VAD-FMK suppressed 2-DG-induced IL-18 release in both cell lines. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (D1, D2) LDH release assays (n = 3) showed Z-VAD-FMK pretreatment reduced 2-DG-induced cytotoxicity in EMT6/4T1 cells. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (E1, E2) Immunoblot analyses (n = 3) showed 2-DG upregulated cleaved caspase-3 (17 kDa, activated), while Z-DEVD-FMK (caspase-3 inhibitor) reduced this activation. Data were mean ± SD, ** P < 0.01, ^^ P < 0.01. (F1, F2) Immunoblot analyses (n = 3) showed Z-DEVD-FMK modulated 2-DG-mediated expression of full-length GSDME (55 kDa) and GSDME-NT (35 kDa). Data were mean ± SD, ** P < 0.01, ^^ P < 0.01.

Techniques: Activation Assay, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Administration of 2-deoxy-D-glucose induces pyroptosis in murine breast cancer cells via cAMP/PKA/HK2 to impair tumor survival

doi: 10.3389/fimmu.2025.1724476

Figure Lengend Snippet: 2-DG inhibits HK2 and activates the cAMP/PKA pathway to cause pyroptosis in EMT6 and 4T1 cells. (A) Top enriched pathways, such as the cAMP signaling system, were found by comparing genes linked to breast cancer (GeneCards) and 2-DG targets (SwissTargetPrediction), which were displayed as a bar plot of -log10(P-values). (B) Western blot analyses (n = 3) show that 2-DG upregulated phosphorylated CREB (p-CREB) and PKA (p-PKA), consistent with pathway activation. Quantification (normalized to β-actin) was presented as mean ± SD, ** P < 0.01. (C) Western blot analyses (n = 3) demonstrating 2-DG downregulated HK2. Quantification (normalized to β-actin) was presented as mean ± SD, ** P < 0.01. (D) Western blot (n = 3) showing H-89 attenuated 2-DG-induced HK2 downregulation and PKA phosphorylation changes. Quantification (β-actin-normalized, mean ± SD, ** P < 0.01). (E) Co-immunoprecipitation (Co-IP) tests (n = 3) verify that HK2 and Caspase-3 interact physically. Protein expression was confirmed by input controls, whereas IgG acted as a negative control. Pyroptosis signals may be modulated by this relationship. (F) Western blot experiments (n = 3) showing how 2-DG and Insulin affect the amounts of Caspase-3, Cleaved-caspase-3, GSDME, and GSDME-NT protein expression. An effective loading control was β-actin. Quantification of relative protein expression is shown in the bar graph. ** P < 0.01. Insulin was used at a concentration of 100 nM to treat EMT6 and 4T1 cells for 48 h, aiming to activate HK2 expression.

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Negative Control, Control, Concentration Assay

Fenbendazole (FBZ) induces pyroptosis in EMT6 breast cancer cells. (A) CCK-8 assay for cell proliferation. Cell lines: EMT6 (top) and HCT116 (bottom, negative control for FBZ sensitivity). Treatment: FBZ at 0.5–8 μM for 24 h (n = 6, mean ± SD). (B) Pyroptotic morphology observation (×200 magnification, scale bar = 50 μm). Phenotypes: CON (Control): Normal cell morphology. FBZ (2 μM, 24 h): Swelling, blistering (red arrows), characteristic of pyroptosis. (C) qPCR analysis of pyroptosis-related genes. Treatment: FBZ (2 μM, 24 h). (n = 3, mean ± SD, normalized to GAPDH). (D) Western Blot validation of pyroptosis execution. Treatment: FBZ (2 μM, 24 h) (n = 3, mean ± SD, normalized to GAPDH). (E) WB analysis of mature IL-1β/IL-18 secretion. Markers: IL-18 (22 kDa, mature), IL-1β (19 kDa, mature). Treatment: FBZ (2 μM, 24 h). (n = 3, mean ± SD, normalized to GAPDH). (F) LDH and cytokine release assays. Indices: LDH (cytotoxicity), IL-18, IL-1β (pyroptotic markers). Treatment: FBZ (2 μM, 24 h). (n = 3, mean ± SD).

Journal: Frontiers in Pharmacology

Article Title: Fenbendazole induces pyroptosis in breast cancer cells through HK2/caspase-3/GSDME signaling pathway

doi: 10.3389/fphar.2025.1596694

Figure Lengend Snippet: Fenbendazole (FBZ) induces pyroptosis in EMT6 breast cancer cells. (A) CCK-8 assay for cell proliferation. Cell lines: EMT6 (top) and HCT116 (bottom, negative control for FBZ sensitivity). Treatment: FBZ at 0.5–8 μM for 24 h (n = 6, mean ± SD). (B) Pyroptotic morphology observation (×200 magnification, scale bar = 50 μm). Phenotypes: CON (Control): Normal cell morphology. FBZ (2 μM, 24 h): Swelling, blistering (red arrows), characteristic of pyroptosis. (C) qPCR analysis of pyroptosis-related genes. Treatment: FBZ (2 μM, 24 h). (n = 3, mean ± SD, normalized to GAPDH). (D) Western Blot validation of pyroptosis execution. Treatment: FBZ (2 μM, 24 h) (n = 3, mean ± SD, normalized to GAPDH). (E) WB analysis of mature IL-1β/IL-18 secretion. Markers: IL-18 (22 kDa, mature), IL-1β (19 kDa, mature). Treatment: FBZ (2 μM, 24 h). (n = 3, mean ± SD, normalized to GAPDH). (F) LDH and cytokine release assays. Indices: LDH (cytotoxicity), IL-18, IL-1β (pyroptotic markers). Treatment: FBZ (2 μM, 24 h). (n = 3, mean ± SD).

Article Snippet: The mouse breast cancer cell line (EMT6) and the mouse mammary epithelial cell (HC11) were purchased from Wuhan Procell Life Science and Technology Co., Ltd (Wuhan, China).

Techniques: CCK-8 Assay, Negative Control, Control, Western Blot, Biomarker Discovery

GSDME mediates FBZ-induced pyroptosis in EMT6 cells. (A) GSDME knockdown efficiency (Western Blot). Groups: CON (Control), NC (Negative Control), siGSDME (GSDME-targeting siRNA). Markers: GSDME (55 kDa), GAPDH (36 kDa, internal control). (P < 0.01 vs. CON/NC, n = 3, normalized to GAPDH). (B) IL-1β/IL-18 activation (2 μM FBZ treatment). Groups: CON, FBZ, siGSDME, siGSDME + FBZ. Markers: IL-18 (22 kDa, mature), IL-1β (19 kDa, mature), GAPDH (36 kDa). (P < 0.01 vs. FBZ, n = 3, normalized to GAPDH). (C) LDH/IL-1β/IL-18 release assays. Indices: LDH (cytotoxicity), IL-1β, IL-18 (pyroptotic markers). Treatment: 2 μM FBZ. (P < 0.01, n = 3). (D) Cell viability (CCK-8 assay). Groups: CON, FBZ, siGSDME, siGSDME + FBZ. (P < 0.01 vs. CON/FBZ, n = 6).

Journal: Frontiers in Pharmacology

Article Title: Fenbendazole induces pyroptosis in breast cancer cells through HK2/caspase-3/GSDME signaling pathway

doi: 10.3389/fphar.2025.1596694

Figure Lengend Snippet: GSDME mediates FBZ-induced pyroptosis in EMT6 cells. (A) GSDME knockdown efficiency (Western Blot). Groups: CON (Control), NC (Negative Control), siGSDME (GSDME-targeting siRNA). Markers: GSDME (55 kDa), GAPDH (36 kDa, internal control). (P < 0.01 vs. CON/NC, n = 3, normalized to GAPDH). (B) IL-1β/IL-18 activation (2 μM FBZ treatment). Groups: CON, FBZ, siGSDME, siGSDME + FBZ. Markers: IL-18 (22 kDa, mature), IL-1β (19 kDa, mature), GAPDH (36 kDa). (P < 0.01 vs. FBZ, n = 3, normalized to GAPDH). (C) LDH/IL-1β/IL-18 release assays. Indices: LDH (cytotoxicity), IL-1β, IL-18 (pyroptotic markers). Treatment: 2 μM FBZ. (P < 0.01, n = 3). (D) Cell viability (CCK-8 assay). Groups: CON, FBZ, siGSDME, siGSDME + FBZ. (P < 0.01 vs. CON/FBZ, n = 6).

Article Snippet: The mouse breast cancer cell line (EMT6) and the mouse mammary epithelial cell (HC11) were purchased from Wuhan Procell Life Science and Technology Co., Ltd (Wuhan, China).

Techniques: Knockdown, Western Blot, Control, Negative Control, Activation Assay, CCK-8 Assay

FBZ triggers GSDME-mediated pyroptosis via the BAX-caspase-3 cascade in EMT6 cells. (A) BAX protein upregulation (2 μM FBZ, 24 h). WB markers: BAX (20 kDa), GAPDH (36 kDa, internal control). (P < 0.05 vs. CON, n = 3, normalized to GAPDH). (B) BAX knockdown efficiency (siRNA). Groups: CON, NC (Negative Control), siBAX. WB markers: BAX (20 kDa), GAPDH (36 kDa). ( P < 0.01 vs. CON/NC, n = 3, normalized to GAPDH). (C) Caspase-3/GSDME activation after BAX silencing (2 μM FBZ, 24 h). WB targets: GSDME (55 kDa), Cleaved-caspase-3 (17 kDa), GSDME-NT (35 kDa). (n = 3, normalized to GAPDH). (D) LDH release assay (2 μM FBZ, 24 h). Groups: CON, FBZ, siBAX, siBAX + FBZ. (P < 0.01 vs. FBZ, n = 3). (E) Screening of Z - DEVD - FMK concentrations. WB markers: Caspase-3 (35 kDa). (n = 3, normalized to GAPDH). (F) GSDME-NT detection after Z-DEVD-FMK + FBZ treatment. WB markers: GSDME-NT (35 kDa), GAPDH (36 kDa). (n = 3, normalized to GAPDH). (G) LDH/IL-1β/IL-18 release with caspase-3 inhibition (2 μM FBZ, 24 h). Groups: CON, FBZ, Z-DEVD-FMK, Z-FMK (inactive control), Z-DEVD-FMK + FBZ. (P < 0.01 vs. FBZ, n = 3).

Journal: Frontiers in Pharmacology

Article Title: Fenbendazole induces pyroptosis in breast cancer cells through HK2/caspase-3/GSDME signaling pathway

doi: 10.3389/fphar.2025.1596694

Figure Lengend Snippet: FBZ triggers GSDME-mediated pyroptosis via the BAX-caspase-3 cascade in EMT6 cells. (A) BAX protein upregulation (2 μM FBZ, 24 h). WB markers: BAX (20 kDa), GAPDH (36 kDa, internal control). (P < 0.05 vs. CON, n = 3, normalized to GAPDH). (B) BAX knockdown efficiency (siRNA). Groups: CON, NC (Negative Control), siBAX. WB markers: BAX (20 kDa), GAPDH (36 kDa). ( P < 0.01 vs. CON/NC, n = 3, normalized to GAPDH). (C) Caspase-3/GSDME activation after BAX silencing (2 μM FBZ, 24 h). WB targets: GSDME (55 kDa), Cleaved-caspase-3 (17 kDa), GSDME-NT (35 kDa). (n = 3, normalized to GAPDH). (D) LDH release assay (2 μM FBZ, 24 h). Groups: CON, FBZ, siBAX, siBAX + FBZ. (P < 0.01 vs. FBZ, n = 3). (E) Screening of Z - DEVD - FMK concentrations. WB markers: Caspase-3 (35 kDa). (n = 3, normalized to GAPDH). (F) GSDME-NT detection after Z-DEVD-FMK + FBZ treatment. WB markers: GSDME-NT (35 kDa), GAPDH (36 kDa). (n = 3, normalized to GAPDH). (G) LDH/IL-1β/IL-18 release with caspase-3 inhibition (2 μM FBZ, 24 h). Groups: CON, FBZ, Z-DEVD-FMK, Z-FMK (inactive control), Z-DEVD-FMK + FBZ. (P < 0.01 vs. FBZ, n = 3).

Article Snippet: The mouse breast cancer cell line (EMT6) and the mouse mammary epithelial cell (HC11) were purchased from Wuhan Procell Life Science and Technology Co., Ltd (Wuhan, China).

Techniques: Control, Knockdown, Negative Control, Activation Assay, Lactate Dehydrogenase Assay, Inhibition

FBZ suppresses aerobic glycolysis in EMT6 cells via the p53-HK2 axis. (A) Network pharmacology of FBZ-breast cancer interactions. (B) PPI (Protein-Protein Interaction) network. (n = 3, FDR-corrected P < 0.05). (C) GO enrichment analysis of FBZ-downregulated proteins. (D) p53 pathway mRNA expression (qPCR, 2 μM FBZ, 24 h). (P < 0.05 vs. CON, n = 3, normalized to GAPDH). (E) p53 protein validation (WB, 2 μM FBZ, 24 h). (n = 3, normalized to GAPDH). (F) Glycolytic enzyme protein levels (WB, 2 μM FBZ, 24 h). (P < 0.05 vs. CON, n = 3, normalized to GAPDH). (G) Glycolytic function assays (2 μM FBZ, 24 h). (n = 6, mean ± SD). (H) p53 knockdown efficiency (siRNA). Groups: CON, NC (Negative Control), sip53. WB markers: p53 (53 kDa), GAPDH (36 kDa). (P < 0.01 vs. CON/NC, n = 3, normalized to GAPDH). (I) HK2 rescue experiment (siRNA + FBZ, 2 μM, 24 h). (n = 3, normalized to GAPDH). (J) Glycolytic parameters after p53 manipulation (2 μM FBZ, 24 h). (n = 6, mean ± SD).

Journal: Frontiers in Pharmacology

Article Title: Fenbendazole induces pyroptosis in breast cancer cells through HK2/caspase-3/GSDME signaling pathway

doi: 10.3389/fphar.2025.1596694

Figure Lengend Snippet: FBZ suppresses aerobic glycolysis in EMT6 cells via the p53-HK2 axis. (A) Network pharmacology of FBZ-breast cancer interactions. (B) PPI (Protein-Protein Interaction) network. (n = 3, FDR-corrected P < 0.05). (C) GO enrichment analysis of FBZ-downregulated proteins. (D) p53 pathway mRNA expression (qPCR, 2 μM FBZ, 24 h). (P < 0.05 vs. CON, n = 3, normalized to GAPDH). (E) p53 protein validation (WB, 2 μM FBZ, 24 h). (n = 3, normalized to GAPDH). (F) Glycolytic enzyme protein levels (WB, 2 μM FBZ, 24 h). (P < 0.05 vs. CON, n = 3, normalized to GAPDH). (G) Glycolytic function assays (2 μM FBZ, 24 h). (n = 6, mean ± SD). (H) p53 knockdown efficiency (siRNA). Groups: CON, NC (Negative Control), sip53. WB markers: p53 (53 kDa), GAPDH (36 kDa). (P < 0.01 vs. CON/NC, n = 3, normalized to GAPDH). (I) HK2 rescue experiment (siRNA + FBZ, 2 μM, 24 h). (n = 3, normalized to GAPDH). (J) Glycolytic parameters after p53 manipulation (2 μM FBZ, 24 h). (n = 6, mean ± SD).

Article Snippet: The mouse breast cancer cell line (EMT6) and the mouse mammary epithelial cell (HC11) were purchased from Wuhan Procell Life Science and Technology Co., Ltd (Wuhan, China).

Techniques: Expressing, Biomarker Discovery, Knockdown, Negative Control

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