Journal: Nucleic acids research

Article Title: Structural basis for the selective methylation of 5-carboxymethoxyuridine in tRNA modification.

doi: 10.1093/nar/gkad668

Figure Lengend Snippet: Figure 2. Crystal structure of E. coli CmoM-tRNA Ser1 -sinefungin complex. ( A ) Sequence of cellularly transcribed tRNA Ser1 (cmo 5 UGA) from cmoM E. coli including known modifications, which was used for crystallography and biochemical assays in this study. ( B ) Overall structure of E. coli CmoM complexed with tRNA Ser1 (cmo 5 UGA) and sinefungin. CmoM and tRNA Ser1 (cmo 5 UGA) are shown in marine and orange ribbons, respecti v ely. Sinefungin is r epr esented in spher e, wher e carbon is sho wn in grey, o xygen in red, and nitrogen in blue. ( C ) Docking site of tRNA (orange) on CmoM is shown, where electrostatic potential is mapped on the surface of the enzyme (positi v e in b lue, negati v e in red, and neutral in white). ( D ) Superposition of reconstituted dimer of CmoM-tRNA complex (marine and orange for CmoM and tRNA, respecti v ely) and tRNA-free CmoM (PDB ID: 4htf, yellow) structures.

Article Snippet: 100 g of the wild-type CmoM (calculated monomer olecular weight = 31.7 kDa) was loaded onto the column ith mobile phase (25 mM HEPES (pH 7.5) and 150 mM aCl) at a flow rate of 0.5 ml / min. Elution of ovalbumin 44 kDa) and MnmC (76 kDa) from the column was also ested under the identical condition for comparison. urification of in vivo transcribed tRNA he plasmid pBSTNAV (Addgene, USA) was used as the RNA e xpression v ector which contains LPP promoter and rnC terminator.

Techniques: Sequencing

Journal: Nucleic acids research

Article Title: Structural basis for the selective methylation of 5-carboxymethoxyuridine in tRNA modification.

doi: 10.1093/nar/gkad668

Figure Lengend Snippet: Figure 3. Intermolecular interactions between CmoM and tRNA. ( A ) Superposed structures of tRNA-complexed (marine) and tRNA-free (PDB ID: 4htf , yello w) CmoM, where the inset highlights a large shift of 1 helix upon binding of tRNA. ( B ) Acti v e site residues of CmoM (blue) that interact with tRNA anticodon loop region (orange). Sinefungin is shown in grey sticks. ( C ) A schematic diagram tRNA showing polar interactions with the enzyme at each r esidue, wher e the phosphate is r epr esented by cir cle, ribose by pentagon, and base by square. Amino acids associated with the phosphate backbone (blue), sugar (orange), and base (black) are grouped in brackets for individual nucleotides. Amino acid residues interacting with nucleobase of ( D ) U33, ( E ) cmo 5 U34 and ( F ) G35 are shown, where dashed lines represent hydrogen bonding interaction. Distance between the amine of sinefungin and the carboxyl group of cmo 5 U34 is labeled in ˚A .

Article Snippet: 100 g of the wild-type CmoM (calculated monomer olecular weight = 31.7 kDa) was loaded onto the column ith mobile phase (25 mM HEPES (pH 7.5) and 150 mM aCl) at a flow rate of 0.5 ml / min. Elution of ovalbumin 44 kDa) and MnmC (76 kDa) from the column was also ested under the identical condition for comparison. urification of in vivo transcribed tRNA he plasmid pBSTNAV (Addgene, USA) was used as the RNA e xpression v ector which contains LPP promoter and rnC terminator.

Techniques: Binding Assay, Labeling

Journal: Nucleic acids research

Article Title: Structural basis for the selective methylation of 5-carboxymethoxyuridine in tRNA modification.

doi: 10.1093/nar/gkad668

Figure Lengend Snippet: Figure 4. Mutagenesis studies of amino acid residues participating in ionic / polar interactions with tRNA. ( A ) The conversion rates of cmo 5 U to mcmo 5 U by the wild-type and mutant CmoM are plotted, which have been determined through in vitro assays using SAM and tRNA Ser1 extracted from E. coli ΔcmoM cells. Error bars r epr esent the standar d de viation from thr ee independent measur ements at each time point. ( B ) Roles of amino acid residues selected for mutagenesis and relati v e acti vities of mutants are summarized, where ++ stands for the wild-type comparable, + for moderate and – for minimal activity.

Article Snippet: 100 g of the wild-type CmoM (calculated monomer olecular weight = 31.7 kDa) was loaded onto the column ith mobile phase (25 mM HEPES (pH 7.5) and 150 mM aCl) at a flow rate of 0.5 ml / min. Elution of ovalbumin 44 kDa) and MnmC (76 kDa) from the column was also ested under the identical condition for comparison. urification of in vivo transcribed tRNA he plasmid pBSTNAV (Addgene, USA) was used as the RNA e xpression v ector which contains LPP promoter and rnC terminator.

Techniques: Mutagenesis, In Vitro, Activity Assay

Journal: Nucleic acids research

Article Title: Structural basis for the selective methylation of 5-carboxymethoxyuridine in tRNA modification.

doi: 10.1093/nar/gkad668

Figure Lengend Snippet: Figure 5. Features of cellularly transcribed tRNA Ser1 . ( A ) Structure of the CmoM-bound tRNA with individual regions shown in different colors; acceptor stem in purple, D-arm in salmon, anticodon arm in red (anticodon in dar k grey), variab le arm in b lue, and T-arm in yellow. ( B ) Superposition of anticodon stem loops of tRNA Ser1 (orange) and S. cerevisiae tRNA Phe (PDB ID: 1EHZ, grey), which highlights the conformational change in tRNA Ser1 , especially the flipping-out cmo 5 U34. ( C ) A close-up view of the long variable arm of tRNA Ser1 . Base pairing interactions are represented in black dashed lines.

Article Snippet: 100 g of the wild-type CmoM (calculated monomer olecular weight = 31.7 kDa) was loaded onto the column ith mobile phase (25 mM HEPES (pH 7.5) and 150 mM aCl) at a flow rate of 0.5 ml / min. Elution of ovalbumin 44 kDa) and MnmC (76 kDa) from the column was also ested under the identical condition for comparison. urification of in vivo transcribed tRNA he plasmid pBSTNAV (Addgene, USA) was used as the RNA e xpression v ector which contains LPP promoter and rnC terminator.

Techniques:

Journal: Nucleic acids research

Article Title: Structural basis for the selective methylation of 5-carboxymethoxyuridine in tRNA modification.

doi: 10.1093/nar/gkad668

Figure Lengend Snippet: Figure 6. A proposed reaction mechanism for the SAM-dependent cmo 5 U methylation by CmoM. Kinetically essential residues such as R26 and W124 are positioned to fine-tune the orientation of the nucleophile and pre v ent the binding of the protonated form to promote the nucleophilicity. Subsequently, the carboxylate of cmo 5 U34 attacks the S -methyl group of SAM to form mcmo 5 U34 and SAH.

Article Snippet: 100 g of the wild-type CmoM (calculated monomer olecular weight = 31.7 kDa) was loaded onto the column ith mobile phase (25 mM HEPES (pH 7.5) and 150 mM aCl) at a flow rate of 0.5 ml / min. Elution of ovalbumin 44 kDa) and MnmC (76 kDa) from the column was also ested under the identical condition for comparison. urification of in vivo transcribed tRNA he plasmid pBSTNAV (Addgene, USA) was used as the RNA e xpression v ector which contains LPP promoter and rnC terminator.

Techniques: Methylation, Binding Assay

Journal: bioRxiv

Article Title: TRPML1 signaling at lysosomes-mitochondria nexus drives triple-negative breast cancer mitophagy, metabolic reprogramming and chemoresistance

doi: 10.1101/2025.09.11.675705

Figure Lengend Snippet: (A) schematic presentation highlighting the question whether lysosomal TRPML1 activation can directly influence mitochondrial Ca 2+ uptake or via an intermediary pathway. (B) Traces representing mitochondrial Ca 2+ uptake in MDA-MB-231 PLKOs, ML-1KD1/ML1-KD3 and MCF-10A in response to ML1-SA1 stimulation in the absence of extracellular Ca 2+ . Traces represent the mean ± SEM. (C) Traces representing mitochondrial Ca 2+ uptake in MDA-MB-231 PLKOs and PLKOs transfected with HA-TRPML1 in response to ML1-SA1 in the absence of extracellular Ca 2+ . Traces represent the mean ± SEM (D 1-4 ) Traces representing mitochondrial Ca 2+ uptake in response to increasing doses of ML1-SA1 in MDA-MB-231 PLKOs and ML1-KD1/ML1-KD3s. (D 5 ) Quantification of maximum mitochondrial Ca 2+ uptake in (D 4 ), error bars represent the mean ± SD, results analyzed by Kruskal-Wallis test with Dunn’s multiple comparison correction. (E) Traces representing cytosolic Ca 2+ uptake in MDA-MB-231 PLKOs vs ML1-KD1/ML1-KD3 in response to ML1-SA1 stimulation in the absence of extracellular Ca 2+ . Traces represent mean ± SEM. (F) Traces representing cytosolic Ca 2+ uptake in MDA-MB-231 PLKOs vs PLKOs transfected with HA-TRPML-1 in response to ML1-SA1 in the absence of extracellular Ca 2+ . Traces represent mean ± SEM. (G) Quantification of (F), results analyzed by Mann Whitney test. (H) Traces representing cytosolic Ca 2+ uptake in MDA-MB-231 PLKOs vs ML1-KD1/ML1-KD3 cells in response to ML1-SA1 stimulation in the presence of extracellular Ca 2+ . Traces represent the mean ± SEM. (I) Quantification of (H), error bars represent the mean ± SD, results analyzed by Kruskal Wallis test with post-hoc Dunn’s multiple comparisons test. (J) Traces to represent mitochondrial Ca 2+ uptake in MDA-MB-231 PLKO vs ML1-KD1/ML1-KD3 cells in response to stimulation with 200 µM ATP. (K) Corresponding quantification of (J), error bars are representative of mean ± SD, results analyzed by Kruskal Wallis test with post-hoc Dunn’s multiple comparisons test. (L) Representative western blot images measuring MCU expression in MDA-MB-231 PLKO, ML-1KD1/ML-1-KD3 and MCF-10A. (M) Quantification of (L) with MCU protein expression normalized to GAPDH loading control, graph represents means ± SD. Results analyzed by one-way ANOVA with post-hoc Tukey’s multiple comparisons test. (N) Quantification of TMRE measurements of MDA-MB-231 PLKO, ML1-KD1/ML1-KD3, and MCF-10A. Results analyzed by one-way ANOVA with post-hoc Tukey’s multiple comparison test. All mitochondrial Ca 2+ measurements were obtained with Rhod-2 AM and normalized to MitoTracker TM Green. All cytosolic Ca 2+ measurements were obtained with Fura-2 AM. All presented experiments represent three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P <0.0001, ns, not significant.

Article Snippet: Membranes were incubated overnight at 4C with the following primary antibodies (all from Cell Signaling Technology, 1:1000 dilution unless otherwise noted): Cell cycle regulators: anti-Cyclin B1 (#4138, anti-Cyclin D1(#2922), anti-p27 (#3686), anti-E2F1 (#3742); Apoptosis markers: anti-Cleaved-Caspase 9(#7237), anti-Caspase 9 (#9502), anti-Cleaved-Caspase7 (#9491), anti-Caspase7 (#9492), anti-Cleaved-Caspase3 (9661), anti-Caspase3 (#9662), anti-p-Bcl-2(S70) (#2827), anti-EndoG(#4969), anti-AIF(#4642), Mitochondrial Dynamics: anti-MFN1 (#14739), anti-MFN2 (#11925), anti-OPA1 (#80471), anti-p-DRP1 (S616) (#3455), anti-p-DRP1 (S637) (#4867), anti-MFF (#86668), Mitochondrial bioenergetics: OXPHOS (#ab110411, Abcam), MCU (#14997), Autophagy regulator: anti-p-P70S6K (Thr389) (#9205, Cell Signaling), anti-P70S6K (#9202, Cell Signaling), anti-p-ULK1 (S757) (#6888, Cell Signaling), anti-p-ULK1 (S555) (#5869, Cell Signaling), anti-ULK1 (#8054, Cell Signaling), anti-LC3 A/B (#12741, Cell Signaling), anti-p-AMPK (Thr 172) (#2531, Cell Signaling), anti-AMPK (#2532, Cell Signaling), Mitophagy Regulators, PINK1 (#6946, Cell Signaling), Parkin (#4211, Cell Signaling), Stress Response: anti-Nrf2 (#12721, Cell Signaling), anti-TFAM (#8076, Cell Signaling) anti-PGC1-alpha (#2178, Cell Signaling).

Techniques: Activation Assay, Transfection, Comparison, MANN-WHITNEY, Western Blot, Expressing, Control

Journal: Biomedicines

Article Title: A Novel Biomarker Driving Poor-Prognosis Liver Cancer: Overexpression of the Mitochondrial Calcium Gatekeepers.

doi: 10.3390/biomedicines8110451

Figure Lengend Snippet: Figure 1. CREB, MCU, MICU1, and MICU2 expression in HCC and its effect on prognosis. (A) Representative images of CREB, MCU, MICU1, and MICU2 immunohistochemistry (IHC) staining in HCC from the Human Protein Atlas (HPA) (B) Plots chart showing higher CREB, MCU, MICU1, and MICU2 expression in HCC patients. Data were obtained from TCGA. (C) Kaplan–Meier curves of overall survival in HCC patients. Survival data were obtained from TCGA. CREB expression was positively correlated with the expression of MCU, MICU1, and MICU2 in TCGA dataset (D). Scale bars 200 µm.

Article Snippet: Four antibodies used to target CREB, MCU, MICU1, and MICU2 were purchased from Biorbyt (Cambridge, UK).

Techniques: Expressing, Immunohistochemistry

Journal: Biomedicines

Article Title: A Novel Biomarker Driving Poor-Prognosis Liver Cancer: Overexpression of the Mitochondrial Calcium Gatekeepers.

doi: 10.3390/biomedicines8110451

Figure Lengend Snippet: Figure 2. CREB was highly expressed and closely correlated with MCU, MICU1, and MICU2 in HCC. (A) Scheme of the human genomic region encompassing the MCU, MICU1, and MICU2 promoter. The pink peaks represent the CREB-binding regions, according to ENCODE. The red square shows the

Article Snippet: Four antibodies used to target CREB, MCU, MICU1, and MICU2 were purchased from Biorbyt (Cambridge, UK).

Techniques: Binding Assay

Journal: Biomedicines

Article Title: A Novel Biomarker Driving Poor-Prognosis Liver Cancer: Overexpression of the Mitochondrial Calcium Gatekeepers.

doi: 10.3390/biomedicines8110451

Figure Lengend Snippet: Figure 3. Immunoreactivity of CREB, MCU, MICU1, and MICU2 in hepatocellular carcinoma (HCC). (A) The representative photomicrographs of CREB, MCU, MICU1, and MICU2 expression for adjacent non-neoplastic, weak (+), and strong (+++) staining in HCC tissues. (B) The IHC scores of CREB, MCU, MICU1 and MICU2 expression in hepatocellular carcinoma tissue and matched adjacent normal liver tissue. *** p < 0.001. Scale bars 200 µm.

Article Snippet: Four antibodies used to target CREB, MCU, MICU1, and MICU2 were purchased from Biorbyt (Cambridge, UK).

Techniques: Expressing, Staining

Journal: Biomedicines

Article Title: A Novel Biomarker Driving Poor-Prognosis Liver Cancer: Overexpression of the Mitochondrial Calcium Gatekeepers.

doi: 10.3390/biomedicines8110451

Figure Lengend Snippet: Figure 4. Kaplan–Meier analysis of clinicopathological parameters, including clinical stage, differential histologic grade, hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, expression of CREB, MCU, MICU1, and MICU2 gene.

Article Snippet: Four antibodies used to target CREB, MCU, MICU1, and MICU2 were purchased from Biorbyt (Cambridge, UK).

Techniques: Virus, Infection, Expressing

Journal: The Journal of Physiology

Article Title: Predominant cause of faster force recovery in females than males after intense eccentric contractions in mouse fast‐twitch muscle

doi: 10.1113/jp281927

Figure Lengend Snippet: Figure 5. Changes in mitochondrial protein contents in males and females at 3 days after eccentric contractions (ECCs) in the superficial region of the gastrocnemius (GAS) muscle The ECC protocol and sample preparation are described in the legends of Figs 1 and 2. A, means (+ SD) of mitochondrial Ca2+ uniporter content (MCU). B, means (+ SD) of cytochrome oxidase IV (COXIV) content. C, means (+ SD) of mitofusin 2 (Mfn2) content. The number of samples are seven and six in males and females, respectively. Individual data from the same mice are connected by grey lines. All data sets showed a normal distribution, as examined by the Shapiro–Wilk normality test. aP < 0.05 versus the female rested group (two-way ANOVA for repeated measure). R: rested muscles; E: eccentrically contracted muscles.

Article Snippet: The primary antibodies used were as follows: antibodies to: RyR1 (mouse IgG;MA3-925, Thermo Fisher Scientific; dilution 1:5000), SR Ca2+-ATPase (mouse IgG; MA3-911, Thermo Fisher Scientific; dilution 1:5000), JP1 (rabbit IgG; 40-5100; Thermo Fisher Scientific; dilution 1:5000), CASQ1 (rabbit IgG; ABN854, Millipore, Massachusetts, MA,USA; dilution 1:5000), COXIV (mouse IgG; ab14744, Abcam, Massachusetts, MA, USA; dilution 1:5000), Mfn2 (rabbit IgG; ab50843, Abcam; dilution 1:2000) and MCU (rabbit IgG; Atlas antibodies, HPA016480, Bromma, Sweden; dilution 1:2000).

Techniques: Sample Prep, Muscles

Journal: Journal of advanced research

Article Title: Sodium citrate targeting Ca 2+ /CAMKK2 pathway exhibits anti-tumor activity through inducing apoptosis and ferroptosis in ovarian cancer.

doi: 10.1016/j.jare.2024.04.033

Figure Lengend Snippet: Fig. 5. Ca2+ chelation by sodium citrate leads to mitochondrial dysfunction. A. Representative traces of mitochondrial Ca2+ dynamics (indicated by fluorescence of 4mt- GCaMP6) following histamine (200 lM) treatment in SKOV3 cells. B. Immunoblot analysis of TCA cycle enzymes was performed after treatment with sodium citrate plus CaCl2 in SKOV3 and A2780 cells. Expression of proteins was normalized to b-actin. Quantitative data were shown as mean ± SEM. C, D. Complex Ⅲand complex V activity in mitochondrial lysates were detected. E. Representative fluorescence images of Mito-LX staining represented H2O2 levels. Scale bar, 10 lm. Quantitative data were shown as mean ± SEM. F, G. Oligomycin, FCCP, antimycin and rotenone were sequentially added to measure OCR in the XFp analyzer from Seahorse Bioscience. H. Immunoblot analysis of CAMKK2 and AMPK activation was performed following different treatments in SKOV3 and A2780 cells. Expression of proteins was normalized to b-actin. Quantitative data were shown as mean ± SEM. I, J. Cell lysates were immunoprecipitated using an anti-MCU antibody and then subjected to immunoblot analysis using an anti-phospho-serine/ threonine antibody and anti-AMPK antibody. K, L. The cell viability was examined by MTT assay after treatment with 15 mM sodium citrate plus 8 lM Ionomycin (Iono) or 150 lM STO-609 for 24 h in SKOV3 and A2780 cells. M, N. MDA and LPO content in SKOV3 and A2780 cells were analyzed following Ionomycin treatment. O. Immunoblot analysis of GPX4 and FTH1 level was performed following Ionomycin treatment in SKOV3 and A2780 cells. Expression of proteins was normalized to b-actin. Quantitative data were shown as mean ± SEM. N = 3 biologically independent replicates. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. The t-test and one-way ANOVA statistical analysis were used.

Article Snippet: Subsequently, 1 lg of MCU antibody (Proteintech, Cat.26312-1-AP) or NCOA4 antibody (Cell Signaling, Cat.66849) and 40 lL of protein A/G-beads (Beyotime, Cat.P2195M) were added to the lysis solution, and the mixture was incubated overnight at 4 C with gentle agitation.

Techniques: Western Blot, Expressing, Activity Assay, Staining, Activation Assay, Immunoprecipitation, MTT Assay

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 2. Intracellular calcium levels are increased by NIST DEP and RSL-3 co-treatment. (A) Fluo4-AM fluorescence images at 20x magnification after 6 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Green: Fluo4-AM positive cells, Blue: nucleus (scale bar =100 μm). (B) Quantitative analysis of Fluo4-AM positive cells was performed based on 10 fluorescent images per condition (mean ± SD). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 3. ER-mitochondria contact sites are increased by NIST DEP and RSL-3 co-treatment. (A) PLA fluorescence images at 40x magnification after 6 hrs exposure to NIST DEP and RSL-3 with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Red: PLA dots, Blue: nucleus (scale bar = 10 μm). (B) Quantification of PLA dots from 50 single cells based on fluorescence images for each condition. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 4. Mitochondrial calcium levels are increased by NIST DEP and RSL-3 co-treatment. (A) Mt-fura-2 fluorescence images at 20x magnification after 6 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Green: Fura 340, Blue: Fura 380 (scale bar = 20 μm). The ratio of fluorescence measured upon excitation at 340/380 nm (F340/F380) is shown in the right panel, ratio range from 0 to 2. (B) Fura340/Fura380 ratio picture of different treatments. Enlarged images of the boxed area were shown in the right panels. (C) Quantitative analysis of F340/F380 was performed based on 10 fluorescent images per condition (mean ± SD). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ***p < 0.001, ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 5. HT22 ferroptotic cell death induced by RSL-3 and NIST DEP is alleviated by inhibition of mitochondrial calcium uptake. (A) PI/Hoechst 33342 double-stained fluorescence images at 40x magnification after 17 hrs exposure to NIST DEP, RSL-3, calcium regulators MCUi4 (5 μM) and CyPPA (10 μM) alone or in combination. Red: dead cells, Blue: nucleus (scale bar =100 μm). (B) Quantitative analysis of PI-positive cells was performed based on 10 fluorescent images per condition (mean ± SD). The percentage of dead cells in the control group was defined as 100 %. (C) MTT assay for the viability of HT22 cells after 17 hrs exposure to NIST DEP, RSL-3, calcium regulators MCUi4 (5 μM) and CyPPA (10 μM) alone or in combination. The cell viability of the control group was defined as 100 %. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Inhibition, Staining, Fluorescence, Control, MTT Assay, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 6. Co-exposure to RSL-3 and NIST DEP induce mitochondrial morphological alteration. (A) Mitotracker Red fluorescence images at 63x magnification after 17 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Red: mito chondrial, Blue: nucleus (scale bar = 20 μm). Enlarged images of the boxed area are shown in the right panels. (B) Mitochondrial skeleton images at 63x magni fication after 17 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). (C-D) Parameters of HT22 mitochondrial network morphology analysis after different treatments: (C) Mitochondrial footprint. 15 single cells were analyzed for each condition. (D) Mean branch length. 10 whole fluorescence images were analyzed for each condition. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 7. Lipid peroxidation induced by RSL-3 and NIST DEP can be alleviated by MCUi4 and CyPPA. (A) BODIPY 581/591 C11fluorescence images at 20x magnification after 6 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Red: C11non-ox, Green: C11ox, Blue: nucleus (scale bar = 100 μm). (B) Quantitative analysis of BODIPY 581/591 C11 mean fluorescence intensity was performed based on 10 fluorescent images per condition (mean ± SD). (C) MDA levels after 6 hrs of different treatments. All conditions were normalized by their corresponding protein concentrations, and the MDA concentration of the control group was defined as 1. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ***p < 0.001, ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Concentration Assay, Control, Comparison