wt hela cells  (Santa Cruz Biotechnology)

Journal: Journal of Human Immunity

Article Title: Complete and partial forms of X-linked MCTS1 deficiency in patients with mycobacterial disease

doi: 10.70962/jhi.20250073

Figure Lengend Snippet: Functional investigation of the new MCTS1 variants. (A) Predicted structure of the MCTS1 protein with the domain of unknown function 1947 (DUF1947) and the pseudouridine synthase and archaeosine transglycosylase (PUA) domains for WT protein, the synthetic LOF variant p. A109D, and the patient variants p.L170* (P1), p.W175* (P2), and predicted p.E60Kfs5* (P4). The numbers annotated for the WT MCTS1 protein correspond to the first and last amino acids of the MCTS1 protein, the DUF1947, and PUA domains. The variants of MCTS1 are shown in red. (B) Western blot of WT and MCTS1-KO HeLa cells after transfection with EV, WT MCTS1 (pWT), or the MCTS1 variants p.E60Kfs5* (pE60Kfs5*), p.L170* (pL170*), p.W175* (pW175*), and p.A109D (pA109D). Details are provided in . Non-transfected HeLa cells (“−”) were used as a negative control. (C) Schematic diagram of the Fluc and lamin B + stuORF reporters used to assess MCTS1 activity. stuORF, synthetic strong Kozak uORF. Modified from . (D) Activity of the MCTS1 variants in the luciferase translation reinitiation assay after the transfection of WT and MCTS1-KO cells with EV, WT MCTS1 or the various MCTS1 variants (p.E60Kfs5* p.L170*, W175*, and p.A109D; see details in ). Bars: Mean and standard deviation of three technical replicates, represented as turquoise dots. This experiment is representative of the three biological replicates. The asterisks indicate the level of significance, as assessed in a one-way ANOVA with Tukey’s test for multiple comparisons and adjustment for multiple testing (*P < 0.05; **P < 0.005). (E) Sanger sequencing confirmation of KI of the MCTS1 W175* variant and silent L174L variants in HEK293T cells. (F) Western blot of HEK293T cells subjected to genome editing to introduce MCTS1 KO in a pooled manner or MCTS1 W175* KI with the indicated single-cell derived clones shown. (G) Quantitation of three independent western blots of the indicated cell lines. Statistical significance was assessed using two-sided Mann–Whitney tests. Source data are available for this figure: .

Article Snippet: WT HeLa cells (Cat# CRM-CCL-2; ATCC, RRID: CVCL_0030) and MCTS1 KO HeLa cells ( ) were cultured in Dulbecco/Vogt modified Eagle’s minimal essential medium (DMEM, Gibco) with 10% fetal-calf serum (FCS) and 100 IU/ml penicillin/streptomycin (Cat# 15140122; Gibco).

Techniques: Functional Assay, Variant Assay, Western Blot, Transfection, Negative Control, Activity Assay, Modification, Luciferase, Standard Deviation, Sequencing, Introduce, Derivative Assay, Clone Assay, Quantitation Assay, MANN-WHITNEY

Journal: Cell Death Discovery

Article Title: Loss of Ezrin triggers mitochondrial dysfunction and oxidative stress, associated with neuronal cell death

doi: 10.1038/s41420-025-02790-5

Figure Lengend Snippet: A Confocal microscopy images showing HeLa WT and EZR −/− cells incubated with the fluorescent dye MitoTracker (magenta) and DAPI (blue) after EGFR-GFP transfection (green). Following EGF stimulation, EGFR translocates to the cytosol and colocalizes with mitochondria in HeLa WT, whereas in HeLa EZR −/− cells remains on the plasma membrane even after EGF stimulation. B Heatmap representing the 82 downregulated genes in HeLa EZR −/− cells (FDR ≤ 0.05) involved in mitochondrial processes. C Bubble plot representing the biological process associated to downregulated genes in HeLa EZR −/− cells. D HeLa WT and EZR −/− cells were lysed and immunoblotted with anti-TOM20 antibody. GAPDH was used as a loading control. The graph shows TOM20 levels relative to GAPDH ± SEM (at least n = 3 experiments). Statistical test: unpaired t-test. E HeLa WT and EZR −/− cells were cultured in 6-well plates for 24 h. They were then fixed and immunostained with the TOM20 antibody (green) and DAPI (blue). The graphs were generated using the Mitochondria Analyzer plugin in ImageJ, allowing for a detailed analysis of mitochondrial structure ± SEM (at least n = 3 experiments). Statistical test: unpaired t-test.

Article Snippet: HeLa WT cell lines were obtained from the American Type Culture Collection (ATCC), while HeLa EZR −/− cells were previously generated in our laboratory [ ].

Techniques: Confocal Microscopy, Incubation, Transfection, Clinical Proteomics, Membrane, Control, Cell Culture, Generated

Journal: Cell Death Discovery

Article Title: Loss of Ezrin triggers mitochondrial dysfunction and oxidative stress, associated with neuronal cell death

doi: 10.1038/s41420-025-02790-5

Figure Lengend Snippet: A Immunofluorescence analysis of WT and EZR −/− HeLa cells stained with the CS antibody (green) and DAPI (blue) reveals a reduction in CS signal in HeLa EZR −/− cells. The graph shows fluorescence values, measured as Fluorescence Intensity/Area, in WT and EZR −/− cells ± SEM (at least n = 3 experiments). Statistical test: unpaired t-test. B Confocal microscopy images showing HeLa WT and EZR −/− cells incubated with the fluorescent dye MitoTracker (magenta) and DAPI (blue) with a reduction of mitochondria activity in HeLa EZR −/− cells. The graph shows the fluorescence intensity of MitoTracker-positive cells ±SEM (at least n = 3 experiments). Statistical test: unpaired t-test. C Immunofluorescence for H2DCFDA. WT and EZR −/− HeLa cells were cultured in 6-well plates for 24 h. They were then incubated with the fluorescent dye H2DCFDA (green) and DAPI (blue). The graph on the right shows the fluorescence intensity of H2DCFDA-positive cells ±SEM (at least n = 3 experiments). Statistical test: unpaired t-test. D WT and EZR −/− HeLa cells were incubated with the fluorescent dye MitoSOX (red) and DAPI (blue). The graph on the right shows the fluorescence intensity (IntDen/Area) of MitoSOX-positive cells ±SEM (at least n = 3 experiments). Statistical test: unpaired t-test. E Representative images of immunofluorescence with JC1 dye (green/red) and DAPI (blue) in WT and EZR −/− HeLa cells. The graph on the right shows data expressed as the red/green fluorescence intensity ratio ±SEM (at least n = 3 experiments). Statistical test: unpaired t-test.

Article Snippet: HeLa WT cell lines were obtained from the American Type Culture Collection (ATCC), while HeLa EZR −/− cells were previously generated in our laboratory [ ].

Techniques: Immunofluorescence, Staining, Fluorescence, Confocal Microscopy, Incubation, Activity Assay, Cell Culture

Journal: Cell Death Discovery

Article Title: Loss of Ezrin triggers mitochondrial dysfunction and oxidative stress, associated with neuronal cell death

doi: 10.1038/s41420-025-02790-5

Figure Lengend Snippet: A WT and EZR −/− HeLa cells were lysed and the proteins incubated with an antibody against OXPHOS. Bottom, the membrane activated prior to antibody incubation is shown as a loading control. The graphs show the quantification of the different OXPHOS complexes relative to the membrane ±SEM (at least n = 3 experiments). Statistical test: unpaired t-test. B The graph shows the relative ATP luminescence levels ± SEM (at least n = 3 experiments) in HeLa WT and HeLa EZR −/− . Statistical test: unpaired t-test. C – I Representative graph of Cell Mito Stress assay performed by Seahorse XFp analyzer in WT and EZR −/− HeLa cells is reported ( C ). In the bar charts, each point in the OCR time courses is the average of three technical replicates. Basal respiration ( D ), maximal respiration ( E ), ATP production ( F ), proton leak ( G ) non-mitochondrial respiration ( H ) and spare respiratory capacity (SRC) ( I ) are reported. The values are expressed as means ± SD.

Article Snippet: HeLa WT cell lines were obtained from the American Type Culture Collection (ATCC), while HeLa EZR −/− cells were previously generated in our laboratory [ ].

Techniques: Incubation, Membrane, Control

Journal: Cell Death Discovery

Article Title: Loss of Ezrin triggers mitochondrial dysfunction and oxidative stress, associated with neuronal cell death

doi: 10.1038/s41420-025-02790-5

Figure Lengend Snippet: A Heatmap representing the 65 upregulated genes in HeLa treated with NSC668394 (FDR ≤ 0.05) involved in cell death and apoptotic processes. B Bubble plot representing the biological process associated to upregulated genes in HeLa treated with NSC668394 . C Co-expression, obtained by GeneMANIA, highlight Ezrin and apoptotic protein binding. D HeLa WT and HeLa EZR −/− were cultured in 6-well plates for 24 h. Subsequently, they were treated with an Annexin V/Propidium Iodide mix for 20 min and DAPI (blu). The images show an increase in cell death in HeLa EZR −/− compared to HeLa WT, while NAC drastically reduces cell death in HeLa EZR −/− . E HeLa WT and EZR −/− cells were cultured in 6-well plates for 24 h. They were then treated with 1.5 mM NAC for 1 h and subsequently trypsinized. A mix of Trypan Blue and cells was prepared and counted using the LUNA cell counter. The graph shows the individual viability percentages for HeLa WT, HeLa WT + NAC, HeLa EZR −/− , and HeLa EZR −/− + NAC ± SEM (at least n = 3 experiments). Statistical test: unpaired t-test. F HeLa WT and EZR −/− cells were cultured in 6-well plates for 24 h. They were then treated with 1.5 mM NAC for 1 h and incubated with the fluorescent dye H2DCFDA (green) and DAPI (blue). The images show ROS reduction when NAC is used in HeLa EZR −/− . G HeLa WT and EZR −/− cells were cultured in 96-well plates for 24 h. They were then treated with Presto blue reagent. The graph shows a reduction in relative absorbance levels percentage in HeLa EZR −/− cells compared to HeLa WT, reflecting a lower cell viability in HeLa EZR −/− cells.

Article Snippet: HeLa WT cell lines were obtained from the American Type Culture Collection (ATCC), while HeLa EZR −/− cells were previously generated in our laboratory [ ].

Techniques: Expressing, Protein Binding, Cell Culture, Incubation