Journal: American Journal of Cancer Research

Article Title: Panoramic analysis of cell death patterns reveals prognostic and immune profiles of head and neck squamous cell carcinoma

doi: 10.62347/PMDA6193

Figure Lengend Snippet: CAAP1 and BCL2L2-PABPN can promote cellular proliferation. (A) Western blot analysis of total cell lysates of HNSCC cell lines demonstrates the expression of CAAP1 and BCL2L2-PABPN. (B) CAAP1 and BCL2L2-PABPN expressions in five paired tumor tissues (T) and their adjacent normal tissues (N). (C) The expression of CAAP1 and BCL2L2-PABPN in FADU and HN8 cells respectively after transferring with sgRNA. Knockdown of CAAP1 and BCL2L2-PABPN inhibited the Invasion and migration of HNSCC cells were test by wound-healing assay (D), transwell assay (E).

Article Snippet: For the purpose of immunoblotting, the following antibodies were employed at a dilution of 1:1000: rabbit anti-CAAP1 antibody (PA5-54977, Thermofisher), rabbit anti-BCL2L2-PABPN1 antibody (NBP2-61706, NOVUS), rabbit anti-caspase 8 antibody (9746S, CST), rabbit anti-caspase 9 antibody (10380-AP, proteintech) and rabbit anti-Vinculin antibody (ab129002, Abcam); and mouse anti-GAPDH (60004-1-Ig, Proteintech).

Techniques: Western Blot, Expressing, Transferring, Migration, Wound Healing Assay, Transwell Assay

Journal: American Journal of Cancer Research

Article Title: Panoramic analysis of cell death patterns reveals prognostic and immune profiles of head and neck squamous cell carcinoma

doi: 10.62347/PMDA6193

Figure Lengend Snippet: CAAP1 inhibit HNSCC intrinsic apoptosis, BCL2L2-PABPN inhibit HNSCC extrinsic apoptosis. (A) Phase-contrast photomicrographs showing Fadu cells treated with ABT-737 for 12 h, analysis with Cell Counting Kit CCK-8 assay (B). (C) Fadu and CAAP1-/- cells were treated with ABT-737 for 10 h, and then stained with Annexin V-FITC and analysed by flow cytometry; Percentage of apoptotic cells was measured right. (D) Western blot of full-length and cleaved caspase 9 for Fadu cell lines treated with ABT-737 (30 μM) for 12 h, (E) Bax level as measured using EIA assay. (F) Cells were photographed 24 h after drug treatment and analysis by CCK8 (G). (H) HN8 and BCL2L2-PABPN-/- cells were treated with TNF-α/CHX for 12 h, and then stained with Annexin V-FITC and analysed by flow cytometry; Percentage of apoptotic cells was measured right. CTRL, control. (I) Western blot of HN8 and BCL2L2-PABPN-/- cell lines treated with TNF-α and cycloheximide (CHX) for 12 h. TNF-α (5 ng/ml), CHX (20 ug/ml). (J) Caspase 8 activity as measured using enzymatic assay and represented by optical density at 450 nm (OD450).

Article Snippet: For the purpose of immunoblotting, the following antibodies were employed at a dilution of 1:1000: rabbit anti-CAAP1 antibody (PA5-54977, Thermofisher), rabbit anti-BCL2L2-PABPN1 antibody (NBP2-61706, NOVUS), rabbit anti-caspase 8 antibody (9746S, CST), rabbit anti-caspase 9 antibody (10380-AP, proteintech) and rabbit anti-Vinculin antibody (ab129002, Abcam); and mouse anti-GAPDH (60004-1-Ig, Proteintech).

Techniques: Cell Counting, CCK-8 Assay, Staining, Flow Cytometry, Western Blot, Enzyme Immunoassay, Activity Assay, Enzymatic Assay

Journal: American Journal of Cancer Research

Article Title: Panoramic analysis of cell death patterns reveals prognostic and immune profiles of head and neck squamous cell carcinoma

doi: 10.62347/PMDA6193

Figure Lengend Snippet: In immune-competent mouse models, inhibition of CAAP1 induces antitumor immunity. (A) A schematic view of the treatment plan. (B) Tumor bodies dissected 15 days after tumor formation in BLAB/C nude mice. (C, D) Summary of tumor weight (C) and volume data (D) of Fadu tumors harvested after euthanizing the mice. (E) A schematic view of the treatment plan and C57 mice received GFP-Luc sg nc, or sg caap1 meer cells were injected with luciferin. (F, G) Summary of luciferase activity (F) and volume data (G) of meer tumors harvested after euthanizing the mice. (H) Differences in the expression of CAAP1, Bax, and Bak in tumor tissues among different groups. (I-K) FACS of CD8+ in CD3+ cells and GZMB+CD8+ in CD3+ TILs from sg nc, or sg caap1 meer xenografts and quantification.

Article Snippet: For the purpose of immunoblotting, the following antibodies were employed at a dilution of 1:1000: rabbit anti-CAAP1 antibody (PA5-54977, Thermofisher), rabbit anti-BCL2L2-PABPN1 antibody (NBP2-61706, NOVUS), rabbit anti-caspase 8 antibody (9746S, CST), rabbit anti-caspase 9 antibody (10380-AP, proteintech) and rabbit anti-Vinculin antibody (ab129002, Abcam); and mouse anti-GAPDH (60004-1-Ig, Proteintech).

Techniques: Inhibition, Injection, Luciferase, Activity Assay, Expressing

Journal: American Journal of Cancer Research

Article Title: Panoramic analysis of cell death patterns reveals prognostic and immune profiles of head and neck squamous cell carcinoma

doi: 10.62347/PMDA6193

Figure Lengend Snippet: CAAP1 and BCL2L2-PABPN can promote cellular proliferation. (A) Western blot analysis of total cell lysates of HNSCC cell lines demonstrates the expression of CAAP1 and BCL2L2-PABPN. (B) CAAP1 and BCL2L2-PABPN expressions in five paired tumor tissues (T) and their adjacent normal tissues (N). (C) The expression of CAAP1 and BCL2L2-PABPN in FADU and HN8 cells respectively after transferring with sgRNA. Knockdown of CAAP1 and BCL2L2-PABPN inhibited the Invasion and migration of HNSCC cells were test by wound-healing assay (D), transwell assay (E).

Article Snippet: For the purpose of immunoblotting, the following antibodies were employed at a dilution of 1:1000: rabbit anti-CAAP1 antibody (PA5-54977, Thermofisher), rabbit anti-BCL2L2-PABPN1 antibody (NBP2-61706, NOVUS), rabbit anti-caspase 8 antibody (9746S, CST), rabbit anti-caspase 9 antibody (10380-AP, proteintech) and rabbit anti-Vinculin antibody (ab129002, Abcam); and mouse anti-GAPDH (60004-1-Ig, Proteintech).

Techniques: Western Blot, Expressing, Transferring, Knockdown, Migration, Wound Healing Assay, Transwell Assay

Journal: American Journal of Cancer Research

Article Title: Panoramic analysis of cell death patterns reveals prognostic and immune profiles of head and neck squamous cell carcinoma

doi: 10.62347/PMDA6193

Figure Lengend Snippet: CAAP1 inhibit HNSCC intrinsic apoptosis, BCL2L2-PABPN inhibit HNSCC extrinsic apoptosis. (A) Phase-contrast photomicrographs showing Fadu cells treated with ABT-737 for 12 h, analysis with Cell Counting Kit CCK-8 assay (B). (C) Fadu and CAAP1-/- cells were treated with ABT-737 for 10 h, and then stained with Annexin V-FITC and analysed by flow cytometry; Percentage of apoptotic cells was measured right. (D) Western blot of full-length and cleaved caspase 9 for Fadu cell lines treated with ABT-737 (30 μM) for 12 h, (E) Bax level as measured using EIA assay. (F) Cells were photographed 24 h after drug treatment and analysis by CCK8 (G). (H) HN8 and BCL2L2-PABPN-/- cells were treated with TNF-α/CHX for 12 h, and then stained with Annexin V-FITC and analysed by flow cytometry; Percentage of apoptotic cells was measured right. CTRL, control. (I) Western blot of HN8 and BCL2L2-PABPN-/- cell lines treated with TNF-α and cycloheximide (CHX) for 12 h. TNF-α (5 ng/ml), CHX (20 ug/ml). (J) Caspase 8 activity as measured using enzymatic assay and represented by optical density at 450 nm (OD450).

Article Snippet: For the purpose of immunoblotting, the following antibodies were employed at a dilution of 1:1000: rabbit anti-CAAP1 antibody (PA5-54977, Thermofisher), rabbit anti-BCL2L2-PABPN1 antibody (NBP2-61706, NOVUS), rabbit anti-caspase 8 antibody (9746S, CST), rabbit anti-caspase 9 antibody (10380-AP, proteintech) and rabbit anti-Vinculin antibody (ab129002, Abcam); and mouse anti-GAPDH (60004-1-Ig, Proteintech).

Techniques: Cell Counting, CCK-8 Assay, Staining, Flow Cytometry, Western Blot, Enzyme Immunoassay, Control, Activity Assay, Enzymatic Assay

Journal: American Journal of Cancer Research

Article Title: Panoramic analysis of cell death patterns reveals prognostic and immune profiles of head and neck squamous cell carcinoma

doi: 10.62347/PMDA6193

Figure Lengend Snippet: In immune-competent mouse models, inhibition of CAAP1 induces antitumor immunity. (A) A schematic view of the treatment plan. (B) Tumor bodies dissected 15 days after tumor formation in BLAB/C nude mice. (C, D) Summary of tumor weight (C) and volume data (D) of Fadu tumors harvested after euthanizing the mice. (E) A schematic view of the treatment plan and C57 mice received GFP-Luc sg nc, or sg caap1 meer cells were injected with luciferin. (F, G) Summary of luciferase activity (F) and volume data (G) of meer tumors harvested after euthanizing the mice. (H) Differences in the expression of CAAP1, Bax, and Bak in tumor tissues among different groups. (I-K) FACS of CD8+ in CD3+ cells and GZMB+CD8+ in CD3+ TILs from sg nc, or sg caap1 meer xenografts and quantification.

Article Snippet: For the purpose of immunoblotting, the following antibodies were employed at a dilution of 1:1000: rabbit anti-CAAP1 antibody (PA5-54977, Thermofisher), rabbit anti-BCL2L2-PABPN1 antibody (NBP2-61706, NOVUS), rabbit anti-caspase 8 antibody (9746S, CST), rabbit anti-caspase 9 antibody (10380-AP, proteintech) and rabbit anti-Vinculin antibody (ab129002, Abcam); and mouse anti-GAPDH (60004-1-Ig, Proteintech).

Techniques: Inhibition, Injection, Luciferase, Activity Assay, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: MKL1/miR-5100/CAAP1 loop regulates autophagy and apoptosis in gastric cancer cells

doi: 10.1016/j.neo.2020.03.001

Figure Lengend Snippet: CAAP1 inhibits apoptosis and promotes autophagy in gastric cancer cells. Transfect CAAP1 expression plasmid or empty vector (control group) into MGC8-3 cells. (A-B): caspase3 and cleaved-caspase3 protein expressions were detected by western blot, and cleaved-caspase3 protein was statistically analyzed ( n = 3), * P < 0.05; (C-D): Cell dysfunction was detected by flow cytometry Death, and statistical analysis of cell apoptosis rate ( n = 3), * P < 0.05. Transfect si-CAAP1 or si-CAAP1 (control group) into AGS cells. (E-F): caspase3 and cleaved-caspase3 protein expressions were detected by western blot, and cleaved-caspase3 protein was statistically analyzed ( n = 3), * P < 0.05; (G-H): Cell dysfunction was detected by flow cytometry Death, and statistical analysis of cell apoptosis rate ( n = 3), * P < 0.05. Transfect CAAP1 or empty vector (control group) into MGC80-3 cells. (I-J): LC3B protein expression was detected by western blot, and the ratio of LC3BⅡ/LC3BⅠ was statistically analyzed ( n = 3), * P < 0.05; (K): Cell autophagy was detected using an autophagosome detection kit. Transfect si-CAAP1 or si-CAAP1 control (control group) into AGS cells. (L-M): LC3B protein expression was detected by western blot, LC3BⅠ and LC3BⅡ proteins were quantified, and the ratio of LC3BⅡ/LC3BⅠ was statistically analyzed ( n = 3), * P < 0.05; N: cells were detected using an autophagosome detection kit.

Article Snippet: The cells were lysed with RIPA Lysis Buffer (Beyotime) and the proteins were quantified with the Enhanced BCA Protein Assay Kit (Beyotime), 40ug of protein per protein lane, then, the protein was transferred to the polyvinylidene fluoride (PVDF) Membrane (Millipore), sealed with 5% skimmed milk powder for 1 h, and incubated overnight with primary antibody at 4 °C, The antibodies used are as follows: Anti-GAPDH (1:2 000, Abclonal), Anti-CAAP1 (1:500, Sigma), Anti-MKL1 (1:1 500, Cell Signaling Technology/CST), Anti-Caspase3 (1:1 500, CST), Anti-LC3B (1:1 500, CST), Incubate secondary antibodies conjugated with named horseradish peroxidase–labeled (HRP) (Santa Cruz) at room temperature for 1 h, Samples were detected by Western fluorescence assay BeyoECL Plus (Beyotime).

Techniques: Expressing, Plasmid Preparation, Western Blot, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: MKL1/miR-5100/CAAP1 loop regulates autophagy and apoptosis in gastric cancer cells

doi: 10.1016/j.neo.2020.03.001

Figure Lengend Snippet: miR-5100 inhibits CAAP1 protein expression by targeting CAAP1 3′UTR. A: Analysis of the TargetScanHuman ( http://www.targetscan.org/vert_72/ ) website shows that miR-5100 can target the 3′UTR of CAAP1. B: miR-5100 targets the base sequence and mutant sequence of CAAP1 3′UTR. C: miR-5100 minic or miR-5100 minic NC (control group) and WT-pmir-CAAP1 or MUT-pmir-CAAP1 were co-transfected into MGC80-3 cells. The luciferase reporter assay was used to detect whether miR-5100 targeted to bind to CAAP1 3′UTR ( n = 3); * P < 0.05, ** P < 0.01. D: miR-5100 minic or miR-5100 minic NC (control group) was transfected into AGSA cells, and CAAP1 protein expression was detected by Western blot ( n = 3), * P < 0.05. E: miR-5100 inhibitor or miR-5100 inhibitor NC (control group) was transfected into MGC80-3 cells, and CAAP1 protein expression was detected by western blot ( n = 3), * P < 0.05. F: MUT format of WT and miR-5100 sequences are shown. G: miR-5100 was pulled down by a CAAP1 probe or a random probe. MiR-5100 levels were analyzed by Northern blot. I indicates input (10% of sample is loaded); and P, precipitation (100% of sample is loaded). H: Cardiomyocytes were transfected with biotinylated WT-miR-5100 (Bio-mir-5100-WT) or a biotinylated mutant miR-5100 (Bio-mir-5100-MUT). Biotinylated microRNA that is not complementary to CAAP1 was used as a negative control (Bio-NC). CAAP1 expression level was analyzed by real-time RT-PCR, * P < 0.05.

Article Snippet: The cells were lysed with RIPA Lysis Buffer (Beyotime) and the proteins were quantified with the Enhanced BCA Protein Assay Kit (Beyotime), 40ug of protein per protein lane, then, the protein was transferred to the polyvinylidene fluoride (PVDF) Membrane (Millipore), sealed with 5% skimmed milk powder for 1 h, and incubated overnight with primary antibody at 4 °C, The antibodies used are as follows: Anti-GAPDH (1:2 000, Abclonal), Anti-CAAP1 (1:500, Sigma), Anti-MKL1 (1:1 500, Cell Signaling Technology/CST), Anti-Caspase3 (1:1 500, CST), Anti-LC3B (1:1 500, CST), Incubate secondary antibodies conjugated with named horseradish peroxidase–labeled (HRP) (Santa Cruz) at room temperature for 1 h, Samples were detected by Western fluorescence assay BeyoECL Plus (Beyotime).

Techniques: Expressing, Sequencing, Mutagenesis, Transfection, Luciferase, Reporter Assay, Western Blot, Northern Blot, Negative Control, Quantitative RT-PCR

Journal: Neoplasia (New York, N.Y.)

Article Title: MKL1/miR-5100/CAAP1 loop regulates autophagy and apoptosis in gastric cancer cells

doi: 10.1016/j.neo.2020.03.001

Figure Lengend Snippet: MKL1 targets the promoter of CAAP1 to promote the expression of CAAP1 protein. A: The base sequence of the CArG box of the wild type (WT-pGL3-CAAP1) and mutant (MUT-pGL3-CAAP1) on the CAAP1 promoter. B: MKL1 or empty vector (control group) was co-transfected with WT-pGL3-CAAP1 or MUT-pGL3-CAAP1 into MGC80-3 cells. The luciferase reporter gene test is used to detect whether MKL1 can target the CAAP1 promoter, * P < 0.05, ** P < 0.01. Transfect MKL1 or empty vector (control group) into AGS cells, (C-E): CAAP1 mRNA expression level was detected by RT-QPCR ( n = 3) ** P < 0.01, and CAAP1 protein expression level was detected by western blot, CAAP1 protein was quantitatively and statistically analyzed ( n = 3), ** P < 0.01. F: Co-transfect MKL1 or empty vector (control group) with WT-pGL3-miR-5100 or MUT-pGL3-miR-5100 into AGS cells and target CAAP1 according to the method described in “Experiment” The ChIP analysis of the primers by PCR showed the amount of DNA in each sample (2% of the input) in the second lane. Immunoprecipitation without primary antibody (No Ab) was used as a mock control, and IgG was used as a negative control.

Article Snippet: The cells were lysed with RIPA Lysis Buffer (Beyotime) and the proteins were quantified with the Enhanced BCA Protein Assay Kit (Beyotime), 40ug of protein per protein lane, then, the protein was transferred to the polyvinylidene fluoride (PVDF) Membrane (Millipore), sealed with 5% skimmed milk powder for 1 h, and incubated overnight with primary antibody at 4 °C, The antibodies used are as follows: Anti-GAPDH (1:2 000, Abclonal), Anti-CAAP1 (1:500, Sigma), Anti-MKL1 (1:1 500, Cell Signaling Technology/CST), Anti-Caspase3 (1:1 500, CST), Anti-LC3B (1:1 500, CST), Incubate secondary antibodies conjugated with named horseradish peroxidase–labeled (HRP) (Santa Cruz) at room temperature for 1 h, Samples were detected by Western fluorescence assay BeyoECL Plus (Beyotime).

Techniques: Expressing, Sequencing, Mutagenesis, Plasmid Preparation, Transfection, Luciferase, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Negative Control

Journal: Neoplasia (New York, N.Y.)

Article Title: MKL1/miR-5100/CAAP1 loop regulates autophagy and apoptosis in gastric cancer cells

doi: 10.1016/j.neo.2020.03.001

Figure Lengend Snippet: miR-5100 inhibits tumorigenesis in nude mice. A: Nude mice injected with two different MGC80-3 cell lines or gavage with paclitaxel after 28 days. The images shown represent results for 3 mice. B: Image shows tumor tissue isolated from nude mice with three mice in each group. C: Tumor growth curves of nude mice injected with two different MGC80-3 cell lines or administered paclitaxel after 28 days, and the tumor volume was estimated using calipers; * P < 0.05. D: The image shows the statistics of the weight of tumor tissue isolated from nude mice; the weight of the tumor tissue is measured with a balance; * P < 0.05. E: Representative H&E-stained sections of the tumor tissues isolated from mice. F: Immunohistochemical analysis of tumor tissue isolated from nude mice, the image shows the results of in-situ analysis of CAAP1 protein. G: Tumor tissues of miR-5100-plko.1 and plko.1 (control group) isolated from nude mice were analyzed for protein expression levels of CAAP1, caspase3, and LC3B by western blot. H: Tumor tissues of miR-5100-plko.1+Taxol group and plko.1+Taxol group (control group) isolated from nude mice were analyzed for protein expression levels of CAAP1, caspase3, and LC3B by western blot.

Article Snippet: The cells were lysed with RIPA Lysis Buffer (Beyotime) and the proteins were quantified with the Enhanced BCA Protein Assay Kit (Beyotime), 40ug of protein per protein lane, then, the protein was transferred to the polyvinylidene fluoride (PVDF) Membrane (Millipore), sealed with 5% skimmed milk powder for 1 h, and incubated overnight with primary antibody at 4 °C, The antibodies used are as follows: Anti-GAPDH (1:2 000, Abclonal), Anti-CAAP1 (1:500, Sigma), Anti-MKL1 (1:1 500, Cell Signaling Technology/CST), Anti-Caspase3 (1:1 500, CST), Anti-LC3B (1:1 500, CST), Incubate secondary antibodies conjugated with named horseradish peroxidase–labeled (HRP) (Santa Cruz) at room temperature for 1 h, Samples were detected by Western fluorescence assay BeyoECL Plus (Beyotime).

Techniques: Injection, Isolation, Staining, Immunohistochemical staining, In Situ, Expressing, Western Blot