Journal: Frontiers in Oncology

Article Title: The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment

doi: 10.3389/fonc.2023.1117262

Figure Lengend Snippet: DHO hydrophilic extract increases the RPA32 S4/8 phosphorylation in response to CPT respect to CPT alone. (A) Western blot analysis of HeLa cells pre-treated, for one hour, with different concentrations (0.5, 1, 3 mg/ml) of DHO extract and DMSO followed by a treatment with 1 μM CPT for additional two hours. Total RPA32 and Lamin A/C are loading as control of pRPA32 S4/8 and total RPA32, respective. The values obtained are reported in the blot images. (B) Immunofluorescence analysis of pRPA32 S4/8 foci intensity in HeLa cells pretreated for one hour with DHO 1mg/ml or DMSO, followed incubation by 1 μM CPT for additional two hours. The histogram reports three independent experiments with standard deviations (SD). Statistically significant differences are indicated with: ***significant (P < 0.001). (C) Western blot analysis of HeLa cells treat with DHO with different concentrations (0.5, 1, 3 mg/ml) and CTP (1 µM) alone and in combination. CHK1 S345 phosphorylation (pCHK1 S345). Total CHK1 and GAPDH are loading as control of pCHK1 S345 and total CHK1, respective. The values obtained are reported in the blot images. (D) Western blot analysis of HeLa cells pre-treated for one hour with different DHO (0.5, 1, 3 mg/ml) or Ascorbic Acid (14.5, 29 and 87 μM) concentrations followed by two hours of CPT treatment. pRPA32 S4/8 was normalized as indicated above.

Article Snippet: HeLa cells were transfected with 2 μg of pCMV-HIS (CV003, Sino Biological) (per 6cm dish) linearized (by ApaI restriction enzyme digestion) and 1 μg of pEGFP-C1 with Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.

Techniques: Western Blot, Control, Immunofluorescence, Incubation

Journal: Frontiers in Oncology

Article Title: The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment

doi: 10.3389/fonc.2023.1117262

Figure Lengend Snippet: DHO hydrophilic extract do not alter cell cycle distribution anh H2AX activation (A) Cell cycle profile of HeLa cells pre-treated with 1 mg/ml of DHO or vehicle (DMSO) for one hours followed by incubation with CPT (1 µM) for additional two hours, was analysed through flow cytometry upon propidium iodide (PI) staining. (B) Immunofluorescence analysis of γH2AX foci intensity in HeLa cells pretreated for one hour with DHO 1mg/ml or vehicle (DMSO), followed incubation by 1 μM CPT for additional two hours. The histogram reports the mean of three independent experiments with standard deviations (SD). Statistically significant differences are indicated with: *significant (P < 0.05). (C) Western blot analysis of HeLa cells pre-treated for one hour with DHO at 1 mg/ml or vehicle (DMSO), followed by two hours of Etoposide (20 μM) incubation. RPA S4/8 and pCHK1 S345 or vehicle (DMSO), followed by two hours of Etoposide (20 μM) incubation. RPA S4/8 and pCHK1 S345 were normalized to those of each respective total RPA32 and CHK1 controls. (D) Immunofluorescence analysis of pRPA32 S4/8 foci intensity in HeLa cells pre-treated with 1mg/ml of DHO followed by incubation with Etoposide 20 µM for additional two hours. The histogram reports the mean of three independent experiments with standard deviations (SD). Statistically significant differences are indicated with: ***significant (P < 0.001).

Article Snippet: HeLa cells were transfected with 2 μg of pCMV-HIS (CV003, Sino Biological) (per 6cm dish) linearized (by ApaI restriction enzyme digestion) and 1 μg of pEGFP-C1 with Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.

Techniques: Activation Assay, Incubation, Flow Cytometry, Staining, Immunofluorescence, Western Blot

Journal: Frontiers in Oncology

Article Title: The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment

doi: 10.3389/fonc.2023.1117262

Figure Lengend Snippet: DHO hydrophilic extract impairs RAD51 chromatin loading (A) HeLa DR-GFP cells were transfected with the coding plasmid for the SceI restriction enzyme in presence of 1 mg/ml of DHO or vehicle (DMSO) and incubated for 48 hours. FACS analysis measurement of GFP levels was used to calculate %HR frequency compared with DMSO alone which was set as 100% (control). Data represent the mean % ± SD obtained from three independent experiments. Statistically significant differences are indicated with: *significant (P < 0.05). (B) Chromatin enhriched purification of HeLa cells pretreated or not with the DHO extract at 1 mg/ml followed by 1 μM CPT treatment for additional two hours. Cells were then lysed to obtain a soluble (S) and a chromatin-enriched (P, as pellet) fraction. Western blotting of RAD51 was performed to analyse the loading onto chromatin of the indicated proteins, involved in the cell response to DNA damage. Total RPA32 and Lamin A/C were used as controls of the supernatant or the chromatin-enriched fraction, respectively. (C) HeLa pimEJ5-GFP cells were transfected with the plasmid encoding the SceI restriction enzyme followed by incubation with 1 mg/ml of DHO extract or vehicle (DMSO) for 48 hours followed by FACS analysis measurement of GFP levels to calculate %NHEJ frequency compared with control cells which were set as 100%. Data represent the mean ± SD obtained from three independent experiments. Statistically significant differences are indicated with: *significant (P < 0.05). (D) HeLa cells were transfected with the pCMV-His empty vector linearized with the ApaI restriction enzyme and pEGFP-C1 in presence of siCTR or si53BP1 followed by incubation with the vehicle (DMSO) or 1 mg/ml DHO hydrophilic extract for 15 days and stained with 2% (w/v) crystal violet solution. Data represent the mean % ± SD obtained from three independent experiments. Statistically significant differences are indicated with: *significant (P < 0.05), ***significant (P < 0.001).

Article Snippet: HeLa cells were transfected with 2 μg of pCMV-HIS (CV003, Sino Biological) (per 6cm dish) linearized (by ApaI restriction enzyme digestion) and 1 μg of pEGFP-C1 with Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Incubation, Control, Purification, Western Blot, Staining

Journal: Frontiers in Oncology

Article Title: The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment

doi: 10.3389/fonc.2023.1117262

Figure Lengend Snippet: DHO hydrophilic extract increases the Single Strand Annealing activity in response to CPT treatment (A) Chromatin enhriched purification of HeLa cells pretreated or not with the DHO extract at 1 mg/ml followed by 1 μM CPT treatment for additional two hours. Cells were then lysed to obtain a soluble (S) and a chromatin-enriched (P, as pellet) fraction. Western blotting was performed to analyse the loading onto chromatin of the Poly (ADP-ribose) polymerase (PARP1) protein, involved in the cell response to DNA damage. Total RPA32 and Lamin A/C were used as controls of the supernatant or the chromatin-enriched fraction, respectively. (B) Chromatin enhriched purification of HeLa cells performed as previously for the analysis of RAD52 chromatin loading. (C) Chromatin enhiched purification of HeLa cells was performed as previously described followed by incubation with ERCC1 antibody. (D) HeLa hprtSAGFP cells were transfected with the plasmid encoding the SceI restriction enzyme followed by incubation with 1 mg/ml of DHO extract or vehicle (DMSO) for 48 hours followed by FACS analysis measurement of GFP levels to calculate %SSA frequency compared with control cells which were set as 100%. Data represent the mean % ± SD. obtained from three independent experiments. Statistically significant differences are indicated with: ***significant (P < 0.001).

Article Snippet: HeLa cells were transfected with 2 μg of pCMV-HIS (CV003, Sino Biological) (per 6cm dish) linearized (by ApaI restriction enzyme digestion) and 1 μg of pEGFP-C1 with Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.

Techniques: Activity Assay, Purification, Western Blot, Incubation, Transfection, Plasmid Preparation, Control

Journal: Frontiers in Oncology

Article Title: The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment

doi: 10.3389/fonc.2023.1117262

Figure Lengend Snippet: Synergistic effect of DHO-CPT combination on different cancer cell lines (A) HeLa cells were pretreated with 1 mg/ml of DHO hydrophilic extract or vehicle (DMSO) followed by different doses of CPT for additional 72 hours and cell viability was analyzed by MTS assay. Results represent the means and SD of three independent experiments, each conducted in triplicate, and are expressed as percentages of cell viability, calculated with respect to the control cells treated with DMSO alone. Combination index was calculated as described in matherial and methods. (B) MDA-MB-231 cells were pretreated with 1mg/ml of DHO or vehicle followed by incubation with different doses of CPT for additional 72 hours and cell viability assay was performed as previously. (C) HeLa cells were pretreated with DHO at 1mg/ml for one hour followed by treatment with CPT at indicated concentrations; at the end of ten days of incubations, Hela cells were stained with crystal violet. (D) MDA-MB-231 cells were incubated with DHO, as described previously, for the cell viabilty assay and cultured for ten days followed by staining with the cristal violet.

Article Snippet: HeLa cells were transfected with 2 μg of pCMV-HIS (CV003, Sino Biological) (per 6cm dish) linearized (by ApaI restriction enzyme digestion) and 1 μg of pEGFP-C1 with Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.

Techniques: MTS Assay, Control, Incubation, Viability Assay, Staining, Cell Culture

Journal: PLoS ONE

Article Title: Cyclophilin B Interacts with Sodium-Potassium ATPase and Is Required for Pump Activity in Proximal Tubule Cells of the Kidney

doi: 10.1371/journal.pone.0013930

Figure Lengend Snippet: A: Yeast two hybrid assay. CDNA for CypB was cloned in frame into the yeast expression vector (pGBKT7) that harbours the GAL4 activation domain. The recombinant plasmids plus Human Kidney Matchmaker cDNA Library were co-transformed into AH109 yeast strain. The co-transformed cells were selected on Ade-/Leu-/His-/Trp-, 10 µM Aminotriazol dropout plates to monitor for growth. The positive control represents co-transformation of p53 and T-antigen in two-hybrid expression vectors, pGBKT7-P53 and pGADT7 (BD Biosciences, Clontech), respectively. B: GST-pull-down assays. CypB was fused in frame with the GST gene. GST and GST-HCypB products were immobilized on Sepharose 4B and incubated with COS-7 cells lysates expressing Na/K-β1 (pCMV-HA-Na/K). The bound proteins were analyzed by immunoblotting with anti-Na/K-β1 rabbit polyclonal antibody. C–E: Co-Immunoprecipitation assays. HK-2 lysates were immunoprecipitated with mouse monoclonal antibodies against Na/K-β1 or Na/K-α1 and rabbit polyclonal antibody against CypB. The immunoprecipitates were subjected to Western blotting analyses, as indicated in the figure. As control, ChromePure mouse IgG and ChromePure rabbit IgG were used. Figures 1B to 1E are representative of at least three independent experiments.

Article Snippet: BD Biosciences, clontech, Palo Alto, CA), was subcloned into the BglII restriction endonuclease site of pCMV-HA mammalian expression vector (BD Biosciences, clontech, Palo Alto, CA) to get the pCMV- Na/K-β1 -HA construct.

Techniques: Y2H Assay, Clone Assay, Expressing, Plasmid Preparation, Activation Assay, Recombinant, cDNA Library Assay, Transformation Assay, Positive Control, Incubation, Western Blot, Immunoprecipitation