Journal: Oxidative Medicine and Cellular Longevity

Article Title: Harpagophytum procumbens Inhibits Iron Overload-Induced Oxidative Stress through Activation of Nrf2 Signaling in a Rat Model of Lumbar Spinal Stenosis

doi: 10.1155/2022/3472443

Figure Lengend Snippet: Harpagophytum procumbens (HP) attenuates FeSO 4 -induced neurotoxicity in primary spinal cord neurons. (a) Schematic illustration of the experimental protocol of primary spinal cord neurons with HP posttreatment. (b and c) CCK-8 results of spinal cord neurons treated with various concentrations of HP for 24 h, without and with exposure to 50 μ M FeSO 4 . (d) Quantification of the number of live and dead cells for the neurotoxicity assay. (e) Cell viability was imaged with confocal microscopy using a live/dead imaging assay kit (live cells, green; dead cells, red). White scale bar = 200 μ m. (f) The percentage of PI-positive neurons from live cell imaging over a 48 h period, with images taken every 30 min. (g) Flow cytometric quantification of necrotic cells (Annexin V-negative/PI-positive), challenged with 50 μ M FeSO 4 . (h) Representative flow cytometric dot plots showing Annexin V ( x -axis) and PI ( y -axis) analysis for cell death type in spinal cord neurons. Data are expressed as the means ± SEM. Significant differences are indicated as #### p < 0.0001 vs. the blank group, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 vs. the FeSO 4 group, according to one-way analysis of variance with Tukey's post hoc test.

Article Snippet: A flow cytometric assay was used to assess apoptosis, using an Annexin V-PE/PI apoptosis detection kit (Abcam) as previously described [ ].

Techniques: CCK-8 Assay, Confocal Microscopy, Imaging, Live Cell Imaging

Journal: bioRxiv

Article Title: GJB3, a gap junction gene, supports cell growth by mediating cystine uptake and regulating cellular stress pathways in SLC7A11 low adenocarcinomas

doi: 10.1101/2024.12.03.626548

Figure Lengend Snippet: A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both GFP-LC3 and RFP-LC3ΔG were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.

Article Snippet: Apoptosis was assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (Elabscience, E-CK-A211) following the manufacturer’s instructions.

Techniques: Knockdown, RNA Sequencing Assay, Expressing, Transformation Assay, Software, Isolation, Control, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Fluorescence, Staining, Activity Assay, Live Cell Imaging

Journal: International Journal of Biological Sciences

Article Title: XPOT Disruption Suppresses TNBC Growth through Inhibition of Specific tRNA Nuclear Exportation and TTC19 Expression to Induce Cytokinesis Failure

doi: 10.7150/ijbs.85006

Figure Lengend Snippet: XPOT ensures successful cytokinesis and resists apoptosis of TNBC cells. (A) Gene ontology analysis based on DEGs in RNA-seq of si-NC and si-XPOT MDA-MB-468 cells in which cytokinesis-related pathways were significantly enriched was conducted. (B) Cell cycle analysis for si-NC and si-XPOT group in MDA-MB-468 and MDA-MB-231 cells. Representative images and quantitative cell count in each phase are shown. Values are means ± SD (Student's t test). (C) Selected frames from Live Cell Imaging of si-NC and si-XPOT/MDA-MB-468 cells. Binucleated cell is indicated by white arrowhead. (D) Representative immunofluorescence staining of β-tubulin and DAPI in si-NC and si-XPOT/MDA-MB-468 cells or MDA-MB-231 cells (left panel). Binucleated cell is indicated by white arrowhead. The binucleated cells ratio was analyzed, as shown on the right panel. Scale bar represents 20 μm. Values are mean ± SD (Student's t test). SD, standard deviation; n, number; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 40,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; PI, propidium iodide; si, small interfering RNA. *P< 0.05; **P < 0.01; ***P< 0.001.

Article Snippet: Cells (3 × 10 5 cells/well) were seeded in a 6-well plate overnight for attachment. siRNAs or plasmids were added to the cells for 48 h, followed by incubating in serum-free medium for another 24 h. To determine the apoptotic cell ratio, the cells were stained using an annexin V-FITC apoptosis detection kit (Dojindo, Kumamoto, Japan) and examined by flow cytometry for analysis.

Techniques: RNA Sequencing, Cell Cycle Assay, Cell Counting, Live Cell Imaging, Immunofluorescence, Staining, Standard Deviation, Small Interfering RNA

Journal: Cell Death & Disease

Article Title: Splice-switching of the oncogenic BCS1L isoform suppresses ovarian cancer progression by disrupting mitochondrial function

doi: 10.1038/s41419-026-08495-6

Figure Lengend Snippet: A GO enrichment of proteins captured by the FLAG antibody from A2780 cells overexpressing BCS1L-L or BCS1L-S with FLAG tags. B Mitochondrial basal OCR, ATP production, Maximal OCR, Proton leak, non-mitochondrial respiration, and spare capacity (SC) were evaluated in A2780 cells with BCS1L-L or BCS1L-S overexpression for 72 h and control cells using an Agilent Seahorse XFe24. C ATP content per 10 5 A2780 cells transfected with control, BCS1L-L, and BCS1L-S overexpression vectors for 72 h. D MMP was measured by JC-1 staining in A2780 cells with control and BCS1L-L or BCS1L-S overexpression for 72 h treated with 500 μM H 2 O 2 for 4 h. Red fluorescence indicated normal MMP, while green fluorescence indicated abnormal MMP. E Apoptotic cells were analyzed by flow cytometry using Annexin V/7-AAD staining in A2780 cells with control and BCS1L-L or BCS1L-S overexpression for 48 h treated with 500 μM H 2 O 2 for 4 h. Statistical analysis of apoptotic cells included early (Q2) and late (Q3) apoptotic cells. F Immunofluorescence of COX4 (red) in A2780 and HEY cells with BCS1L knockdown and control cells showing mitochondria morphology. DAPI was used to visualize the nuclei. Scale bars, 10 µm. G and H Mitochondrial basal OCR, maximal OCR, and SC (spare capacity) were evaluated in A2780 and HEY cells with BCS1L knockdown for 72 h using an Agilent Seahorse XFe96. I ATP content per 10 5 cells in three ovarian cancer cell lines with BCS1L knockdown for 48 h (three independent experiments). J MMP was measured by JC-1 staining in A2780 and HEY cells with BCS1L knockdown for 72 h. Red fluorescence indicated normal MMP, while green fluorescence indicated abnormal MMP. K Apoptotic cells were analyzed by flow cytometry using Annexin V/7-AAD staining in A2780 and HEY cells with BCS1L knockdown for 72 h. Statistical analysis of apoptotic cells included early (Q3) and late (Q2) apoptotic cells. All results were repeated in three independent experiments and evaluated by a two-tailed unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are shown as the mean ± SD.

Article Snippet: An Annexin V-FITC Apoptosis Detection Kit (Vazyme, A213-01) was used to measure the rate of both early and late-stage apoptotic cells.

Techniques: Over Expression, Control, Transfection, Staining, Fluorescence, Flow Cytometry, Immunofluorescence, Knockdown, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Splice-switching of the oncogenic BCS1L isoform suppresses ovarian cancer progression by disrupting mitochondrial function

doi: 10.1038/s41419-026-08495-6

Figure Lengend Snippet: A Transmission electron microscopy in control and A2780 cells with USP39 knockdown. Scale bars, 2 µm. Morphometric analysis of mitochondrial cristae was performed in a blinded fashion on at least five mitochondria per cell from six randomly selected cells, and maximal cristae width was measured using Image J. B Silver staining of BN-PAGE indicated mitochondrial OXPHOS assembly in control and USP39 knockdown A2780 cells. C and D Electron transport chain Complex III activities in control and USP39 knockdown A2780 cells were evaluated by substrate-uncoupler-inhibitor-titration (SUIT) on a high-resolution Oxygraph-2k respirometer. Complex activities were normalized to that of CS ( n = 3). Quantification of OCR measurements in A2780 cells ( E ) and HEY cells ( F ) after USP39 knockdown with the pharmacological inhibitors of metabolism (left). Individual parameters of mitochondrial function, including basal respiration, ATP production, proton leak, and spare respiratory capacity, were calculated according to the manufacturer’s protocol with non-mitochondrial oxygen consumption adjustment (right). Rot/AA, rotenone/antimycin A. G Quantification of ATP levels in A2780 cells for investigating the potential of BCS1L to rescue the loss of USP39. H and I ROS distribution was measured by flow cytometry in A2780 cells transfected with siUSP39 and BCS1L overexpression vector for 48 h. J Measurements and quantification of MMP (by JC-1) in A2780 cells with USP39 knockdown and BCS1L overexpression for 72 h. K Apoptotic A2780 cells after USP39 depletion together with BCS1L overexpression for 48 h were analyzed by flow cytometry using Annexin V/7-AAD staining. Three biological replicates were conducted in all functional experiments, and the p -value was calculated by two-tailed Student’s unpaired t -test ( A , C–G , I–K ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: not significant. Data are shown as the mean ± SD.

Article Snippet: An Annexin V-FITC Apoptosis Detection Kit (Vazyme, A213-01) was used to measure the rate of both early and late-stage apoptotic cells.

Techniques: Transmission Assay, Electron Microscopy, Control, Knockdown, Silver Staining, Titration, Flow Cytometry, Transfection, Over Expression, Plasmid Preparation, Staining, Functional Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Splice-switching of the oncogenic BCS1L isoform suppresses ovarian cancer progression by disrupting mitochondrial function

doi: 10.1038/s41419-026-08495-6

Figure Lengend Snippet: A The schematic diagram for designing ASOs targeting the alternative splicing of BCS1L . B and C The inclusion of BCS1L exon 2 in response to three ASOs was analyzed by RT-PCR in A2780 cells (200 nM ASOs) and HEY cells (100 nM ASOs) for 48 h. D and E Isoform-specific primers were used in qPCR to determine the ratio of BCS1L-L to BCS1L-S in A2780 and HEY cells after treatment with the three ASOs. F and G The IC 50 of ASO3 after treatment for 120 h was calculated in A2780 cells (30.78 nM ASO3) and HEY cells (22 nM ASO3). H and I The cell proliferation assay was performed using an Incucyte S3 live-cell imaging and analysis system in A2780 and HEY cells treated with 200 nM ASO3 and 80 nM ASO3, respectively, compared to corresponding controls ( n = 3) for five days. Cell proliferation was compared to the initial state (time = 1 day). The shaded areas indicate the mean ± SEM. The p -value was calculated by two-way ANOVA. J Apoptotic cells stained with Annexin V-PE/7-AAD were detected by flow cytometry after ASO3 treatment (400 nM ASO3 for A2780 cells and 200 nM ASO3 for HEY cells) for 48 h. The percentage of apoptotic cells, including both early and late apoptotic cells. Three biological replicates were conducted in all functional experiments. K ASO3 and ASO-control in PBS were injected intratumorally into subcutaneous tumor xenografts using A2780 cells ( n = 5 NOD-SCID mice per group). The arrow indicates the injection time. The tumor weight ( L ) and volume ( M ) were measured for each group. The p-values were determined by two-tailed unpaired Student’s t -test ( D , E , J , L , M ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: not significant. Data are shown as the mean ± SD.

Article Snippet: An Annexin V-FITC Apoptosis Detection Kit (Vazyme, A213-01) was used to measure the rate of both early and late-stage apoptotic cells.

Techniques: Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Proliferation Assay, Live Cell Imaging, Staining, Flow Cytometry, Functional Assay, Control, Injection, Two Tailed Test

Journal: Cancers

Article Title: CLEFMA Induces the Apoptosis of Oral Squamous Carcinoma Cells through the Regulation of the P38/HO-1 Signalling Pathway

doi: 10.3390/cancers14225519

Figure Lengend Snippet: CLEFMA induced apoptosis in OSCC. ( A ) Cells were treated with various CLEFMA concentrations and then subjected to apoptosis assay through flow cytometry with annexin V/PI staining for 24 h. The quantification results of apoptosis assay of ( B ) HSC-3 and ( C ) SCC-9 cells are shown in the bar graph. Data represent the average ± standard deviation (SD) from three independent experiments. * p < 0.05 compared with the DMSO control group.

Article Snippet: Apoptotic cell death was measured with BD Annexin V-FITC Apoptosis Detection Kit (BD Bioscience, Becton Dickinson Co., Franklin Lakes, NJ, USA).

Techniques: Apoptosis Assay, Flow Cytometry, Staining, Standard Deviation

Journal: Cancers

Article Title: CLEFMA Induces the Apoptosis of Oral Squamous Carcinoma Cells through the Regulation of the P38/HO-1 Signalling Pathway

doi: 10.3390/cancers14225519

Figure Lengend Snippet: Elevated effects of CLEFMA on caspase-3 activation and HO-1 expression in human OSCC cells. ( A ) HSC-3 cells were incubated for 24 h with 8 μM CLEFMA and compared with untreated lysate (DMSO control group), and the cell lysates were used to detect 35 different apoptosis-related proteases using the human proteome profiler apoptosis array. ( B ) The quantification of cleaved caspase-3, HO-1, and cIAP-1 from human apoptosis array. ( C ) Live cell imaging apoptosis dye for caspase-3 activity used to detect apoptotic death using fluorescence microscopy with the corresponding filter (green channel). Western blot was performed on HO-1 and cIAP-1 using β-actin as the internal control in ( D ) HSC-3 and (E) SCC-9 cells. Quantitative data were acquired from three independent experiments and expressed as mean ± SD. * represent p < 0.05 as compared with the DMSO vehicle.

Article Snippet: Apoptotic cell death was measured with BD Annexin V-FITC Apoptosis Detection Kit (BD Bioscience, Becton Dickinson Co., Franklin Lakes, NJ, USA).

Techniques: Activation Assay, Expressing, Incubation, Live Cell Imaging, Activity Assay, Fluorescence, Microscopy, Western Blot

Journal: Cancers

Article Title: CLEFMA Induces the Apoptosis of Oral Squamous Carcinoma Cells through the Regulation of the P38/HO-1 Signalling Pathway

doi: 10.3390/cancers14225519

Figure Lengend Snippet: CLEFMA induced the apoptosis of human OSCC via the regulation of p38/ HO-1 signalling cascade. ( A , B ) HSC-3 and ( C , D ) SCC-9 cells were pre-treated with UO126, JNK in 8, or SB203580 for 2 h before CLEFMA (8 μM) treatment for 24 h and untreated cells. Western blot assay was conducted. The quantification data are shown in the bar graph using densitometric measurement (ratio to β-actin). * p < 0.05 compared with the control group; # p < 0.05 compared with the 8 μM CLEFMA treated group.

Article Snippet: Apoptotic cell death was measured with BD Annexin V-FITC Apoptosis Detection Kit (BD Bioscience, Becton Dickinson Co., Franklin Lakes, NJ, USA).

Techniques: Western Blot