Journal: Cells

Article Title: Mitochondrial Dynamics Participate in an Early Metabolic Adaptation of Glioblastoma Multiforme T98G Cells to Doxorubicin-Induced Chemotherapeutic Stress

doi: 10.3390/cells15100899

Figure Lengend Snippet: Metabolic mobilization of DOX-induced PGCs. ( a ) Morphology (left) and size (right) of pulse DOX-treated (1 µM; 48 h) T98G cells 14 days after DOX removal (averaged cell volume and surface projection area expressed as a % of control. Note the hypertrophy of DOX-treated T98G cells. Statistical significance was assessed by non-parametric Mann–Whitney test; * p < 0.05 versus control. Bars represent SD. Data are representative of ≥50 single cells in three independent biological replicates. Scale bars—100 µm. ( b ) Reverse correlation between nuclear DOX localization and IC 50 values estimated for U87 and T98G cells. PCC values represent the degree of DOX- and DNA-specific fluorescence colocalization (Hoechst 33258; representative for >50 cells in 3 independent replicates). Scale bars—20 µm. ( c ) Heatmaps of metabolic proteins significantly upregulated in DOX_R 14d cells as determined by LC–MS/MS proteomics and classified according to Gene Ontology (GO) and KEGG pathway annotations. Heatmap colors reflect fold-increase versus control (red = highest; green = lowest). ( d ) Interactome of ATP-dependent proteins significantly increased in DOX_R 14d cells (Fold change ≥ 1.2). Interaction networks were generated using the STRING database; high-confidence interaction score threshold (0.700). The table below the network assigns individual proteins to biological processes according to GO. Statistical significance was assessed by Student’s t -test with Benjamini–Hochberg FDR set to 0.05 ( p < 0.05 vs. control). Data are representative of three independent biological replicates. ( e ) Extra-mitochondrial localization of DOX in T98G cells on the 14th day after DOX removal estimated by confocal microscopy. An exemplary PCC value illustrates a low degree of DOX- and MitoTracker Green-specific fluorescence co-localization. Data are representative of ≥50 single cells in three independent biological replicates. Scale bar—50 µm. Note the concomitant extra-nuclear and extra-mitochondrial DOX sequestration, mobilization of mitochondrial catabolism, and mobilization of cell anabolic machinery.

Article Snippet: Human glioblastoma multiforme (GBM) T98G and U87-MG (ATCC, CRL-1690 and HTB-14, respectively; not listed as commonly misidentified cell lines by the International Cell Line Authentication Committee) cells were maintained under standard culture conditions in high-glucose (4500 mg/L) DMEM medium (Sigma-Aldrich, St. Louis, MO, USA; No. D6429; up to 25 passages), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA; No. A3840402) and 1% Antibiotic-Antimycotic Solution (Merck, No. A5955), as described previously [ , ].

Techniques: Control, MANN-WHITNEY, Fluorescence, Liquid Chromatography with Mass Spectroscopy, Generated, Confocal Microscopy

Journal: Cells

Article Title: Mitochondrial Dynamics Participate in an Early Metabolic Adaptation of Glioblastoma Multiforme T98G Cells to Doxorubicin-Induced Chemotherapeutic Stress

doi: 10.3390/cells15100899

Figure Lengend Snippet: Mitochondrial dynamics in pulse DOX-treated T98G cells. ( a , e ) Heatmaps of mitochondrial matrix/inner membrane ( a ), oxidative phosphorylation and mitochondrial stress proteins ( e ) significantly upregulated in DOX_R 14d cells and determined by LC–MS/MS proteomics and classified according to Gene Ontology (GO) and KEGG pathway annotations. Heatmap colors reflect fold-increase versus control (red = highest; green = lowest). Statistical significance was assessed by ANOVA with permutation FDR < 0.05 and Tukey post hoc ( p < 0.05 vs. control). Data are representative of three independent biological replicates. ( b ) Structural variants of mitochondria (HNs, LTs and mitochondria-on-a-string; MOAS) visualized in control and DOX_R 14d cells by CellROX DeepRed staining (upper panel) and the quantification of mitochondrial length and aspect ratio estimated with custom Python pipeline based on the Frangi tubeness filter (lower panel). Scale bars—20 and 25 µm ( c ) Ultrastructure of mitochondria in DOX_R 14d c ells visualized via transmission electron microscopy (TEM). Scale bars—500 nm ( d ) NADH and ATP levels in DOX_R 14d cells quantified with ATP content assay. ( f ) Mosaic distribution of mitochondrial membrane potential along tubular networks visualized with JC-1 staining and a custom Python pipeline. Pix PCC values indicate co-localization of green/red fluorescence within individual pixels. Scale bars—5 µm. Statistical significance of the differences in ( b , d ) was assessed by non-parametric Mann–Whitney test; * p < 0.05 vs. control. Bars represent SD. Data are representative of three independent biological replicates and ≥50 single cells. Note the signs of mitochondrial adaptation in PGCs under DOX-induced stress.

Article Snippet: Human glioblastoma multiforme (GBM) T98G and U87-MG (ATCC, CRL-1690 and HTB-14, respectively; not listed as commonly misidentified cell lines by the International Cell Line Authentication Committee) cells were maintained under standard culture conditions in high-glucose (4500 mg/L) DMEM medium (Sigma-Aldrich, St. Louis, MO, USA; No. D6429; up to 25 passages), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA; No. A3840402) and 1% Antibiotic-Antimycotic Solution (Merck, No. A5955), as described previously [ , ].

Techniques: Membrane, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Control, Staining, Transmission Assay, Electron Microscopy, Fluorescence, MANN-WHITNEY

Journal: Cells

Article Title: Mitochondrial Dynamics Participate in an Early Metabolic Adaptation of Glioblastoma Multiforme T98G Cells to Doxorubicin-Induced Chemotherapeutic Stress

doi: 10.3390/cells15100899

Figure Lengend Snippet: Mitofusin 2 up-regulation in pulse DOX-treated T98G cells. ( a , b , d ) MFN1 ( a ), MFN2 ( b ) and MFF ( d ) levels in pulse DOX-treated T98G cells (1 µM; 48 h) estimated with quantitative fluorimetry at the 14th day after DOX removal. Data are representative of 3 independent biological replicates. Bars represent SD. Statistical significance of the differences was assessed by non-parametric Mann–Whitney U test; * p < 0.05 vs. control ( a , b , d ). Scale bars—50 µm. ( c ) MFN2 and MFF levels visualized in pulse DOX-treated T98G cells with (1 µM; 48 h) estimated with immunoblotting. Numerical values represent results of densitometric analyses, normalized against housekeeping protein levels (α-tubulin) and compared to control. ( e ) Interactome of ATP/GTP conversion proteins significantly up-regulated in DOX_R 14d cells as estimated by LC–MS/MS proteomics (Fold change ≥ 1.2). Interaction networks were generated using the STRING database (high-confidence interaction score threshold—0.700). The table below assigns individual proteins to biological processes according to GO. Statistical significance was assessed by Student’s t -test with Benjamini–Hochberg FDR set to 0.05 ( p < 0.05 vs. control) from experimental triplicates (n = 3). Note the adaptive up-regulation of MFN2 in PGCs.

Article Snippet: Human glioblastoma multiforme (GBM) T98G and U87-MG (ATCC, CRL-1690 and HTB-14, respectively; not listed as commonly misidentified cell lines by the International Cell Line Authentication Committee) cells were maintained under standard culture conditions in high-glucose (4500 mg/L) DMEM medium (Sigma-Aldrich, St. Louis, MO, USA; No. D6429; up to 25 passages), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA; No. A3840402) and 1% Antibiotic-Antimycotic Solution (Merck, No. A5955), as described previously [ , ].

Techniques: MANN-WHITNEY, Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Cells

Article Title: Mitochondrial Dynamics Participate in an Early Metabolic Adaptation of Glioblastoma Multiforme T98G Cells to Doxorubicin-Induced Chemotherapeutic Stress

doi: 10.3390/cells15100899

Figure Lengend Snippet: The effect of MFN1/2 down-regulation on mitochondrial homeostasis in T98G cells. ( a ) Experimental approach to the estimation of the consequences of MFN1/2 silencing in T98G cells. Cells were treated with the relevant esiRNAs for 16 h and cultivated for 48 h before endpoint analyses. ( b ) MFN1 or MFN2 levels following the treatment of T98G cells with esiRNA estimated by quantitative fluorimetry. Scale bars—100 µm. ( c , d ) Effect of MFN1 or MFN2 down-regulation in T98G cells on the morphology of their mitochondria ( c ) ( left ), the fraction of viable T98G cells (Trypan blue assay); ( c ) ( right ), and metabolic profile ( d ). Statistical significance was assessed by non-parametric Mann–Whitney test; * p < 0.05 vs. control. Data are representative of 3 independent biological replicates and/or ≥50 single cells. ( e ) Mitochondrial protein levels in T98G cells undergone MFN2 down-regulation quantified by LC–MS/MS; proteins with fold change >1.2 were classified into functional clusters in the STRING database according to Gene Ontology. Statistical significance was assessed by Student’s t -test with Benjamini–Hochberg FDR set to 0.05 ( p < 0.05 vs. control). Data are representative of 3 independent biological replicates. Note a slight deterioration of T98G welfare accompanied by respiratory mobilization of T98G cells following MFN2 down-regulation.

Article Snippet: Human glioblastoma multiforme (GBM) T98G and U87-MG (ATCC, CRL-1690 and HTB-14, respectively; not listed as commonly misidentified cell lines by the International Cell Line Authentication Committee) cells were maintained under standard culture conditions in high-glucose (4500 mg/L) DMEM medium (Sigma-Aldrich, St. Louis, MO, USA; No. D6429; up to 25 passages), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA; No. A3840402) and 1% Antibiotic-Antimycotic Solution (Merck, No. A5955), as described previously [ , ].

Techniques: esiRNA, MANN-WHITNEY, Control, Liquid Chromatography with Mass Spectroscopy, Functional Assay

Journal: Cells

Article Title: Mitochondrial Dynamics Participate in an Early Metabolic Adaptation of Glioblastoma Multiforme T98G Cells to Doxorubicin-Induced Chemotherapeutic Stress

doi: 10.3390/cells15100899

Figure Lengend Snippet: MFN2 involvement in the mitochondrial homeostasis of T98G cells following their pulse DOX treatment. ( a ) Effect of MFN1 or MFN2 down-regulation on the morphology of their mitochondria following pulse DOX (48 h) treatment (measured as a fraction of T98G cells that display the presence of integral/non-fragmented mitochondria; Python pipeline-assisted analysis; cf. ). Scale bars—10 µm ( b ) Effect of MFN1 or MFN2 down-regulation on mitochondrial organization in T98G cells ( left ), the fraction of viable T98G cells following a pulse DOX treatment (estimated with Trypan blue assay; middle) (and surface area; right ). Statistical significance was assessed by non-parametric Mann–Whitney test; * p < 0.05 vs. control and # p < 0.05 vs. DOX-treated variant. Data are representative of ≥50 single cells in 3 independent biological replicates. ( c ) Heatmap of upregulated “mitochondrion organization” proteins estimated with LC-MS/MS proteomics in DOX_R 14d cells (red = highest increase within each group; green = lowest increase; fold change > 1.2). ( d ) BCS1 (BCS1L) levels in T98G cells following their pulse DOX treatment at specific time points, estimated with LC-MS/MS. ( e ) An enriched interactome between BCS1L (red dashed circle) and its closest physical interactors generated using the STRING database and a high-confidence interaction score threshold (0.700). The diagram on the right represents Gene Ontology-based functional enrichment analysis of the abovementioned proteins, which identified a coherent group of biological processes collectively associated with mitochondrial bioenergetics regulation. Statistical significance was assessed by ANOVA with permutation FDR < 0.05 and Tukey post hoc ( p < 0.05 vs. control). Data are representative of 3 independent biological replicates. Note the presence of tubular mitochondria in minute T98G cell sub-populations that survived a combined MFN2 down-regulation/DOX treatment, accompanied by the up-regulation of other regulators of mitochondrial organization.

Article Snippet: Human glioblastoma multiforme (GBM) T98G and U87-MG (ATCC, CRL-1690 and HTB-14, respectively; not listed as commonly misidentified cell lines by the International Cell Line Authentication Committee) cells were maintained under standard culture conditions in high-glucose (4500 mg/L) DMEM medium (Sigma-Aldrich, St. Louis, MO, USA; No. D6429; up to 25 passages), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA; No. A3840402) and 1% Antibiotic-Antimycotic Solution (Merck, No. A5955), as described previously [ , ].

Techniques: MANN-WHITNEY, Control, Variant Assay, Liquid Chromatography with Mass Spectroscopy, Generated, Functional Assay

Journal: Cells

Article Title: Mitochondrial Dynamics Participate in an Early Metabolic Adaptation of Glioblastoma Multiforme T98G Cells to Doxorubicin-Induced Chemotherapeutic Stress

doi: 10.3390/cells15100899

Figure Lengend Snippet: MFN2 and the activity of the drug-relocation/retention system in DOX-resistant cells. ( a ) Lysosomal DOX retention (red) in pulse DOX-treated (1 µM; 48 h) T98G cells visualized by LysoTracker and confocal microscopy on the 14th day after DOX removal (representative images for 3 independent biological replicates). ( b ) Effect of MFN2 silencing on lysosomal DOX retention in T98G cells. ( c ) Immunolocalization of selected ABC transporters in T98G cells; accompanying values show the percentage of cells exhibiting nuclear localization for each ABC protein. ( d ) V-type proton ATPase levels in T98G cells following their pulse DOX treatment estimated with LS-MS. Statistical significance was assessed by ANOVA with permutation FDR < 0.05 and Tukey post hoc ( p < 0.05 vs. control) from experimental triplicates (N = 3). ( e ) Effect of metabolic inhibitors, MFN1/2 silencing and ABC/V-type proton ATPase inhibitors on the formation of PGCs in T98G populations under DOX-induced stress. Scale bars = 2 ( a ), 20 and 50 ( b ) and 100 µm ( c , e ). Statistical significance was calculated with non-parametric Mann–Whitney U test, * p < 0.05 vs. control ( e ). All data are representative of 3 independent biological replicates. Error bars represent SD values. Note the lysosomal accumulation of DOX in PGCs and impairment of the PGC program following the Baf A1 administration.

Article Snippet: Human glioblastoma multiforme (GBM) T98G and U87-MG (ATCC, CRL-1690 and HTB-14, respectively; not listed as commonly misidentified cell lines by the International Cell Line Authentication Committee) cells were maintained under standard culture conditions in high-glucose (4500 mg/L) DMEM medium (Sigma-Aldrich, St. Louis, MO, USA; No. D6429; up to 25 passages), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA; No. A3840402) and 1% Antibiotic-Antimycotic Solution (Merck, No. A5955), as described previously [ , ].

Techniques: Activity Assay, Confocal Microscopy, Control, MANN-WHITNEY