Journal: bioRxiv

Article Title: The p53 Protein is a Suppressor of Atox1 Copper Chaperon in Tumor Cells Under Genotoxic Effects

doi: 10.1101/2023.07.25.550476

Figure Lengend Snippet: Dependence of Atox1 and p53 level in HCT116 and A549 cell lines with different TP53 status: A - immunoblotting with antibodies to p53, p21, and Atox1; beta-actin was used as a normalization. A densitometric analysis of the obtained data is shown below. B – RT-qPCR analysis with primers for TP53, CDKN1A, and ATOX1 genes; GAPDH gene was used as a reference. C - immunofluorescence staining with primary antibodies to Atox1, secondary antibodies with AlexaFluor488. DAPI was used for nuclei staining. WT – wild type cells, TP53 -/- – cells without TP53. For all experiments: n = 3, mean +/− SEM, paired Student t-test, p < 0,05.

Article Snippet: Transformed human cell lines used: HCT116 (colon adenocarcinoma) with intact p53; HCT116p53 -/- with a deletion of both alleles of the TP53 genes, as well as the A549 line with wild (A549) and knockout p53 (A549p53 -/- ) by the CRISPR-Cas9 method, acquired at ATCC.

Techniques: Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The p53 Protein is a Suppressor of Atox1 Copper Chaperon in Tumor Cells Under Genotoxic Effects

doi: 10.1101/2023.07.25.550476

Figure Lengend Snippet: Influence of cytotoxic agents on the activity of Atox1 at different status (WT and KO) of the TP53 gene in A549 and HCT116 cell lines, 24h after drugs exposure. A - immunoblotting with antibodies to p53, p21, and Atox1; beta-actin was used as a normalization. A densitometric analysis of the obtained data is shown below. B – RT-qPCR analysis with primers for TP53, CDKN1A and ATOX1 genes; GAPDH gene was used as a reference, C - immunofluorescence staining with primary antibodies to Atox1, secondary antibodies with AlexaFluor488. DAPI was used for nuclei staining. DOX – doxorubicin (0,1μM), CIS – cisplatin (35μM), PMA – phorbol-12-myristate- 13-acetate (80nM), H 2 O 2 – hydrogen peroxide (450μM), BLE – bleomycin (10μM). WT – wild type cells, TP53 -/- – cells without TP53. For all experiments: n = 3, mean +/− SEM, two-way ANOVA, p < 0,05.

Article Snippet: Transformed human cell lines used: HCT116 (colon adenocarcinoma) with intact p53; HCT116p53 -/- with a deletion of both alleles of the TP53 genes, as well as the A549 line with wild (A549) and knockout p53 (A549p53 -/- ) by the CRISPR-Cas9 method, acquired at ATCC.

Techniques: Activity Assay, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The p53 Protein is a Suppressor of Atox1 Copper Chaperon in Tumor Cells Under Genotoxic Effects

doi: 10.1101/2023.07.25.550476

Figure Lengend Snippet: Influence of ionizing radiation on the activity of Atox1 at different status (WT and KO) of the TP53 gene in A549 and HCT116 cell lines, 24h after ionizing irradiation (10Gy) exposure. A - immunoblotting with antibodies to p53, p21, and Atox1; beta-actin was used as a normalization. A densitometric analysis of the obtained data is shown below. B – RT-qPCR analysis with primers for TP53, CDKN1A and ATOX1 genes; GAPDH gene was used as a reference. The value of WT 0Gy (control) was taken as a 1.0 for all genes and is not shown in the graphs. For all experiments: n = 3, mean +/− SEM, two-way ANOVA, p < 0,05.

Article Snippet: Transformed human cell lines used: HCT116 (colon adenocarcinoma) with intact p53; HCT116p53 -/- with a deletion of both alleles of the TP53 genes, as well as the A549 line with wild (A549) and knockout p53 (A549p53 -/- ) by the CRISPR-Cas9 method, acquired at ATCC.

Techniques: Activity Assay, Irradiation, Western Blot, Quantitative RT-PCR, Control

Journal: iScience

Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

doi: 10.1016/j.isci.2025.114592

Figure Lengend Snippet: The IDR of TRIP12 is responsible for the formation of chromatin condensates (A) Prediction of TRIP12 3D-structure by AlphaFold model. Predicted local distance difference test (pLDDT) indicates the confidence level of the predicted structure. Dark blue, light blue, orange, and yellow represent very high, confident, low, and very low model confidence, respectively. (B) Graphical representation of TRIP12 different domains fused to the GFP reporter protein. Dark blue rectangles locate endogenous or artificial NLSs. IDR, intrinsically disordered region; ARM, armadillo domain; WWE, tryptophan-tryptophan-glutamate-rich domain; HECT, homologous to E6-AP carboxyl terminus; and GFP, green fluorescent protein. (C) Representative images of TRIP12-domains fused to GFP expressing HeLa S3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Insertion of an artificial NLS in ARM-WWE-GFP, ΔIDR/ARM-WWE/HECT-GFP, HECT-GFP, and ΔIDR-GFP constructs allowed for nuclear localization with a faint presence in the cytoplasm. Scale bars represent 2 μm. (D) Determination of DNA granularity relative to GFP expression level in TRIP12-domains fused to GFP expressing HeLa S3 cells. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on over 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated. (E) Representative images of chromatin condensates induced by TRIP12-IDR overexpression in HCT-116, U2OS, and hTert-RPE1 cell lines by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 2 μm. (F) Comparison of isoelectric point (pI) and capacity to form chromatin condensates (slope) of the different TRIP12-GFP constructs. The pI of the different TRIP12 fragments was determined using ProtParam on Expasy website. The capacity to form chromatin condensates is indicated by the slope values obtained in D, 2F, and C. A Spearman r coefficient test and a two tailed-p value are indicated. (G) Representative images of DNA organization in IDR-GFP deletion constructs in high expressing HeLa S3 cells (left). The cytoplasmic expression of 325-445-GFP constructs is explained by the loss of NLS sequences. Determination of DNA granularity relative to GFP expression level for the different IDR-GFP deletion constructs (right). The DAPI granularity and GFP expression were determined as described in “ ” on more than 40 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated. (H) Comparison of isoelectric point (pI), capacity to form chromatin condensates (slope), and the length (in aa) of the different IDR-GFP deletion constructs. The pI and the length of the different TRIP12 fragments were determined using ProtParam on Expasy website. The capacity to form chromatin condensates was assessed from the slope values obtained in G. A Spearman r coefficient test and a two tailed-p value are indicated. (I) Representative image of cellular fractionation of HeLa S3 cells expressing 1-107-GFP, 108-207-GFP, 208-324-GFP, and 325-445-GFP constructs. GFP expression in soluble and chromatin-bound fractions was determined by western blot analysis. The level of HSP90 and PanH3 protein expression were used as loading and enrichment controls. The graph represents the percentage of GFP expression in the different fraction. Results are expressed as mean ± SEM of four different experiments. (J) Electric net charge of [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR at pH 7.4 determined by Prot pi|Protein Tool (left graph). Percentage of basic, acidic, and uncharged residues in [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR determined by Prot pi|Protein Tool (right graph). (K) Graphical representation of MED1-IDR fused to GFP protein. The dark blue rectangle indicates artificial NLSs. Representative images of MED1-IDR-GFP expressing HeLa S3 cells obtained by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on more than 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is represented in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated.

Article Snippet: HeLa S3 (CCL-2TM, female), HEK-293T (CRL-3216TM, female), U2OS (HTB-96 TM , female), hTERT RPE-1 (CRL-4000 TM , female) and HCT-116 (CCL-247 TM , male) cell lines were procured from ATCC ( https://www.atcc.org ).

Techniques: Expressing, Immunofluorescence, Construct, Transfection, Two Tailed Test, Over Expression, Comparison, Cell Fractionation, Western Blot

Journal: PLoS ONE

Article Title: Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase

doi: 10.1371/journal.pone.0167385

Figure Lengend Snippet: (a,b) Images of live synchronized HCT116 cells released from APH block without (a) and with (b) BrdU labeling (100 μM, 4 h), stained with HXT (1 μM, 30 min). Scale bar is 50 μm. (c) Representative examples of HXT fluorescence decays (data trace) for individual pixels in selected nuclei (indicated by circles on (b)) showing mono- and double-exponential fittings. (d) Average τ m (left) and intensity (right) signals for no BrdU (red, n = 12) and +BrdU (blue, n = 15) nuclei. Asterisks indicate significant difference between groups (p < 0.05): *—p < 0.001, **—p < 0.00001. Error bars show the standard deviation.

Article Snippet: Tumor spheroids were formed by seeding HCT116 cells on a Lipidure-coat TM plate (Amsbio, UK) at concentration of 200 cells/ well, and by growing them for 4 days.

Techniques: Blocking Assay, Labeling, Staining, Fluorescence, Standard Deviation

Journal: PLoS ONE

Article Title: Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase

doi: 10.1371/journal.pone.0167385

Figure Lengend Snippet: ( a) Asynchronous and synchronized live HCT116 cells were incubated with BrdU (100 μM, 4 h) and stained with HXT (1 μM, 30 min). Immediately after FLIM cells were fixed with 4% paraformaldehyde and stained with anti-BrdU antibody. Scale bar is 50 μm. (b) Average (n = 5) distributions of τ m . Black arrow indicates threshold τ m , which differentiates between S phase and non S-phase cells. (c) Cell proliferation rates calculated by the different methods. Bar chart shows fractions of total cell numbers and standard deviation for +BrdU cells (S-phase). The mean values were calculated from five different images of the asynchronous and synchronized cell cultures.

Article Snippet: Tumor spheroids were formed by seeding HCT116 cells on a Lipidure-coat TM plate (Amsbio, UK) at concentration of 200 cells/ well, and by growing them for 4 days.

Techniques: Incubation, Staining, Standard Deviation