AZD7762 (S1532) was purchased from Selleck (Shanghai, China); DDP was purchased from MCE (Shanghai, China). This study used antibodies against caspase-3 (Servicebio, Wuhan, China), cleaved caspase-3 (Servicebio, Wuhan, China), and Ki67 (Servicebio, Wuhan, China).
Cleaved caspase 3
Manufactured by Wuhan Servicebio Technology
Sourced in China
Cleaved caspase-3 is a laboratory reagent used in cellular and biochemical research. It is an enzyme fragment that plays a role in the process of apoptosis, or programmed cell death. The product can be used to study various cellular mechanisms and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Azd7762 s1532, by Selleck Chemicals (1 mentions)
Caspase 3, by Wuhan Servicebio Technology (1 mentions)
Topo 1, by Proteintech (1 mentions)
P akt, by Affinity Biosciences (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Dab plus kit, by Beijing Strong Biotechnologies (1 mentions)
Ultrasensitive sp mouse rabbit ihc kit, by Beijing Strong Biotechnologies (1 mentions)
Edta antigen retrieval solution, by Wuhan Servicebio Technology (1 mentions)
Nephrin, by Thermo Fisher Scientific (1 mentions)
Podocin, by Proteintech (1 mentions)
Cd2ap, by Thermo Fisher Scientific (1 mentions)
Beclin1, by Wuhan Servicebio Technology (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
One step tunel apoptosis assay kit, by Roche (1 mentions)
Dab substrate kit, by Agilent Technologies (1 mentions)
Diaminobenzidine dab, by Wuhan Servicebio Technology (1 mentions)
Mayers hematoxylin, by Wuhan Servicebio Technology (1 mentions)
Erk1 2, by Wuhan Servicebio Technology (1 mentions)
13 protocols using cleaved caspase 3
1
Evaluating AZD7762 and DDP Effects
2
Tumor Regression with CPT-Loaded Nanoparticles
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The HCT116 tumor-bearing mice were randomly divided into 4 groups (n = 7 for each group). After the tumor size reached about 100 mm3, the mice were intravenously injected twice a week with DMSO, free CPT, P-CON-CPT, and P-LPK-CPT (Equivalent CPT dose of 5 mg/kg). The tumors were measured by a vernier caliper twice a week. The weights of the mice were recorded. Then, the mice were sacrificed at the 28th day. The tumors were dissected and weighted. The tumor volumes were calculated to evaluate the therapeutic efficacy.
PDX mice were also randomly divided into four groups (n = 8) and injected with DMSO, free CPT, P-CON-CPT, and P-LPK-CPT (Equivalent CPT dose of 3 mg/kg) twice a week. The tumors were measured every other day and harvested at 26th day. The tumor tissues were immune-stained for Topo-I (Proteintech, Wuhan, China, Cat No.20705-1-AP, 1:100) and CAIX (Proteintech, Wuhan, China, Cat No. 11071-1-AP, 1:100), Ki67 (Servicebio, Wuhan, China, Cat No. GB111499, 1:500) and cleaved caspase-3 (Servicebio, Wuhan, China, Cat No. GB11532, 1:500). Mice intestinal tissues were collected, processed, and stained for cleaved caspase-3.
PDX mice were also randomly divided into four groups (n = 8) and injected with DMSO, free CPT, P-CON-CPT, and P-LPK-CPT (Equivalent CPT dose of 3 mg/kg) twice a week. The tumors were measured every other day and harvested at 26th day. The tumor tissues were immune-stained for Topo-I (Proteintech, Wuhan, China, Cat No.20705-1-AP, 1:100) and CAIX (Proteintech, Wuhan, China, Cat No. 11071-1-AP, 1:100), Ki67 (Servicebio, Wuhan, China, Cat No. GB111499, 1:500) and cleaved caspase-3 (Servicebio, Wuhan, China, Cat No. GB11532, 1:500). Mice intestinal tissues were collected, processed, and stained for cleaved caspase-3.
3
Molecular Mechanisms of PPII-Induced Apoptosis
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Cells were treated with different doses of PPII (1.85 µg/mL for U87, 5 µg/mL for U251) for 24 h, and washed twice in cold PBS. Then the treated cells were collected and lysed in RIPA lysis buffer, bicinchoninic acid (BCA) kit was used to determine protein concentration, all the protein samples were quantified to be the same concentration. Cell lysates (50 µg) were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. Then incubated with the primary antibody at 4 ˚C overnight after blocked with 5% non-fat dry milk, and then incubated again with the secondary antibody in a dark place for 1 h. The protein level was corrected using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). P-AKT (Affinity Biosciences, Inc., USA, product code: AF0908), AKT (Servicebio Inc., China, product code: GB13427), BAX (Servicebio Inc., China, product code: GB11690), caspase 3 (Servicebio Inc., China, product code: GB11767C), cleaved-caspase 3 (Servicebio Inc., China, product code: GB11009), cytochrome C (Servicebio Inc., China, product code: GB11080), BCL2 (Wanleibio Co, China, product code: WL0234), GAPDH (Santa Cruz Biotechnology, Inc., USA, product code: sc-32233).
4
Immunohistochemical analysis of BRD4, Ki-67, and Cleaved Caspase-3
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Immunohistochemical staining was carried out using Ultra-Sensitive SP (Mouse/Rabbit) IHC Kit (MXB Biotechnologies, China) and the DAB Plus Kit (MXB Biotechnologies, China). BRD4 (cat. No. 13440s, Cell Signaling Technology), Ki-67 (cat. No. GB111499, Servicebio), and cleaved-caspase 3 (cat. No. GB11009-1, Servicebio, Boston, MA, USA) antibodies were used.
5
Immunohistochemical Analysis of Tumor Tissue
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The paraffinized sections of tumor tissue were dewaxed with xylene and immersed in gradient ethanol before being rehydrated with water. The tumor tissue was repaired with EDTA antigen retrieval solution (pH 9.0) (Servicebio) in a microwave. After cooling at 25°C, the slides were placed in PBS (pH 7.4) and shaken on a decolorizing shaker for 5 min three times. The slides were immersed in 3% hydrogen peroxide solution for 25 min at RT before washing for 5 min on a decolorizing shaker three times. The slides were incubated with 3% BSA at 25°C for 30 min before incubating with primary antibody, including mouse antibody against CD8 and cleaved caspase‐3. After incubating with secondary antibody for 50 min. The primary antibodies involved in this assay were Cleaved‐caspase3 (Servicebio, GB11532) and CD8 (Cell Signaling Technology, #98941); and the HRP Goat anti‐rabbit secondary antibody was used. DAB chromogenic fluid was used for color development. Images were visualized using fluorescence microscopy.
6
Kidney Podocyte Protein Expression Analysis
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Kidney tissues and podocytes were first lysed with RIPA, followed by electrophoresis, membrane transfer, blocking, probing with primary antibodies nephrin (1:500, Invitrogen, PA5-106921), podocin (1:500, Proteintech, 20384-1-AP), cd2ap (1:500, Invitrogen, PA5-51894), Bax (1:500, Abcam, ab216494), Bcl-2 (1:500, Abcam, ab196495), Cleaved-caspase3 (1:500, Servicebio, GB11532), p-mTOR (1:1000,CST,5536 T), mTOR (1:1000,CST,2983 T), Beclin1(1:500, Servicebio, GB112053), p62(1:500, Servicebio, GB11239-1), and LC3(1:1000, Proteintech, 14600-1-AP) overnight at 4 °C, and incubation with secondary antibodies for 1 h at room temperature. Finally, the immunoreactive bands were developed by chemiluminescence and analyzed using Image J software.
7
Apoptosis and Nrf2 Expression Analysis
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Fresh mice tissues were fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin-embedded block tissues were cut into 4 μm sections and the positive expression of the cleaved caspase-3 (Servicebio, Wuhan, China) and nuclear factor erythroid 2-related factor 2 (Nrf2) (Servicebio, Wuhan, China) were detected using a DAB Substrate kit (DAKO, Hangzhou, China), and the apoptosis level was detected using a One-Step TUNEL Apoptosis Assay Kit (Roche, Shanghai, China).
8
Immunohistochemical Analysis of Tissue Samples
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Tissue was also fixed in 10% formaldehyde. According to the standard protocol, the paraffin sections were deparaffinized. After repairing antigen and blocking endogenous peroxidase act, tissue sections were blocked in 3% BSA. Tissue sections were incubated with primary antibodies (Ki67, cleaved caspase3, cleaved PARP1, Servicebio, Wuhan, China) at 4 °C overnight, followed by conjugated secondary antibodies (Servicebio, Wuhan, China) and diaminobenzidine (DAB, Servicebio, Wuhan, China). Then nuclei counterstaining was performed with Mayers hematoxylin (Servicebio, Wuhan, China). Finally, the tissue sections were dehydrated and sealed.
9
Quantifying Cell Proliferation in GH3 Tumors
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20 µm thick coronal sections were prepared from GH3 tumor and pituitary gland tissues, permeabilized with 0.5% Triton X-100, blocked with 10% horse serum, and incubated with the primary antibodies against Ki67 (Affinity BioReagents, Golden, CA), phospho-histone H3 (Proteintech, Tokyo, Japan) and cleaved caspase-3 (Servicebio, Wuhan, China). Then the sections were incubated with DAPI (4',6-diamidino-2-phenylindole). Sections were imaged using a Nikon Optiphot fuorescent microscope (Tokyo, Japan). Ki67-, phospho-histone H3- and DAPI-positive cells were counted using Image-J software.
10
Xenograft Tumor Model and ADR Treatment
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Animal experiments were conducted following the procedures approved by the Animal Experiments Committee of Fudan University, and according to institutional guidelines for the Care and Use of Laboratory Animals. The stably reconstituted LM2-4175 cells were subcutaneously injected into the flanks of 6-week-old BALB/c female nude mice to generate xenograft tumors. When the tumor volume in one of groups exceeded 100 mm3, mice were treated with 3 mg/kg ADR intraperitoneally twice a week. Tumor volume and growth kinetics were measured twice a week and calculated according to the formula of 0.5 × length × width2. After the mice were euthanized, the tumors were carefully removed and weighted. Immunohistochemical (IHC) staining of cleaved caspase-3 was performed by Servicebio.
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