Journal: World Journal of Surgical Oncology

Article Title: Identification of a novel circ_0018289/miR-183-5p/TMED5 regulatory network in cervical cancer development

doi: 10.1186/s12957-021-02350-y

Figure Lengend Snippet: Circ_0018289 expression was increased in cervical cancer. A qRT-PCR analysis of circ_0018289 in 50 primary tumor samples and 50 paired normal tissues from the same patients. B Circ_0018289 expression by qRT-PCR analysis in Ect1/E6E7, SiHa, and HeLa cells. C RNase R assay in SiHa and HeLa cells. D Subcellular localization assay in SiHa and HeLa cells. * P < 0.05

Article Snippet: Human ectocervical Ect1/E6E7 cells, SiHa and HeLa cervical cancer cells, and human umbilical vein endothelial cells (HUVECs) were originally from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated at 37 °C in 5% CO 2 under standard conditions provided by ATCC.

Techniques: Expressing, Quantitative RT-PCR

Journal: World Journal of Surgical Oncology

Article Title: Identification of a novel circ_0018289/miR-183-5p/TMED5 regulatory network in cervical cancer development

doi: 10.1186/s12957-021-02350-y

Figure Lengend Snippet: Circ_0018289 silencing suppressed cervical carcinogenesis and angiogenesis in vitro. SiHa and HeLa cells were transfected with si-circ_0018289 or si-NC. A qRT-PCR analysis of circ_0018289 in transfected cells. B CCK-8 assay showing cell proliferation ability. C Representative images depicting a cell proliferation assay and cell proliferation by Edu assay. Scale bars, 100 μm. D Representative images depicting a cell apoptosis assay and cell apoptosis by flow cytometry. E Western blot showing the levels of Bax and Bcl-2 in transfected cells. F Representative images depicting a tube formation assay performed with human HUVECs pre-treated with the conditional medium of transfected cells. 100 × magnification. G Western blot showing the expression levels of VEGFA and FGF2 in transfected cells. * P < 0.05

Article Snippet: Human ectocervical Ect1/E6E7 cells, SiHa and HeLa cervical cancer cells, and human umbilical vein endothelial cells (HUVECs) were originally from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated at 37 °C in 5% CO 2 under standard conditions provided by ATCC.

Techniques: In Vitro, Transfection, Quantitative RT-PCR, CCK-8 Assay, Proliferation Assay, EdU Assay, Apoptosis Assay, Flow Cytometry, Western Blot, Tube Formation Assay, Expressing

Journal: World Journal of Surgical Oncology

Article Title: Identification of a novel circ_0018289/miR-183-5p/TMED5 regulatory network in cervical cancer development

doi: 10.1186/s12957-021-02350-y

Figure Lengend Snippet: Circ_0018289 targeted miR-183-5p. A Sequence of miR-183-5p, the complementary sites for miR-183-5p in circ_0018289, and the mutant of the seed sites. B qRT-PCR analysis of miR-183-5p in miR-183-5p mimic- or miR-NC mimic-transfected SiHa and HeLa cells. C Dual-luciferase reporter assays using circ_0018289 wild-type (circ_0018289-WT) or mutant-type (circ_0018289-MUT) reporter constructs in SiHa and HeLa cells. D qRT-PCR analysis of miR-183-5p in si-circ_0018289- or si-NC-transfected SiHa and HeLa cells. E qRT-PCR analysis showing the expression of miR-183-5p in 50 primary tumor samples and 50 paired normal tissues from the same patients. F Relative miR-183-5p expression in Ect1/E6E7, SiHa and HeLa cells by qRT-PCR analysis. G Correlation between circ_0018289 and miR-183-5p levels in tumor samples analyzed by Pearson’s correlation coefficients. * P < 0.05

Article Snippet: Human ectocervical Ect1/E6E7 cells, SiHa and HeLa cervical cancer cells, and human umbilical vein endothelial cells (HUVECs) were originally from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated at 37 °C in 5% CO 2 under standard conditions provided by ATCC.

Techniques: Sequencing, Mutagenesis, Quantitative RT-PCR, Transfection, Luciferase, Construct, Expressing

Journal: World Journal of Surgical Oncology

Article Title: Identification of a novel circ_0018289/miR-183-5p/TMED5 regulatory network in cervical cancer development

doi: 10.1186/s12957-021-02350-y

Figure Lengend Snippet: Silencing of circ_0018289 impeded cervical carcinogenesis and angiogenesis by up-regulating miR-183-5p. A qRT-PCR analysis of miR-183-5p in SiHa and HeLa cells transfected with anti-miR-183-5p or anti-miR-NC. CCK-8 ( B ) and Edu ( C ) assays for cell proliferation, flow cytometry for cell apoptosis ( D ), western blot analysis for Bax and Bcl-2 levels ( E ), in SiHa and HeLa cells transfected with si-circ_0018289, si-NC, si-circ_0018289 + anti-miR-NC or si-circ_0018289 + anti-miR-183-5p. F Tube formation assay performed with human HUVECs pre-treated with the conditional medium of SiHa and HeLa cells transfected with si-circ_0018289, si-NC, si-circ_0018289 + anti-miR-NC or si-circ_0018289 + anti-miR-183-5p. G Western blot showing the levels of VEGFA and FGF2 in SiHa and HeLa cells transfected with si-circ_0018289, si-NC, si-circ_0018289 + anti-miR-NC or si-circ_0018289 + anti-miR-183-5p. * P < 0.05

Article Snippet: Human ectocervical Ect1/E6E7 cells, SiHa and HeLa cervical cancer cells, and human umbilical vein endothelial cells (HUVECs) were originally from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated at 37 °C in 5% CO 2 under standard conditions provided by ATCC.

Techniques: Quantitative RT-PCR, Transfection, CCK-8 Assay, Flow Cytometry, Western Blot, Tube Formation Assay

Journal: World Journal of Surgical Oncology

Article Title: Identification of a novel circ_0018289/miR-183-5p/TMED5 regulatory network in cervical cancer development

doi: 10.1186/s12957-021-02350-y

Figure Lengend Snippet: TMED5 was a direct target of miR-183-5p. A Sequence of miR-183-5p, the complementary sites for miR-183-5p in TMED5 3′UTR and the mutant of the seed sites. B Correlation between TMED5 level and the prognosis of patients with cervical cancer shown in the GEPIA database. C Dual-luciferase reporter assays using TMED5 3′UTR wild-type (TMED5 3′UTR-WT) and mutant (TMED5 3′UTR-MUT) reporter constructs in SiHa and HeLa cells. D , E TMED5 mRNA and protein levels in SiHa and HeLa cells transfected with anti-miR-NC, anti-miR-183-5p, miR-NC mimic, or miR-183-5p mimic by qRT-PCR and western blot assays, respectively. F , G TMED5 mRNA and protein levels in primary tumor samples and paired normal tissues from the same patients. H , I TMED5 mRNA and protein levels by qRT-PCR and western blot assays, respectively, in Ect1/E6E7, SiHa, and HeLa cells. J , K Correlation between TMED5 mRNA level and miR-183-5p expression or circ_0018289 expression in tumor samples analyzed by Pearson’s correlation coefficients. * P < 0.05

Article Snippet: Human ectocervical Ect1/E6E7 cells, SiHa and HeLa cervical cancer cells, and human umbilical vein endothelial cells (HUVECs) were originally from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated at 37 °C in 5% CO 2 under standard conditions provided by ATCC.

Techniques: Sequencing, Mutagenesis, Luciferase, Construct, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: World Journal of Surgical Oncology

Article Title: Identification of a novel circ_0018289/miR-183-5p/TMED5 regulatory network in cervical cancer development

doi: 10.1186/s12957-021-02350-y

Figure Lengend Snippet: MiR-183-5p-mediated inhibition of TMED5 impeded cervical cancer development and angiogenesis in vitro. A , B TMED5 mRNA and protein levels by qRT-PCR and western blot assays, respectively, in SiHa and HeLa cells transfected with pcDNA-TMED5 or negative control pcDNA. SiHa and HeLa cells were transfected with miR-183-5p mimic, miR-NC mimic, miR-183-5p mimic + pcDNA, or miR-183-5p mimic + pcDNA-TMED5, followed by the assessment of cell proliferation by CCK-8 ( C ) and Edu ( D ) assays, cell apoptosis by flow cytometry ( E ), and Bax and Bcl-2 levels by western blot ( F ). G Tube formation assay performed with human HUVECs pre-treated with the conditional medium of SiHa and HeLa cells transfected with miR-183-5p mimic, miR-NC mimic, miR-183-5p mimic + pcDNA, or miR-183-5p mimic + pcDNA-TMED5. H Western blot showing VEGFA and FGF2 levels in SiHa and HeLa cells transfected with miR-183-5p mimic, miR-NC mimic, miR-183-5p mimic + pcDNA, or miR-183-5p mimic + pcDNA-TMED5. * P < 0.05

Article Snippet: Human ectocervical Ect1/E6E7 cells, SiHa and HeLa cervical cancer cells, and human umbilical vein endothelial cells (HUVECs) were originally from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated at 37 °C in 5% CO 2 under standard conditions provided by ATCC.

Techniques: Inhibition, In Vitro, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, CCK-8 Assay, Flow Cytometry, Tube Formation Assay

Journal: World Journal of Surgical Oncology

Article Title: Identification of a novel circ_0018289/miR-183-5p/TMED5 regulatory network in cervical cancer development

doi: 10.1186/s12957-021-02350-y

Figure Lengend Snippet: Circ_0018289 modulated TMED5 expression through binding to miR-183-5p. SiHa and HeLa cells were transfected with si-circ_0018289, si-NC, si-circ_0018289 + anti-miR-NC or si-circ_0018289 + anti-miR-183-5p. A qRT-PCR analysis of TMED5 mRNA level in transfected cells. B Western blot showing TMED5 protein level in transfected cells. * P < 0.05

Article Snippet: Human ectocervical Ect1/E6E7 cells, SiHa and HeLa cervical cancer cells, and human umbilical vein endothelial cells (HUVECs) were originally from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated at 37 °C in 5% CO 2 under standard conditions provided by ATCC.

Techniques: Expressing, Binding Assay, Transfection, Quantitative RT-PCR, Western Blot

Journal: Molecules

Article Title: Annona coriacea Mart. Fractions Promote Cell Cycle Arrest and Inhibit Autophagic Flux in Human Cervical Cancer Cell Lines

doi: 10.3390/molecules24213963

Figure Lengend Snippet: IC 50 values for A. coriacea compounds and cisplatin in cervical cancer cell lines.

Article Snippet: CaSki, HeLa, SiHa cells (ATCC catalog number CRL-1550, CCL-2, and HTB-35, respectively), and one normal keratinocytes cell line (HaCaT) were kindly provided by Dr. Luisa Villa.

Techniques:

Journal: Molecules

Article Title: Annona coriacea Mart. Fractions Promote Cell Cycle Arrest and Inhibit Autophagic Flux in Human Cervical Cancer Cell Lines

doi: 10.3390/molecules24213963

Figure Lengend Snippet: IC 50 values and selectivity index for the C3 and C5 fractions of cisplatin to tumor cells as compared with HaCaT.

Article Snippet: CaSki, HeLa, SiHa cells (ATCC catalog number CRL-1550, CCL-2, and HTB-35, respectively), and one normal keratinocytes cell line (HaCaT) were kindly provided by Dr. Luisa Villa.

Techniques:

Journal: Molecules

Article Title: Annona coriacea Mart. Fractions Promote Cell Cycle Arrest and Inhibit Autophagic Flux in Human Cervical Cancer Cell Lines

doi: 10.3390/molecules24213963

Figure Lengend Snippet: Cytotoxicity in SiHa cells. ( A ) Cell viability measured after 24 h of exposure in SiHa cells. ( B ) Cell cytotoxicity measured after 24 h of exposure in SiHa cells. There was an increase in cytotoxicity and a decrease in viability in a dose-dependent manner ( p < 0.0001). C3: Ethyl acetate fraction; C5: Fraction enriched in acetogenin; Cis: cisplatin. *** Indicates a statistical difference between groups. UFR: Relative unit of fluorescence.

Article Snippet: CaSki, HeLa, SiHa cells (ATCC catalog number CRL-1550, CCL-2, and HTB-35, respectively), and one normal keratinocytes cell line (HaCaT) were kindly provided by Dr. Luisa Villa.

Techniques: Fluorescence

Journal: Molecules

Article Title: Annona coriacea Mart. Fractions Promote Cell Cycle Arrest and Inhibit Autophagic Flux in Human Cervical Cancer Cell Lines

doi: 10.3390/molecules24213963

Figure Lengend Snippet: Cell proliferation and invasion upon C3 and C5 treatment (5µg/mL) in SiHa cells ( A ) Western blotting of phospho-AKT (protein kinase B) upon C3 and C5 treatment. ( B ) Number of colonies in the soft agar assay performed for 45 days. ( C ) BrdU incorporation after C3 and C5 treatment ( p < 0.0001) in SiHa cells. ( D ) Densitometry of p-AKT. ( E ) Invasion inhibition through C3 and C5 treatments in SiHa cells. ( F ) Percentage of invasion cells in SiHa cells (*** p < 0.0001; * p < 0.05). * Indicates statistical difference between the treatments). C3: Ethyl acetate fraction; C5: Fraction enriched in acetogenin; Cis: cisplatin.

Article Snippet: CaSki, HeLa, SiHa cells (ATCC catalog number CRL-1550, CCL-2, and HTB-35, respectively), and one normal keratinocytes cell line (HaCaT) were kindly provided by Dr. Luisa Villa.

Techniques: Western Blot, Soft Agar Assay, BrdU Incorporation Assay, Inhibition

Journal: Molecules

Article Title: Annona coriacea Mart. Fractions Promote Cell Cycle Arrest and Inhibit Autophagic Flux in Human Cervical Cancer Cell Lines

doi: 10.3390/molecules24213963

Figure Lengend Snippet: Cell cycle alterations in SiHa cells after exposure to C3 and C5 compounds ( A ) Western blot of p21 in SiHa cells upon C3, C5, and cisplatin treatments. ( B ) Densitometry of p21. ( C ) Cell cycle profile in SiHa cells. ( D ) Cell cycle phase distribution after treatment with C3 and C5. (*** p < 0.0001; * p < 0.05). C3: Ethyl acetate fraction; C5: Fraction enriched in acetogenin; Cis: cisplatin; DMSO: dimethylsulfoxide.

Article Snippet: CaSki, HeLa, SiHa cells (ATCC catalog number CRL-1550, CCL-2, and HTB-35, respectively), and one normal keratinocytes cell line (HaCaT) were kindly provided by Dr. Luisa Villa.

Techniques: Western Blot

Journal: Molecules

Article Title: Annona coriacea Mart. Fractions Promote Cell Cycle Arrest and Inhibit Autophagic Flux in Human Cervical Cancer Cell Lines

doi: 10.3390/molecules24213963

Figure Lengend Snippet: Apoptosis evaluation in SiHa cells upon C3 and C5 compounds. ( A ) Western blot of PARP (Poly (ADP-ribose) polymerase), caspase 3, and H2AX (H2A histone family member X) proteins ( B ) Flow cytometry for SiHa cells. ( C ) Comparison of apoptotic cells upon C3 and C5 treatment. There was a significant increase for cells in apoptosis only for cisplatin (CIS) * p = 0.0282 ( D ) Depolarization of the mitochondrial membrane after treatment with C3 and C5 and cisplatin in the SiHa cell line. C3: Ethyl acetate fraction; C5: Fraction enriched in acetogenin; Cis: cisplatin; DMM: Depolarized mitochondrial membrane; PMM: Polarized mitochondrial membrane. p < 0.05). C3: Ethyl acetate fraction; C5: Fraction enriched in acetogenin; Cis: cisplatin.

Article Snippet: CaSki, HeLa, SiHa cells (ATCC catalog number CRL-1550, CCL-2, and HTB-35, respectively), and one normal keratinocytes cell line (HaCaT) were kindly provided by Dr. Luisa Villa.

Techniques: Western Blot, Flow Cytometry, Comparison, Membrane

Journal: Molecules

Article Title: Annona coriacea Mart. Fractions Promote Cell Cycle Arrest and Inhibit Autophagic Flux in Human Cervical Cancer Cell Lines

doi: 10.3390/molecules24213963

Figure Lengend Snippet: Analysis of the involvement of A. coriacea fractions in autophagy. ( A ) Analysis of the expression of proteins involved in the autophagic flux in SiHa cells. ( B ) Densitometry of p62. ( C ) Densitometry of LC3 B/A (Microtubule-associated protein 1A/1B-light chain 3). ( D ) Acridine orange staining in SiHa cells. There was a reduction in the percentage of formation of acid vesicles, evidenced by a reduction of the fluorescent green signal after treatment with the fractions ( p < 0.05). HBSS: Hank’s balanced salt solution; EBSS: Earle’s balanced salt solution; C3: Ethyl acetate fraction; C5: Fraction enriched in acetogenin; Cis: cisplatin. BAF: Bafilomycin.

Article Snippet: CaSki, HeLa, SiHa cells (ATCC catalog number CRL-1550, CCL-2, and HTB-35, respectively), and one normal keratinocytes cell line (HaCaT) were kindly provided by Dr. Luisa Villa.

Techniques: Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: (+)-Catechin in a 1:2 Complex with Lysine Inhibits Cancer Cell Migration and Metastatic Take in Mice

doi: 10.3389/fphar.2017.00869

Figure Lengend Snippet: (+)-Catechin derivatives and epigallocatechin-3-gallate (EGCG) have similar general antioxidant effects in SiHa-F3 cancer cells. (A–E) Reactive oxygen species (ROS) level in SiHa-F3 cells was measured after a 1 h treatment with increasing doses of EGCG ( N = 2, n = 8–16), (+)-catechin ( N = 2, n = 16), (+)-catechin in a 1:1 complex with lysine ( N = 3, n = 24–32), and (+)-catechin in a 1:2 complex with lysine ( N = 2, n = 16–24). The five panels show the same sets of data with different statistical analyses to compare individual treatments to reference compound EGCG (A) or to vehicle treatment set as 100% (B–E) . (A) The graph shows the dose-response curves of the four treatments for comparison. (B) Dose-response curve for EGCG. (C) Dose-response curve for (+)-catechin. (D) Dose-response curve for (+)-catechin:lysine 1:1. (E) Dose-response curve for (+)-catechin:lysine 1:2. (F) ROS level in SiHa-F3 cells treated for 1 h with increasing doses of D/L -lysine monohydrochloride ( N = 2, n = 16). All data are shown as means ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.005 compared to a same dose of EGCG using two-way ANOVA; ## P < 0.01, ### P < 0.005 compared to vehicle treatment using one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Wild-type B16F10 mouse melanoma and SiHa human cervix adenocarcinoma cells were from ATCC.

Techniques: Comparison

Journal: Frontiers in Pharmacology

Article Title: (+)-Catechin in a 1:2 Complex with Lysine Inhibits Cancer Cell Migration and Metastatic Take in Mice

doi: 10.3389/fphar.2017.00869

Figure Lengend Snippet: (+)-Catechin and its complexes with lysine are less cytotoxic for SiHa-F3 cancer cells than EGCG. (A–E) SiHa-F3 cell viability was measured by crystal violet staining following a 16 h treatment with increasing doses of EGCG ( N = 2, n = 24), (+)-catechin ( N = 2, n = 16–24), (+)-catechin in a 1:1 complex with lysine ( N = 2, n = 15–16), and (+)-catechin in a 1:2 complex with lysine ( N = 2, n = 16–24). The five panels show the same sets of data with different statistical analyses to compare individual treatments to reference compound EGCG (A) or to vehicle treatment set as 100% (B–E) . (A) The graph shows the dose-response curves of the four treatments for comparison. (B) Dose-response curve for EGCG. (C) Dose-response curve for (+)-catechin. (D) Dose-response curve for (+)-catechin:lysine 1:1. (E) Dose-response curve for (+)-catechin:lysine 1:2. (F) SiHa-F3 cell viability was measured by crystal violet staining following a 16 h treatment with increasing doses of D/L -lysine monohydrochloride ( N = 2, n = 15–16). All data are shown as means ± SEM. ∗∗∗ P < 0.005 compared to a same dose of EGCG using two-way ANOVA; ### P < 0.005 compared to vehicle treatment using one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Wild-type B16F10 mouse melanoma and SiHa human cervix adenocarcinoma cells were from ATCC.

Techniques: Staining, Comparison

Journal: Frontiers in Pharmacology

Article Title: (+)-Catechin in a 1:2 Complex with Lysine Inhibits Cancer Cell Migration and Metastatic Take in Mice

doi: 10.3389/fphar.2017.00869

Figure Lengend Snippet: (+)-Catechin and its complexes with lysine are less metabolically toxic for SiHa-F3 cancer cells than EGCG. (A–E) , The activity of NAD(P)H-dependent oxidoreductases in SiHa-F3 cells was measured with a MTT assay following a 16-h treatment with increasing doses of EGCG ( N = 2, n = 12), (+)-catechin ( N = 2, n = 12), (+)-catechin in a 1:1 complex with lysine ( N = 2, n = 16), and (+)-catechin in a 1:2 complex with lysine ( N = 2, n = 12). The five panels show the same sets of data with different statistical analyses to compare individual treatments to reference compound EGCG (A) or to vehicle treatment set as 100% (B–E) . (A) The graph shows the dose-response curves of the four treatments for comparison. (B) Dose-response curve for EGCG. (C) Dose-response curve for (+)-catechin. (D) Dose-response curve for (+)-catechin:lysine 1:1. (E) Dose-response curve for (+)-catechin:lysine 1:2. All data are shown as means ± SEM. ∗∗∗ P < 0.005 compared to a same dose of EGCG using two-way ANOVA; ## P < 0.01, ### P < 0.005 compared to vehicle treatment using one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Wild-type B16F10 mouse melanoma and SiHa human cervix adenocarcinoma cells were from ATCC.

Techniques: Metabolic Labelling, Activity Assay, MTT Assay, Comparison

Journal: Frontiers in Pharmacology

Article Title: (+)-Catechin in a 1:2 Complex with Lysine Inhibits Cancer Cell Migration and Metastatic Take in Mice

doi: 10.3389/fphar.2017.00869

Figure Lengend Snippet: (+)-Catechin:lysine complexes and EGCG, but not (+)-catechin, inhibit SiHa-F3 cancer cell migration. (A–E) SiHa-F3 cancer cell migration was evaluated with a Boyden chamber assay using FBS (0.15%) as chemoattractant and 16 h as an endpoint. Cells were treated with increasing doses of EGCG ( N = 2, n = 6–18), (+)-catechin ( N = 2, n = 6–12), (+)-catechin in a 1:1 complex with lysine ( N = 2, n = 11–12), and (+)-catechin in a 1:2 complex with lysine ( N = 3, n = 17–18). The five panels show the same sets of data with different statistical analyses to compare individual treatments to reference compound EGCG (A) or to vehicle treatment set as 100% (B–E) . (A) The graph shows the dose-response curves of the four treatments for comparison. (B) Dose-response curve for EGCG. (C) Dose-response curve for (+)-catechin. (D) Dose-response curve for (+)-catechin:lysine 1:1. (E) Dose-response curve for (+)-catechin:lysine 1:2. (F) SiHa-F3 cancer cell migration was evaluated with a Boyden chamber assay using FBS (0.15%) as chemoattractant and 16 h as an endpoint. Cells were treated with increasing doses of D/L -lysine monohydrochloride ( N = 2, n = 12). All data are shown as means ± SEM. ∗∗∗ P < 0.005 compared to a same dose of EGCG using two-way ANOVA; # P < 0.05, ### P < 0.005 compared to vehicle treatment using one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Wild-type B16F10 mouse melanoma and SiHa human cervix adenocarcinoma cells were from ATCC.

Techniques: Migration, Boyden Chamber Assay, Comparison

Journal: Frontiers in Pharmacology

Article Title: (+)-Catechin in a 1:2 Complex with Lysine Inhibits Cancer Cell Migration and Metastatic Take in Mice

doi: 10.3389/fphar.2017.00869

Figure Lengend Snippet: Activities of catechin derivatives on SiHa-F3 cancer cells.

Article Snippet: Wild-type B16F10 mouse melanoma and SiHa human cervix adenocarcinoma cells were from ATCC.

Techniques: Concentration Assay, Inhibition

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, HCT116 (p53 Wt and p53 Null ) lines were treated with the indicated dose of P5091 (0 to 80 µM) for 24 and 48 hrs, and cell viability was checked by MTT assay. Inset graph representing change in mean IC-50 value of both cell lines at different time points. B, Protein levels of USP7, PARP, Cleaved PARP, Caspase3, Cleaved Caspase3, p53, MDM2 and p21 were determined by immunoblotting (IB) where HCT116 (p53 Wt and p53 Null ) cell lines were treated with USP7 inhibitor P5091 (20µM) for 24 hrs. GAPDH was kept as loading control. C, Following treatment of HCT116 (p53 Null ) cells with P5091 in different doses (1, 5, 10µM) for 24 hrs, the increase in caspase 3/7 activity was determined using a fluorescence plate reader. Data represent mean ± SD of three independent biological replicates. D, Number of colonies formed by HCT116 (p53 Wt ), HepG2 (p53 Null ), and Huh7 (p53 Mut ) cells after treatment with P5091 (20 µM) for 24 hrs; colonies were counted after 15 days. E & F, Identification of USP7 and XIAP interacting proteins by Mass Spectrometry. Silver stained SDS-PAGE gels containing elute from respective pull-down as indicated. MS analysis identifies specific peptides of XIAP and USP7 respectively from GST-USP7 and GST-XIAP pull-down lanes by using HEK cell lysates. G, Identification of USP7 target proteome in HCT116 cells by label-free comparative proteomics analysis upon USP7 inhibition with P5091 (15µM for 24 hrs), figure represents the workflow of label-free quantitation (LTQ) by nano-LC−MS/M.S. on a Q-exactive followed by SIEVE TM processing. H, Graphical representation of calculated protein ratios of P5091 treated samples with respect to the vehicle control (left panel). Venn diagram showing identified number of Up-regulated, Unchanged and Down regulated proteins in HCT116 p53 wt and HCT116 p53 null cell lines upon P5091 treatment with respect to control (right panel). I & J, Whole-cell lysates were prepared from MCF7, MD-AMB 231, MD-AMB 468, T47D, C6, U87, LN18, HCT116 (p53 Wt ), HCT116 (p53 Null ), SW480, SW620, HeLa, SiHa, LnCap, A549, RAW, HEK293T, and HEK293 cells. IB analysis was performed using respective antibodies for USP7, XIAP and β-Actin. Normalized values were plotted to show a strong positive correlation between USP7 and XIAP where p value < 0.0001, n=2. Error bars in all the indicated sub-figures represent mean ± SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P<0.05 is represented as *, otherwise non-significant (ns).

Article Snippet: Human colorectal cancer (CRC) cell lines [HCT 116 (p53 Wt and p53 Null ), SW-480, HT-29, SW-620]; human embryonic kidney cell line [HEK-293 and HEK-293T] and other cell lines (RAW, C6, U87, HeLa, SiHa, HepG2, Huh7) were used (ATCC.

Techniques: MTT Assay, Western Blot, Control, Activity Assay, Fluorescence, Mass Spectrometry, Staining, SDS Page, Inhibition, Quantitation Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, HEK cells transfected with 2µG of EV (pGZ) (lane-1&3), pGZ-USP7 (lane-2), and consecutively inactive form of USP7 mutant pGZ-USP7 C223S (lane-4); 48 hrs post-transfection, USP7 and XIAP protein levels were checked by IB. Additionally, three different shRNAs targeting USP7 and scramble shRNA were transfected (lanes-5,6,7&8) and the levels of XIAP and USP7 were analyzed by IB. GAPDH was kept as loading control. B, qRT-PCR analysis showing relative mRNA fold change upon USP7 inhibition by P5091 (15µM) for 24 hrs. Relative mRNA fold change of XIAP upon USP7 knockdown (using siUSP7) and USP7 over-expression. Protein expression of XIAP from the same experiment was also represented by IB, where GAPDH was used as an internal loading control. C, Multiple cancer cell lines such as HCT116, C6, SH-SY5Y, SiHa, and SW620 were treated with 15µM of USP7 inhibitor (P5091-lane 2, 4, 6, 8, 10; P22077-lane 11) for 24 hrs and XIAP was analyzed by IB and compared with respective control lanes (lane 1, 3, 5, 7, 9). IB analysis of XIAP protein level in HCT116 cells transfected with GFP-USP7 (4 µG, lane 13), pGZ-USP7 C223S (4 µG, lane 14), or siUSP7 (lane 16). EV (lane 12), control Si (lane 15) were kept for control. D, Huh7 and HT29 cells were treated with USP7 inhibitor P22077 in a dose-dependent manner (5, 10, 15, 20 µM) for 24 hrs. XIAP was checked by IB. GAPDH was kept as an internal loading control. E, HCT116 cells were transfected with either Control Si (upper left panel) or siUSP7 (Upper right panel) and HEK cells were transfected with either EV (Lower left panel) or GFP-USP7 (Lower right panel) before treatment with cycloheximide (CHX: 50µG/ml). XIAP protein levels at indicated time points were analyzed by IB as shown in the figure (left panels). Graph showing the change in XIAP half-life was determined in USP7 knockdown and overexpression conditions. Error bars in all the indicated sub-figures represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P<0.05 is represented as *, otherwise non-significant (ns).

Article Snippet: Human colorectal cancer (CRC) cell lines [HCT 116 (p53 Wt and p53 Null ), SW-480, HT-29, SW-620]; human embryonic kidney cell line [HEK-293 and HEK-293T] and other cell lines (RAW, C6, U87, HeLa, SiHa, HepG2, Huh7) were used (ATCC.

Techniques: Transfection, Mutagenesis, shRNA, Control, Quantitative RT-PCR, Inhibition, Knockdown, Over Expression, Expressing

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, HCT 116 (p53 Wt ) cells were transfected with indicated plasmids and treated with Doxorubicin for 24 hrs to induced apoptosis, prepared the lysates and analyzed by IB using the panel of indicated antibodies. B, Similarly, HEK cells were transfected with indicated plasmids and treated with Doxorubicin (1 µM) for 24hrs to induced apoptosis, prepared the lysates and analyzed by IB using the panel of indicated antibodies. C, Huh7 (p53 Mut ) and HepG2 cells (p53 Wt ) cells were transfected with Flag-USP7 and GFP-XIAP for the indicated time points, prepared the lysates and analyzed by IB. D, HCT116 (p53 Null ) cells were transfected with either Flag-USP7 or GFP-XIAP. After 48 hrs, cells were treated with P5091 (15µM) or Doxorubicin (1µM). Prepared the cell lysates and analyzed by IB using the indicated antibodies. XIAP, USP7, and Cleaved Caspase 3 were plotted after normalization against GAPDH. E, HCT116 (p53 Null - upper panel) and HCT116 (p53 Wt - lower panel) cells were treated with either XIAP inhibitor Embellin or USP7 inhibitor P5091 for the indicated doses. Prepared the lysates and analyzed by IB. XIAP expression was plotted after normalization against GAPDH. F, HEK and C6 cells were seeded in a 6-well plate and treated with P5091 (15µM and 20 µM for HEK and, 15 µM for C6) for 24 hrs. Treated cells were stained as per protocol to detect TUNNEL positivity. G, Cell cycle profile of HCT116 (p53 WT ) cells were analyzed as per protocol after treatment with an increasing dose of P5091 (5µM, 10µM and 15µM 24 hrs). H, Scratch assay was performed to evaluate the migration of HepG2 (p53 Wt ) and Huh7 (p53 Mut ) cells pre-treated with P5091 (20µM). I, percent of cell population in different apoptosis phases of HCT116 (p53 WT ) cells were analyzed as per protocol after treatment with indicated dose of P5091 for 24 hrs. Error bars in all the indicated sub-figures represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P<0.0001 is represented as *, otherwise non-significant (ns).

Article Snippet: Human colorectal cancer (CRC) cell lines [HCT 116 (p53 Wt and p53 Null ), SW-480, HT-29, SW-620]; human embryonic kidney cell line [HEK-293 and HEK-293T] and other cell lines (RAW, C6, U87, HeLa, SiHa, HepG2, Huh7) were used (ATCC.

Techniques: Transfection, Expressing, Staining, Wound Healing Assay, Migration