Journal: Journal of Translational Medicine

Article Title: Metabolic reprogramming of poly(morpho)nuclear giant cells determines glioblastoma recovery from doxorubicin-induced stress

doi: 10.1186/s12967-024-05541-9

Figure Lengend Snippet: Two-phasic pattern of T98G adaptation to the pulse DOX-induced stress. ( a ) Long-term dynamics of mesenchymal cell morphology (values show % of mesenchymal cells classified according to the AR values > 2.5) following a pulse DOX-induced treatment of T98G cells, estimated with NIC microscopy-assisted morphometry (cf. Fig. a). ( b ) Nuclear Snail-1 (upper panel) and p-Snail(Ser246; lower panel) visualized and quantified with fluorescence microscopy and fluorimetry. ( c ) Long-term dynamics of T98G hypertrophy following a pulse DOX-induced treatment of T98G cells, estimated with volumetric approach. ( d - f ) The dynamics of T98G viability (left) and spreading (right) following a pulse DOX-treatment, estimated with trypan blue assay ( d ) and microscopy-assisted morphometric approach, respectively ( e , f ). ( g ) Experimental approach towards the identification of the processes underlying long-term GBM adaptation to the pulse DOX treatment. Scale bars = 50 μm ( a , c ), 25 μm ( b ) and 100 μm ( g ). Statistical significance was calculated with ANOVA and Tukey’s post hoc ( a - c ) and t-student test ( e ). Bars represent SEM or minimum/maximum values ( e ). Data representative for 3 independent biological replicates or ≥ 50 cells in 3 replicates. Note GMT of pulse DOX-treated T98G cells, followed by their long-term phenotypic diversification

Article Snippet: Specimens were incubated for 45 min. in the presence of the following primary antibodies (in 2% BSA/0.01% Tween): polyclonal goat anti-SNAI1 (N-terminal, Sigma; No. SAB2501370; 1:300), polyclonal rabbit anti-Cx43 (Sigma; No. C6219; 1:500), monoclonal IgG mouse anti-α-tubulin (Sigma; No. T6199; 1:300), polyclonal rabbit anti vimentin (GeneTex; No. GTX100619; 1:200) and polyclonal rabbit anti phospho-Snail-1-PSer246 (Sigma; No. SAB4504319; 1:200) and polyclonal rabbit anti-Ki67 (Sigma; No. SAB5700770; 1:300).

Techniques: Microscopy, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Metabolic reprogramming of poly(morpho)nuclear giant cells determines glioblastoma recovery from doxorubicin-induced stress

doi: 10.1186/s12967-024-05541-9

Figure Lengend Snippet: Heterogeneity of T98G populations recovering from DOX-induced stress. ( a ) Nuclear Ki67 (insert) and Snail-1 (green) localization in PGCs at the 7th day after DOX removal and the dynamics of their nuclear polymorphism following a pulse DOX-treatment. ( b ) Seahorse XFp analyses of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR, left) and ATP accumulation (right) in PGCs following a pulse DOX-treatment. ( c ) β-galactosidase activity in PGCs and SECs visualized (as x-gal fluorescence) at 14th day after DOX-removal with fluorimetry. ( d , e ) Cx43 gap junctions ( d ) and GJIC ( e ) between PGCs and adjacent clusters of SECs visualized with immunofluorescence and calcein microinjection/transfer, respectively. Single cells are marked according to the signature of the relevant calcein transfer curve. Red arrow indicates the dye injection site. ( f ) Motility of SECs estimated with time-lapse video-microscopy at the population level (cf. Fig. b in Supplementary data). Scale bar = 25 μm ( a , c ), 10 μm ( d ) and 50 μm ( e ). Statistical significance was calculated with ANOVA and Tukey’s post hoc test ( a , c ), t-student ( b ) and non-parametric Mann-Whitney test ( f ), * p < 0.05 vs. control. Error bars represent SD ( b , c ) and SEM ( f ). Data representative for n > 30 single cells and/or 3 independent biological replicates. Note the metabolic cooperation of dormant PGCs and SECs

Article Snippet: Specimens were incubated for 45 min. in the presence of the following primary antibodies (in 2% BSA/0.01% Tween): polyclonal goat anti-SNAI1 (N-terminal, Sigma; No. SAB2501370; 1:300), polyclonal rabbit anti-Cx43 (Sigma; No. C6219; 1:500), monoclonal IgG mouse anti-α-tubulin (Sigma; No. T6199; 1:300), polyclonal rabbit anti vimentin (GeneTex; No. GTX100619; 1:200) and polyclonal rabbit anti phospho-Snail-1-PSer246 (Sigma; No. SAB4504319; 1:200) and polyclonal rabbit anti-Ki67 (Sigma; No. SAB5700770; 1:300).

Techniques: Activity Assay, Fluorescence, Immunofluorescence, Microinjection, Injection, Microscopy, MANN-WHITNEY, Control