human cervical adenocarcinoma c33a cells  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: C -terminal amino acid Ala459 residue cooperates in the regulation of CAIX catalytic function: ( a ) Immunoblot analysis of cell lysates from C33a cells transfected with mock control, CAIX wild type (wt) or mutant (A459G) cultured 48 h in hypoxia. CAIX was detected using mouse monoclonal antibody M75 diluted 1:10 and β-actin was detected using mouse monoclonal antibody (CS3700, Cell Signaling Technology, Danvers, Massachusetts) diluted 1:5000. HRP-conjugated anti-mouse antibody (Dako Agilent, Santa Clara, California) diluted 1:5000 was used as a secondary antibody. ( b ) Fluorescence staining of CAIX in non-fixed and non-permeabilized C33a-CAIX wild type as well as A459G transfectants measured by flow cytometry. CAIX was detected using PG-domain specific mouse monoclonal antibody M75 diluted to 1 µg/mL and AlexaFluor 488-conjugated anti-mouse secondary antibody (Invitrogen, Carlsbad, California) diluted 1:1000. Results clearly demonstrate plasma membrane localization of CAIX and showed that 42.7% of C33a-CAIX-wt and 50.9% of C33a-CAIX-A459G transfectants expressed CAIX protein. C33a cells transfected with mock control plasmid were used as a negative control. The data are presented as the mean, n = 2. ( c ) Effect of Ala459 mutation on CAIX-mediated extracellular acidification. The graph shows the differences between pHe values (ΔpH) of culture media from CAIX wt or A459G-transfected and mock-transfected cells cultured 48 h in hypoxia. A459G mutant reduced acidification of extracellular pHe when compared to control wild type C33a-CAIX transfectants. The data are presented as the mean ± s.d., n = 5. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( d ) Effect of Ala459 mutation on migration capacity of C33a cells. The graph depicts the results of the wound healing assay given as a % of the area covered by cells migrating to close the wound at 30 h after the scratch, measured at various positions along the wounds. Area covered by C33a cells expressing CAIX-wt was set as 100%. C33a cells expressing CAIX with mutated Ala459 exhibited slower migration. The data are presented as the mean ± s.d., n = 10. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( e ) Accumulation of the fluorescent sulfonamide (FITC-CA-i) occurred in hypoxic MDCK cells expressing the CAIX-wt, whereas it was diminished in hypoxic MDCK cell transfected with the CAIX-A459G mutant. Images were taken using objective 10×. ( f ) In situ detection of the interaction between CAIX and AE2 using a proximity ligation assay (PLA). Analysis was performed in C33a cells transiently transfected with CAIX-wt and A459G mutant and cultured in hypoxia for an additional 48 h. Red PLA signal indicates the existence of interaction or close proximity localization also between CAIX with mutated Ala459 and AE2. C33a-mock transfectants served as a negative assay control due to a lack of one target protein. CAIX protein was post-labelled in green, nuclei are blue. Images were taken using objective 40× and zoom 3.

Article Snippet: Human C33a cervical carcinoma cells (ATCC HTB-31), HCT116 (ATCC CCL-247) and RKO (ATCC CRL-2577) colorectal carcinoma cells, MDA-MB-231 breast carcinoma cells (ATCC HTB-26), A549 lung carcinoma cells (ATCC CCL-185), Panc-1 (ATCC CLR-1469) pancreatic carcinoma and MDCK canine epithelial cells (ATCC CCL-34) were cultured under standard conditions in Dulbecco’s modified Eagle’s medium (BioSera, Nuaille, France) supplemented with 10% fetal calf serum (BioWhittaker, Basel, Switzerland) and gentamicine (Sandoz, Holzkirchen, Germany) in humidified air containing 21% O 2 , 5% CO 2 at 37 °C and in hypoxic conditions at anaerobic workstation (1% O 2 , 2% H 2 , 5% CO 2 , 91% N 2 , Ruskinn Technology, Bridgend, United Kingdom).

Techniques: Residue, Western Blot, Transfection, Control, Mutagenesis, Cell Culture, Fluorescence, Staining, Flow Cytometry, Clinical Proteomics, Membrane, Plasmid Preparation, Negative Control, Migration, Wound Healing Assay, Expressing, In Situ, Proximity Ligation Assay

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: Protein L-isoaspartyl methyltransferase interacts with carbonic anhydrase IX: ( a ) Coomassie staining of purified GST and GST-CAIX-IC-wt separated by SDS-PAGE (left panel). Immunoblot analysis of GST-CAIX-IC-wt immunoprecipitates and lysates from different cancer cell lines: C33a, Panc-1, A549, RKO and MDA-MB-231 (right panel). GST immunoprecipitates and beads after lysate preclearing was used as negative control. PIMT signal was detected using monoclonal antibody (sc-100977, Santa Cruz Biotechnology, Dallas, Texas) diluted 1:1000. ( b ) In situ detection of the interaction between PIMT and CAIX protein using the proximity ligation assay. Analysis was performed in C33a cells transiently transfected with full length CAIX-wt, CAIX-A459G or mock control and cultured in hypoxia for an additional 48 h. The green PLA signal indicating the interaction of CAIX with PIMT was clearly visible in the cells expressing wild type CAIX, while in CAIX-A459G the extent and intensity of the signal was considerably lower. C33a-mock transfectants served as a negative assay control due to a lack of one target protein. CAIX protein was post-labelled in red, nuclei are blue. Images were taken using objective 40× and zoom 3.

Article Snippet: Human C33a cervical carcinoma cells (ATCC HTB-31), HCT116 (ATCC CCL-247) and RKO (ATCC CRL-2577) colorectal carcinoma cells, MDA-MB-231 breast carcinoma cells (ATCC HTB-26), A549 lung carcinoma cells (ATCC CCL-185), Panc-1 (ATCC CLR-1469) pancreatic carcinoma and MDCK canine epithelial cells (ATCC CCL-34) were cultured under standard conditions in Dulbecco’s modified Eagle’s medium (BioSera, Nuaille, France) supplemented with 10% fetal calf serum (BioWhittaker, Basel, Switzerland) and gentamicine (Sandoz, Holzkirchen, Germany) in humidified air containing 21% O 2 , 5% CO 2 at 37 °C and in hypoxic conditions at anaerobic workstation (1% O 2 , 2% H 2 , 5% CO 2 , 91% N 2 , Ruskinn Technology, Bridgend, United Kingdom).

Techniques: Staining, Purification, SDS Page, Western Blot, Negative Control, In Situ, Proximity Ligation Assay, Transfection, Control, Cell Culture, Expressing

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: Functional role of PIMT binding to CAIX: ( a ) Effect of Pcmt1 transient silencing on CAIX-mediated extracellular acidification. The graph shows the differences between pH values (ΔpH) measured in culture medium at the beginning and after 48 h in hypoxia normalized to protein concentration. Results clearly showed that transient Pcmt1 silencing in C33a-CAIX cells (stable CAIX transfectants) resulted in the alkalinization of extracellular pHe when compared to scramble control (scr). Measurements were performed after 72 h post-transfection of siRNAs. The data are presented as the mean ± s.d., n = 6. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( b ) Efficacy of transient silencing was checked in parallel at the protein level using immunoblot analysis depicted on a representative image. ( c ) Activation of CAIX enzymatic function under hypoxic conditions. Posttranslational modification of the intracellular tail is necessary for full activation of CAIX enzymatic function. Phosphorylation of Thr443 was identified previously. In this work we found out a novel condition necessary for CAIX activation—interaction between the IC tail of CAIX and PIMT facilitated by terminal Ala459. Hypoxia can affect the activity of CAIX in several ways: it upregulates cAMP levels through ADCY6 and ADCY7, leading to higher levels of cAMP, PKA activation and Thr443 phosphorylation, and also enhances the expression of PIMT, allowing its interaction with the IC tail of CAIX.

Article Snippet: Human C33a cervical carcinoma cells (ATCC HTB-31), HCT116 (ATCC CCL-247) and RKO (ATCC CRL-2577) colorectal carcinoma cells, MDA-MB-231 breast carcinoma cells (ATCC HTB-26), A549 lung carcinoma cells (ATCC CCL-185), Panc-1 (ATCC CLR-1469) pancreatic carcinoma and MDCK canine epithelial cells (ATCC CCL-34) were cultured under standard conditions in Dulbecco’s modified Eagle’s medium (BioSera, Nuaille, France) supplemented with 10% fetal calf serum (BioWhittaker, Basel, Switzerland) and gentamicine (Sandoz, Holzkirchen, Germany) in humidified air containing 21% O 2 , 5% CO 2 at 37 °C and in hypoxic conditions at anaerobic workstation (1% O 2 , 2% H 2 , 5% CO 2 , 91% N 2 , Ruskinn Technology, Bridgend, United Kingdom).

Techniques: Functional Assay, Binding Assay, Protein Concentration, Control, Transfection, Western Blot, Activation Assay, Modification, Phospho-proteomics, Activity Assay, Expressing

Journal: Bioscience Reports

Article Title: Study of the biological function of LncRNA LUCAT1 on cervical cancer cells by targeting miR-199b-5p

doi: 10.1042/BSR20200422

Figure Lengend Snippet: ( A ) Comparison of LUCAT1 expression levels in different CC cells, * represents comparison with C33A cells, P <0.050; # represents comparison with HeLa cells, P <0.050; @ represents comparison with AV3 cells, P <0.050. ( B ) Proliferation of C33A cells after transfection with LUCAT1. ( C ) Proliferation of AV3 cells after transfection with LUCAT1. ( D ) Apoptosis rate and flow cytometry of C33A and AV3 cells after transfection with LUCAT1. ( E ) Invasion ability of C33A and AV3 cells after transfection with LUCAT1. ( F ) Protein expression and protein imprinting map of C33A cells after transfection with LUCAT1. ( G ) Protein expression and protein imprinting map of AV3 cells after transfection with LUCAT1.

Article Snippet: Human cervical carcinoma cells C33A (BNCC341097), HeLa (BNCC342189), AV3 (BNCC340836) and normal cervical epithelial cells H8 (BNCC340657) were all purchased from BeNa Culture Collection, a subsidiary agency of ATCC.

Techniques: Comparison, Expressing, Transfection, Flow Cytometry

Journal: Bioscience Reports

Article Title: Study of the biological function of LncRNA LUCAT1 on cervical cancer cells by targeting miR-199b-5p

doi: 10.1042/BSR20200422

Figure Lengend Snippet: ( A ) Comparison of miR-199b-5p expression levels in different CC cells, * represents comparison with C33A cells, P <0.050; # represents comparison with HeLa cells, P <0.050; @ represents comparison with AV3 cells, P <0.050. ( B ) Proliferation of C33A cells after transfection with miR-199b-5p. ( C ) Proliferation of AV3 cells after transfection with miR-199b-5p. ( D ) Apoptosis rate and flow cytometry of C33A and AV3 cells after transfection with miR-199b-5p. ( E ) Invasion ability of C33A and AV3 cells after transfection with miR-199b-5p. ( F ) Protein expression and protein imprinting map of C33A cells after transfection with miR-199b-5p. ( G ) Protein expression and protein imprinting map of AV3 cells after transfection with miR-199b-5p.

Article Snippet: Human cervical carcinoma cells C33A (BNCC341097), HeLa (BNCC342189), AV3 (BNCC340836) and normal cervical epithelial cells H8 (BNCC340657) were all purchased from BeNa Culture Collection, a subsidiary agency of ATCC.

Techniques: Comparison, Expressing, Transfection, Flow Cytometry

Journal: Bioscience Reports

Article Title: Study of the biological function of LncRNA LUCAT1 on cervical cancer cells by targeting miR-199b-5p

doi: 10.1042/BSR20200422

Figure Lengend Snippet: ( A ) Dual luciferase reporter enzyme; ( B ) RIP experiment; ( C ) RNA pull-down experiment; ( D ) proliferation of C33A cells; ( E ) proliferation of AV3 cells; ( F ) apoptosis rate; ( G ) cells invasion, * P <0.050.

Article Snippet: Human cervical carcinoma cells C33A (BNCC341097), HeLa (BNCC342189), AV3 (BNCC340836) and normal cervical epithelial cells H8 (BNCC340657) were all purchased from BeNa Culture Collection, a subsidiary agency of ATCC.

Techniques: Luciferase

Journal: Bioengineering & Translational Medicine

Article Title: Targeting HPV‐infected cervical cancer cells with PEGylated liposomes encapsulating siRNA and the role of siRNA complexation with polyethylenimine

doi: 10.1002/btm2.10022

Figure Lengend Snippet: Apoptosis of HPV‐18‐positive HeLa cervical cancer cells and HPV‐negative C33A cervical cancer cells after incubation for 24 hr with 2.5 nM of complexed siRNA/PEI (N:P = 6) encapsulated in targeted and non‐targeted liposomes or delivered free in solution. 2.5 nM of uncomplexed siRNA (N:P = 0) was also delivered to cells encapsulated in targeted and non‐targeted liposomes. Other controls included empty targeted liposomes (750 nM lipids), targeted liposomes encapsulating 2.5 nM of a control siRNA complexed with PEI at N:P = 6 and untreated cells. All liposomes were PEGylated and the targeted liposomes were prepared with 5 mol% AG86. Data are presented as the mean ± SE ( n = 3, performed in quintuplicate). The targeted liposomes encapsulating siRNA/PEI (N:P = 6) were the only formulation that was statistically different from all other samples, and only when delivered to HeLa cells (* p < 0.01)

Article Snippet: HPV‐negative human cervical carcinoma C33A cells (ATCC, Manassas, VA) and HPV‐18‐positive HeLa cells were subcultured at 30,000 cells/well on fibronectin‐coated 20 mm glass coverslips (Neuvitro, Vancouver, WA) placed into clear 12‐well plates in 1 ml of Dulbecco's Modified Eagle Medium (DMEM) (ThermoFisher Scientific, Waltham, MA) supplemented with 10% FBS, 100 units/ml penicillin and 0.1 mg/ml streptomycin and incubated for 24 hr at 37 °C and 5% CO 2 .

Techniques: Incubation, Liposomes, Control, Formulation