Journal: Journal of visualized experiments : JoVE

Article Title: Quantification of protein phosphorylation using single molecule pull-down

doi: 10.3791/63665

Figure Lengend Snippet: Table of Materials

Article Snippet: Mix-n-Stain™ CF® Dye Antibody Labeling Kits , Biotium , 92245 , Suggested conjugation kit.

Techniques: Electron Microscopy, Lysis, Recombinant, Bicinchoninic Acid Protein Assay, Convection, Protease Inhibitor, Plasmid Preparation, Antibody Labeling, Conjugation Assay, Binding Assay, Microscopy, Cell Counting, Transferring, Staining, Software

Journal: Acta Pharmacologica Sinica

Article Title: The ceramide pathway is involved in the survival, apoptosis and exosome functions of human multiple myeloma cells in vitro

doi: 10.1038/aps.2017.118

Figure Lengend Snippet: Effect of ceramide pathway on the apoptosis of MM cells. (A) Representative pictures of Hoechst 33342 stain in MM cells which were treated with DMSO only (control), 10 μmol/L C6-cer and 10 μmol/L GW4869 for 24 h; (B) Summarized data of positive cells in Hoechst 33342 stain assay, shown as mean±SEM; n=3/group (*P<0.05, compared to control); (C) Western blots with anti-caspase 3, anti-cleaved caspase 3, anti-caspase 9, anti-cleaved caspase 9, anti-PARP and anti-cleaved PARP antibodies. β-Actin is used as normalization control for the total lysate. The data shown were representatives of three independent experiments that gave similar results; (D) Summarized data of Western blots (white bar: control; black bar: C6-cer), shown as mean±SEM; n=3/group. *P<0.05, **P<0.01 compared to control.

Article Snippet: A Hoechst 33342 staining kit (BD, USA) was used to detect cell apoptosis.

Techniques: Staining, Western Blot

Journal: Acta Pharmacologica Sinica

Article Title: The ceramide pathway is involved in the survival, apoptosis and exosome functions of human multiple myeloma cells in vitro

doi: 10.1038/aps.2017.118

Figure Lengend Snippet: The effect of ceramide pathway on the exosome functions and miR levels of targeting MM cells. (A) Markers for MM exosomes that were examined by using Western blot. (B) The effects of exosomesC6-cer and exosomesGW4869 on MM cell growth; (C) The effect of exosomesC6-cer or exosomesGW4869 on MM apoptosis by using Hoechst 33342 stain assay; (D) Expressions of caspase pathway after the treatment of exosomesC6-cer or exosomesGW4869; (E, F) The levels of miR 202, miR 16, miR 29b and miR 15a were measured by qRT-PCR in MM cells treated by exosomesC6-cer or exosomesGW4869. *P<0.05, compared to Exosomes Control, n=3/group.

Article Snippet: A Hoechst 33342 staining kit (BD, USA) was used to detect cell apoptosis.

Techniques: Western Blot, Staining, Quantitative RT-PCR

Journal: The FASEB Journal

Article Title: Hypoxia‐inducible factor‐1α can reverse the Adriamycin resistance of breast cancer adjuvant chemotherapy by upregulating transferrin receptor and activating ferroptosis

doi: 10.1096/fj.202401119R

Figure Lengend Snippet: The differences in ferroptosis‐related factors between breast cancer and normal breast tissue. ( A–D) Transferrin receptor (TFRC), iron‐responsive element‐binding protein 2 (IREB2), ferritin heavy chain 1 (FTH1), and hypoxia‐inducible factor‐1α (HIF1α) shows positive expression in breast cancer. (E–H) TFRC, IREB2, FTH1, and HIF1α shows negative expression in normal breast tissue (Immunohistochemistry EnVision method, ×200, consecutive slices of the same tissue in the same field of view). (I and J) Protein expression of TFRC, IREB2, and FTH1 in breast cancer and normal breast tissue (Western blot electrophoretogram and Western blot histograms). (K) mRNA expression of TFRC, IREB2, and FTH1 in breast cancer and normal breast tissue (qRT‐PCR histograms). (L) OD values of iron ion (Fe 2+ ), malondialdehyde (MDA), and glutathione (GSH) in breast cancer and normal breast tissue. The tissue samples were collected from 150 patients that were included in this study. Protein expression in tissue samples was detected using Western blot and mRNA expression in tissue samples was detected using RT‐PCR. Fe 2+ , MDA, and GSH in breast cancer and normal breast tissue were detected using respective detection kit. All experiments were performed in triplicate with at least three independent samples. * p < .05, ** P < .01, *** P < .001 are statistically significant.

Article Snippet: Fe 2+ , MDA (lipid peroxidation product), and GSH detection kits, FerroOrange fluorescence staining kits, cell proliferation and cell counting kit‐8 (CCK‐8) reagent kits, Fer‐1 (ferroptosis inhibitor) reagent kits, and Erastin (ferroptosis activator) reagent kits were purchased from GlpBio, United States.

Techniques: Binding Assay, Expressing, Immunohistochemistry, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Hypoxia‐inducible factor‐1α can reverse the Adriamycin resistance of breast cancer adjuvant chemotherapy by upregulating transferrin receptor and activating ferroptosis

doi: 10.1096/fj.202401119R

Figure Lengend Snippet: Correlation of Fe 2+ , MDA, and GSH expression with breast cancer as well as normal breast tissue.

Article Snippet: Fe 2+ , MDA (lipid peroxidation product), and GSH detection kits, FerroOrange fluorescence staining kits, cell proliferation and cell counting kit‐8 (CCK‐8) reagent kits, Fer‐1 (ferroptosis inhibitor) reagent kits, and Erastin (ferroptosis activator) reagent kits were purchased from GlpBio, United States.

Techniques: Expressing, Multiple Displacement Amplification

Journal: The FASEB Journal

Article Title: Hypoxia‐inducible factor‐1α can reverse the Adriamycin resistance of breast cancer adjuvant chemotherapy by upregulating transferrin receptor and activating ferroptosis

doi: 10.1096/fj.202401119R

Figure Lengend Snippet: Differences in ferroptosis‐related factors between the sensitive and resistant groups in breast cancer TAC treatment. ( A) Transferrin receptor (TFRC) shows positive expression in the sensitive group. (B) Iron‐responsive element‐binding protein 2 (IREB2) exhibits positive expression in the sensitive group. (C) Ferritin heavy chain 1 (FTH1) demonstrates negative expression in the sensitive group. (D) Hypoxia‐inducible factor‐1α (HIF1α) displays positive expression in the sensitive group. (E) TFRC shows weak positive expression in the resistant group. (F) IREB2 exhibits positive expression in the resistant group. (G) FTH1 demonstrates negative expression in the resistant group. (H) HIF1α displays weak positive expression in the resistant group (Immunohistochemistry EnVision method, ×200, consecutive slices of the same tissue in the same field of view). (I) In breast cancer, the correlation between HIF1α and the expression of TFRC, IREB2, and FTH1. (J–L) mRNA and protein expression of TFRC, IREB2, FTH1, and HIF1α in the sensitive and resistant groups of breast cancer TAC treatment (qRT‐PCR histograms, Western blot electrophoresis, and Western blot histograms). (M) OD values of iron ion (Fe 2+ ), malondialdehyde (MDA), and glutathione (GSH) in the sensitive and resistant groups of breast cancer TAC treatment. (N) Correlation between tumor regression rate and the expression of TFRC, IREB2, FTH1, and HIF1α. (O) Correlation between tumor regression rate and the expression of Fe 2+ , MDA, and GSH. The tissue samples were collected from 150 patients that were included in this study. Protein expression in tissue samples was detected using Western blot and mRNA expression in tissue samples was detected using RT‐PCR. Fe 2+ , MDA, and GSH in breast cancer were detected using respective detection kit. All experiments were performed in triplicate with at least three independent samples. * p < .05, ** P < .01, *** P < .001 are statistically significant.

Article Snippet: Fe 2+ , MDA (lipid peroxidation product), and GSH detection kits, FerroOrange fluorescence staining kits, cell proliferation and cell counting kit‐8 (CCK‐8) reagent kits, Fer‐1 (ferroptosis inhibitor) reagent kits, and Erastin (ferroptosis activator) reagent kits were purchased from GlpBio, United States.

Techniques: Expressing, Binding Assay, Immunohistochemistry, Quantitative RT-PCR, Western Blot, Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Hypoxia‐inducible factor‐1α can reverse the Adriamycin resistance of breast cancer adjuvant chemotherapy by upregulating transferrin receptor and activating ferroptosis

doi: 10.1096/fj.202401119R

Figure Lengend Snippet: Cell experiments validate the effects of Hypoxia‐inducible factor‐1α (HIF1α), transferrin receptor (TFRC), and ferroptosis on Adriamycin (ADR) resistance in breast cancer. (A) The expression levels of HIF1α in six cell lines. (B) The chemical sensitivity of six cell lines to ADR (dose–response curve). (C) The protein and mRNA expression levels of HIF1α, TFRC, and iron‐responsive element‐binding protein 2 (IREB2) in MCF‐7 and BT‐549 cells (Western blot electrophoresis, Western blot histograms, and qRT‐PCR histograms). (D) The optical density (OD) values of iron ion (Fe 2+ ), malondialdehyde (MDA), and glutathione (GSH) in MCF‐7 and BT‐549 cells. (E) Double luciferase assay confirmed the direct binding of HIF1α to TFRC. (F) Cell viability and chemosensitivity to ADR (dose–response curve) in each group of MCF‐7 cells. (G) Cell viability and chemosensitivity to ADR (dose–response curve) in each group of BT‐549 cells. Cells were incubated with culture medium containing different concentrations of ADR drugs for 24 h before cell counting kit‐8 (CCK‐8) assay was performed. All experiments were performed in triplicate with at least three independent samples. * p < .05, ** P < .01, *** P < .001 are statistically significant.

Article Snippet: Fe 2+ , MDA (lipid peroxidation product), and GSH detection kits, FerroOrange fluorescence staining kits, cell proliferation and cell counting kit‐8 (CCK‐8) reagent kits, Fer‐1 (ferroptosis inhibitor) reagent kits, and Erastin (ferroptosis activator) reagent kits were purchased from GlpBio, United States.

Techniques: Expressing, Binding Assay, Western Blot, Electrophoresis, Quantitative RT-PCR, Luciferase, Incubation, Cell Counting, CCK-8 Assay

Journal: The FASEB Journal

Article Title: Hypoxia‐inducible factor‐1α can reverse the Adriamycin resistance of breast cancer adjuvant chemotherapy by upregulating transferrin receptor and activating ferroptosis

doi: 10.1096/fj.202401119R

Figure Lengend Snippet: The average OD values of Fe 2+ , MDA, and GSH in various groups of MCF‐7 cell lines [ x ¯ ± s ].

Article Snippet: Fe 2+ , MDA (lipid peroxidation product), and GSH detection kits, FerroOrange fluorescence staining kits, cell proliferation and cell counting kit‐8 (CCK‐8) reagent kits, Fer‐1 (ferroptosis inhibitor) reagent kits, and Erastin (ferroptosis activator) reagent kits were purchased from GlpBio, United States.

Techniques: Control

Journal: The FASEB Journal

Article Title: Hypoxia‐inducible factor‐1α can reverse the Adriamycin resistance of breast cancer adjuvant chemotherapy by upregulating transferrin receptor and activating ferroptosis

doi: 10.1096/fj.202401119R

Figure Lengend Snippet: The average OD values of Fe 2+ , MDA, and GSH in various groups of BT‐549 cell lines [ x ¯ ± s ].

Article Snippet: Fe 2+ , MDA (lipid peroxidation product), and GSH detection kits, FerroOrange fluorescence staining kits, cell proliferation and cell counting kit‐8 (CCK‐8) reagent kits, Fer‐1 (ferroptosis inhibitor) reagent kits, and Erastin (ferroptosis activator) reagent kits were purchased from GlpBio, United States.

Techniques: Control