Journal: bioRxiv

Article Title: Transcriptional Regulation of the P-bodies Associated Psoriasis Candidate Gene CCHCR1

doi: 10.1101/2023.04.08.535951

Figure Lengend Snippet: ( a ) RT-qPCR for the expression of CCHCR1 and TCF19 in HeLa cells transfected with E2F activators (E2F1, E2F2 or E2F3). pEGFP-N1 was the empty vector control. Gene expression level was normalized to the expression level of RPL13A . Results were mean ± SD of three replicates. Statistically significant differences of the gene expression levels were compared to empty vector control and were analyzed by paired t-test (** = P ≤ 0.01). ( b ) Down-regulation of CCHCR1 and TCF19 mRNA by E2F1 knockdown. Cells were transfected with siE2F1 and were harvested for the time indicated (time post-transfection). mRNA expressions (normalized to RPL13A gene expression) were analyzed by RT-qPCR. Results were mean ± SD of three replicates. Statistically significant differences of the gene expression levels were compared to 0 h post-siE2F1 transfection and were analyzed by paired t-test (*** = P ≤ 0.001).

Article Snippet: Human cervical cancer HeLa cells (ATCC ® CRM-CCL-2 TM ) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Control, Gene Expression, Knockdown

Journal: bioRxiv

Article Title: Transcriptional Regulation of the P-bodies Associated Psoriasis Candidate Gene CCHCR1

doi: 10.1101/2023.04.08.535951

Figure Lengend Snippet: ( a ) HeLa cells were synchronized and arrested in G1/S transition by a double thymidine block. The cells were released and collected at different time points, stained with propidium iodide (PI). The percentage of cells in different cell cycle phases in each time point (G1/S, S, G2/M, and G1) were analyzed by flow cytometry. ( b ) Percentage of cells in different cell cycles after double thymidine block and release. (C) Relative expression of CCHCR1 and TCF19 in different cell cycle phases. CCHCR1 and TCF19 gene expression were normalized to RPL13A gene expression. The time points after release from the double thymidine block were indicated for different cell cycle phases; i.e. 0 h: G1/S, 2 h: S, 8 h: G2/M and 14 h: G1. Results were mean ± SD of three replicates. Statistically significant differences of the gene expressions were compared to 0 h after double thymidine release (G1/S transition) and were analyzed by paired t-test (** = P ≤ 0.01 and *** = P ≤ 0.001)

Article Snippet: Human cervical cancer HeLa cells (ATCC ® CRM-CCL-2 TM ) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Blocking Assay, Staining, Flow Cytometry, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Transcriptional Regulation of the P-bodies Associated Psoriasis Candidate Gene CCHCR1

doi: 10.1101/2023.04.08.535951

Figure Lengend Snippet: ( a ) HeLa cells were transfected with the corresponding siRNAs. Non-viable cells were counted using trypan blue exclusion assay. siCTL was Silencer select negative control #1 (Ambion, Life Technologies, USA). Results are mean ± SD of five replicates. Statistically significant differences of the cell counts were compared to the untransfected samples in the corresponding time point, and were analyzed by paired t-test (* = ≤0.05 and ** = P ≤ 0.01). ( b ) Up-regulation of p21 mRNA was observed after knockdown of CCHCR1 . siRNAs were transfected into HeLa cells and were collected for analysis 4 days post-transfection. Gene expressions (normalized to RPL13A gene expression) were analyzed by RT-qPCR. Results are mean ± SD of three replicates. Statistically significant differences of the gene expressions were compared to untransfected sample and were analyzed by paired t-test (* = ≤0.05, ** = P ≤ 0.01 and *** = P ≤ 0.001).

Article Snippet: Human cervical cancer HeLa cells (ATCC ® CRM-CCL-2 TM ) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Transfection, Trypan Blue Exclusion Assay, Negative Control, Knockdown, Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Transcriptional Regulation of the P-bodies Associated Psoriasis Candidate Gene CCHCR1

doi: 10.1101/2023.04.08.535951

Figure Lengend Snippet: ( a ) HeLa cells were treated with 0.1% DMSO (vehicle control) or 2 μM CPT for 24 hours. The percentage of cells in different cell cycle phases were analyzed by flow cytometry. ( b ) Relative expression of CCHCR1 and TCF19 (normalized to RPL13A gene expression) in HeLa cells after the treatment with CPT for 24 hours. Results are mean ± SD of three replicates. Statistically significant differences of the gene expression level were compared to 0.1% DMSO treatment and were analyzed by paired t-test (* = ≤0.05 and ** = P ≤ 0.01).

Article Snippet: Human cervical cancer HeLa cells (ATCC ® CRM-CCL-2 TM ) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Control, Flow Cytometry, Expressing, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells

doi: 10.3390/ijms21207445

Figure Lengend Snippet: IC 50 values, maximum percentage mean suppression and relative gene expression profile of human cervical cancer cells.

Article Snippet: Human cervical cancer cell lines HeLa, ME-180, C-33A and SiHa (ATCC numbers CCL-2, HTB-33, HTB-31, HTB-35, respectively) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells

doi: 10.3390/ijms21207445

Figure Lengend Snippet: Growth inhibition of BCA-M and ADI on human cervical cancer cells. The results of cell proliferation assay are represented by the mean % absorbance and S.D. ( A ) BCA-M induced growth inhibition on human cervical cancer cells. ( B ) C-33A and SiHa cells were susceptible to ADI treatment. ( C ) HeLa, CC3 and ME-180 cells were resistant to ADI treatment even at a 10-fold higher maximum tested concentration, as in the susceptible cell lines.

Article Snippet: Human cervical cancer cell lines HeLa, ME-180, C-33A and SiHa (ATCC numbers CCL-2, HTB-33, HTB-31, HTB-35, respectively) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Inhibition, Proliferation Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells

doi: 10.3390/ijms21207445

Figure Lengend Snippet: BCA-M induced cell cycle arrest in SiHa, HeLa and ME-180 cells. The effects of BCA-M on cell cycle phase distribution were determined using flow cytometric analysis with propidium iodide (PI) staining and RNase digestion. The results for percentage of cells in G 1 , G 2 /M and S phases were shown as means and S.D. One-way ANOVA indicated significant phase arrestment at S phase for SiHa cells, G 2 /M and S phases for HeLa cells and G 2 /M phase for ME-180 cells with post hoc Dunnett’s test showing p values for comparison between control and treatment as: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human cervical cancer cell lines HeLa, ME-180, C-33A and SiHa (ATCC numbers CCL-2, HTB-33, HTB-31, HTB-35, respectively) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Staining, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells

doi: 10.3390/ijms21207445

Figure Lengend Snippet: Effect of BCA-M on relative gene expression levels of various cyclins in SiHa, HeLa and ME-180 cells. The relative gene expression levels were determined using real-time PCR and normalized with the expression levels of the internal control (GAPDH, data not shown) and that of the respective genes in drug treatment control. The results were shown as means and S.D. One-way ANOVA with post hoc Dunnett’s test showing p values for comparison between control and treatment as: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human cervical cancer cell lines HeLa, ME-180, C-33A and SiHa (ATCC numbers CCL-2, HTB-33, HTB-31, HTB-35, respectively) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells

doi: 10.3390/ijms21207445

Figure Lengend Snippet: Fluorescent micrographs showing green punctate signals that represent autophagosome formation in ( A ) HeLa and ( B ) SiHa cells treated with BCA-M. Rapamycin was used as positive control. Quantification of the average number of LC3 puncta per cell was performed manually by counting obvious and large LC3 puncta ( n = 3).

Article Snippet: Human cervical cancer cell lines HeLa, ME-180, C-33A and SiHa (ATCC numbers CCL-2, HTB-33, HTB-31, HTB-35, respectively) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells

doi: 10.3390/ijms21207445

Figure Lengend Snippet: Growth inhibitory effect of BCA-M was enhanced synergistically by its combination to CQ on HeLa and SiHa human cervical cancer cells. The results of cell proliferation assay were represented by the mean % absorbance and S.D. ( A ) For SiHa cells, BCA-M was 2-fold serially diluted from 0.625 U/mL with or without combination to CQ starting at 6.25 μM and serially diluted along with BCA-M to maintain a constant ratio between the drugs. ( B ) For HeLa cells, constant drug ratio approach was also used with the starting concentration of BCA-M at 5 U/mL and CQ at 25 μM. In both cases, the combination of CQ enhanced the growth inhibitory effect of BCA-M.

Article Snippet: Human cervical cancer cell lines HeLa, ME-180, C-33A and SiHa (ATCC numbers CCL-2, HTB-33, HTB-31, HTB-35, respectively) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Proliferation Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells

doi: 10.3390/ijms21207445

Figure Lengend Snippet: IC 50 values and combination index (C.I.) for the treatment of BCA-M and chloroquine.

Article Snippet: Human cervical cancer cell lines HeLa, ME-180, C-33A and SiHa (ATCC numbers CCL-2, HTB-33, HTB-31, HTB-35, respectively) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: