tris  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a) Oxygen consumption rate (OCR) was measured at various time points with consecutive injections of oligomycin (1 µM), FCCP (two injections at 2 µM and 6 µM), and Rotenone/antimycin A (1 µM each), and normalized to cell number. The left panel shows a representative OCR curve with the calculations used to determine the different components of OCR in Seahorse Mitostress experiments. The right panel shows the mean OCR curves of p27 +/+ (n = 7) and p27 -/- (n = 13) MEFs. (b-g) Basal respiration (b), Maximal respiration (c), Spare capacity (d), ATP-linked respiration (e), Non-mitochondrial OCR (f) and Proton leak (g) of p27 +/+ (n = 7) and p27 -/- (n = 13) MEFs, derived from Seahorse Mitostress experiments (a, right panel). Graphs show means ± SEM. ns: p > 0.05; *: p < 0.05; **: p < 0.01. (h) Mitochondrial Mass was determined by flow cytometry analysis of Mitotracker Green FM (MTG)-stained p27 +/+ and p27 -/- MEFs. Graph shows mean MTG intensity ± SEM from 5 independent experiments. ns: p > 0.05. (i) Extracellular acidification rate (ECAR) of p27 +/+ (n = 7) and p27 -/- (n = 13) MEFs. Graph shows means ± SEM. ****: p < 0.0001. (j) OCR / ECAR ratio of p27 +/+ (n = 7) and p27 -/- (n = 13) MEFs from Seahorse Mitostress experiments. Graphs show means ± SEM. ***: p < 0.001. (k) Energy map of p27 +/+ (n = 7) and p27 -/- (n = 13) MEFs from Seahorse Mitostress experiments. (l, m) ATP production in p27 +/+ (n = 7) and p27 -/- (n = 13) MEFs (k), and Glycolytic ATP/Mitochondrial ATP production ratio (l) based on Seahorse Real-time ATP rate Assays. Graphs show means ± SEM. ns: p > 0.05; ****: p < 0.0001.

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques: Derivative Assay, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a) Extracellular acidification rate (ECAR) was measured at various time points with consecutive injections of glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM) and normalized to cell number. The left panel shows a representative ECAR curve with the calculations used to determine the different components of ECAR in Seahorse Glycolysis Stress experiments. The right panel shows the mean ECAR curves of p27 +/+ (n = 8) and p27 -/- (n = 8) MEFs. (b-f) Basal ECAR (b), Glycolysis (c), Glycolytic capacity (d), Glycolytic reserve (e) and Non-glycolytic acidification (f) of p27 +/+ (n = 8) and p27 -/- (n = 8) MEFs, derived from Seahorse Glycolysis Stress experiments (a, right panel). Graphs show means ± SEM. ns: p > 0.05; *: p < 0.05; **: p < 0.01. (g) ATP quantification using Firefly Luciferase ATP Assay kit of p27 +/+ and p27 -/- MEFs (n = 11) treated with either vehicle, Oligomycin + Antimycin A (OAA) (10 µM each) or 2-DG (50 mM). Graph shows means ± SEM. ns: p > 0.05; *: p < 0.05; **: p < 0.01.

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques: Derivative Assay, Luciferase, ATP Assay

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a, b) Quantitative analyses of metabolic alterations between p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs (metabolome) (a) or in the corresponding culture media (exometabolome) (b) of cells grown for 24 h in full medium. In the volcano plots (left panels), all the metabolites significantly altered [log2(Fold Change) > 0.585 or Log2(FC) < −0.585, corresponding to a fold change of ± 1.5 x; and False Discovery Rate (FDR) < 0.05] between p27 +/+ and p27 -/- MEFs are named. The heatmaps (left panels) show only the metabolites (a) or exometabolites (b) significantly altered between p27 +/+ and p27 -/- MEFs. (c-f) Similar experiments were performed on p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown for 24 h in either glucose starvation medium (c, d) or in amino acid starvation medium (e, f).

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques:

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: Dendrogram (left panel) and principal component analysis (right panel) of cellular metabolites (a) and exometabolites (b) in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs after 24 h of culture in full medium (a, b) , or 24 h of culture in absence of glucose (c, d) , or 24 h of culture in absence of amino acids (e, f) .

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques:

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a) Example of genome viewer capture of the p27 gene locus from transcriptomics data of p27 +/+ and p27 -/- MEFs. (b) Volcano plot showing genes differentially expressed between p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs. Genes were considered differentially expressed when −0.6 < log2(FC) < 0.6 and p < 0.05. (c, d) Gene ontology analyses of RNA-Seq data from p27 +/+ and p27 -/- MEFs cultivated in full medium for 24 h. The pathways most affected are shown for genes downregulated (c) and upregulated (d) in p27 -/- MEFs. (e) Data integration of transcriptomics and metabolomics datasets showing the ontology pathways most deregulated between p27 +/+ and p27 -/- MEFs cultivated in full medium for 24 h. (f) Graphical representation of glycolysis showing metabolites (black), enzymes (red) and corresponding genes (orange). (g) Gene expression of the 10 glycolytic enzymes in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs from RNA-Seq data expressed as Count Per Million (CPM), normalized to p27 +/+ levels. Graph shows means ± SEM. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. (h) RT-qPCR validation of glycolytic enzymes expression in p27 +/+ and p27 -/- MEFs. mRNA levels were normalized to p27 +/+ . Graph shows means ± SEM from five independent experiments. **: p < 0.01; ***: p < 0.001. (i) Immunoblots for Hexokinase 1 (HK1), p27 and actin (loading control) in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of HK1 levels normalized to actin (p27 +/+ n = 5; p27 -/- n = 6) (right panel). **: p < 0.01. (j) Immunoblots for Aldolase (ALDOA), p27 and actin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of ALDOA levels normalized to actin (n = 5) (right panel). *: p < 0.05. (k) Immunoblots for Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), p27 and actin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of GAPDH levels normalized to actin (p27 +/+ n = 5; p27 -/- n = 6) (right panel). *: p < 0.05. (l) Immunoblots for Enolase 1 (ENO1), p27 and actin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of ENO1 levels normalized to actin (p27 +/+ n = 5; p27 -/- n = 6) (right panel). *: p < 0.05. (m) Immunoblots for Pyruvate Kinase (PKM1/2), p27 and actin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of PKM1/2 levels normalized to actin (n = 5) (right panel). *: p < 0.05.

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques: Transcriptomics, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a) Graphical representation of pyruvate fate after glycolysis. (b, c) Lactate levels in the exometabolome of p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown in full medium (b) or glucose starvation medium (c) for 24 h. Data is presented as box plot, means are represented by yellow squares. ns: p > 0.05; ***: p < 0.001. (d) LDHA gene expression in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown in full medium for 24 h from RNA-Seq data expressed as Count Per Million, normalized to p27 +/+ levels. Graph shows means ± SEM. ****: p < 0.0001. ( e ) LDHA mRNA levels in p27 +/+ and p27 -/- MEFs by RT-qPCR, normalized to p27 +/+ levels. Graph shows means ± SEM from five independent experiments. ***: p < 0.001. ( f ) Immunoblots for LDHA, p27 and actin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of LDHA levels normalized to actin (n = 5) (right panel). *: p < 0.05. ( g ) LDHB gene expression in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown in full medium for 24 h from RNA-Seq data expressed as Count Per Million, normalized to p27 +/+ levels. Graph shows means ± SEM. ****: p < 0.0001. ( h ) LDHB mRNA levels in p27 +/+ and p27 -/- MEFs by RT-qPCR, normalized to p27 +/+ levels. Graph shows means ± SEM from five independent experiments. **: p < 0.01. ( i ) Immunoblots for LDHB, p27, and β-tubulin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of LDHB levels normalized to β-tubulin or actin (n = 8) (right panel). ****: p < 0.0001. (j) PDK1 gene expression in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown in full medium for 24 h from RNA-Seq data expressed as Count Per Million, normalized to p27 +/+ levels. Graph shows means ± SEM. ****: p < 0.0001. (k) PDK1 mRNA level in p27 +/+ and p27 -/- MEFs by RT-qPCR, normalized to p27 +/+ levels. Graph shows means ± SEM from 5 independent experiments. **: p < 0.01. ( l ) Immunoblots for PDK1, p27 and actin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of PDK1 levels normalized to actin (n = 6) (right panel). *: p < 0.05. (m) Immunoblots for Lys18-lactylated Histone H3 (HH3-K18-La), p27 and actin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of HH3-K18-La levels normalized to actin (n = 6) (right panel). **: p < 0.01. (n) ATP quantification in p27 +/+ and p27 -/- MEFs treated with either vehicle or Sodium Oxamate (50 mM) for 1 h. Graph shows means ± SEM from four independent experiments. *: p < 0.05; ***: p < 0.001.

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques: Gene Expression, RNA Sequencing, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a) Glucose quantification in fresh culture medium (grey) (n = 3), and media from p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown for 24 h in full medium from exometabolome data. Data is presented as box plot, means are represented by yellow squares. ns: p > 0.05; *: p < 0.05. (b) Glucose quantification in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown for 24 h in full medium from metabolome data. Data is presented as box plot, means are represented by yellow squares. ns: p > 0.05. (c) GLUT1 gene expression in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs from RNA-Seq data expressed as Count Per Million, normalized to p27 +/+ levels. Graph shows means ± SEM. ****: p < 0.0001 . (d) GLUT1 mRNA levels in p27 +/+ and p27 -/- MEFs by RT-qPCR, normalized to p27 +/+ levels. Graph shows means ± SEM from five independent experiments. **: p < 0.01. (e) Immunoblot for GLUT1, p27 and actin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of GLUT1 levels normalized to Actin (n = 6) (right panel). *: p < 0.05. (f) Representative images of GLUT1 immunostaining (green) in p27 +/+ and p27 - /- MEFs. DNA was stained with H33342 (blue). (g) Graphical representation of glycolysis with metabolites from metabolome data in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown for 24 h in full medium. Data is presented as box plots, means are represented by yellow squares. P values are indicated beside each graph.

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques: Gene Expression, RNA Sequencing, Quantitative RT-PCR, Western Blot, Immunostaining, Staining

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a, b) Graphical representation of glycolysis showing cell metabolites analyzed in the metabolome of p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs after 24 h in glucose starvation conditions (a) or after 24 h in amino acid starvation conditions. Data is presented as box plot, means are represented by yellow squares. ns: p > 0.05; *: p < 0.05; **: p < 0.01. p values are indicated beside each box plot.

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques:

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a) Glutamine quantification in fresh culture medium (grey) (n = 3), and media from p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown for 24 h in full medium from exometabolome data. Data is presented as box plot, means are represented by yellow squares. ns: p > 0.05; *: p < 0.05; **: p < 0.01. (b) Glutamine quantification in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown for 24 h in full medium from metabolome data. Data is presented as box plot, means are represented by yellow squares. *: p < 0.05. (c) SLC1A5 gene expression in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs from RNA-Seq data expressed as Count Per Million, normalized to p27 +/+ levels. Graph shows means ± SEM. ****: p < 0.0001. (d) SLC1A5 mRNA level in p27 +/+ and p27 -/- MEFs by RT-qPCR, normalized to p27 +/+ levels. Graph shows means ± SEM from five independent experiments. **: p < 0.01. (e) GLS2 gene expression in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs from RNA-Seq data expressed as Count Per Million, normalized to p27 +/+ levels. Graph shows means ± SEM. ****: p < 0.0001. (f) GLS2 mRNA level in p27 +/+ and p27 -/- MEFs by RT-qPCR, normalized to p27 +/+ levels. Graph shows means ± SEM from five independent experiments. **: p < 0.01. (g) Immunoblot for GLS2, p27 and actin in p27 +/+ and p27 -/- MEFs (left panel). Graph shows means ± SEM of the quantification of GLS2 levels normalized to Actin (n = 8) (right panel). ****: p < 0.0001. ( h ) Graphical representation of TCA cycle related metabolites in p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs grown for 24 h in full medium from metabolome data. Data is presented as box plots, means are represented by yellow squares. P values are indicated beside each graph. (i) Glutamine dependency for mitochondrial respiration in p27 +/+ and p27 -/- MEFs from Seahorse fuel dependency experiments (left panel). Oxygen consumption rate (OCR) was measured at various time points with consecutive injections of CB-839 (3 µM), and UK5099 (4 µM)/Etomoxir (2 µM), and normalized to cell number. From these experiments, the Glutamine dependency for mitochondrial respiration of p27 +/+ and p27 -/- MEFs was calculated (right panel). Graphs show means ± SEM from four independent experiments. *: p < 0.05.

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques: Gene Expression, RNA Sequencing, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a, b) Graphical representation of TCA cycle-related metabolites analyzed in the metabolome of p27 +/+ (n = 8) and p27 -/- (n = 4) MEFs after 24 h in glucose starvation conditions (a) or after 24 h in amino acids starvation conditions (b). Data is presented as box plot, means are represented by yellow squares. ns: p > 0.05; *: p < 0.05. (c) Oxygen consumption rate (OCR) curve showing the calculation used to determine glutamine dependency for mitochondrial respiration in Seahorse fuel dependency experiments. Measurements were performed after injections of CB-839 (Glutaminase inhibitor; 3 µM), and UK5099 (inhibits pyruvate entry to mitochondria; 4µM) / Etomoxir (inhibits long-fatty acid entry to mitochondria; 2 µM)

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques:

Journal: bioRxiv

Article Title: Loss of p27 Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

doi: 10.64898/2026.02.06.703945

Figure Lengend Snippet: (a) Oxygen consumption rate (OCR) was measured at various time points with consecutive injections of oligomycin (1 µM), FCCP (two injections at 2 µM and 6 µM), and Rotenone/antimycin A (1 µM each), and normalized to cell number. Graph shows the mean OCR curves of hTert-RPE1 transfected with control siRNA (siCTL) or p27 siRNA (sip27) (n = 5). (b) Extracellular acidification rate (ECAR) of p27 siRNA transfected hTert-RPE1 normalized to control siRNA condition (n = 5). Graph shows means ± SEM. *: p < 0.05. ( c ) Energy map of hTert-RPE1 transfected with control siRNA or p27 siRNA from Seahorse Mitostress experiments (n = 5). ( d ) Immunoblots for Hexokinase 1 (HK1), p27 and actin (loading control) in hTert-RPE1 transfected with control siRNA or p27 siRNA (left panel). Graph shows means ± SEM of the quantification of HK1 levels normalized to actin (n = 4) (right panel). *: p < 0.05. ( e ) Immunoblots for GAPDH, p27 and actin (loading control) in hTert-RPE1 transfected with control siRNA or p27 siRNA (left panel). Graph shows means ± SEM of the quantification of GAPDH levels normalized to actin (n = 4) (right panel). *: p < 0.05. ( f ) Immunoblots for Enolase 1 (ENO1), p27 and actin in hTert-RPE1 transfected with control siRNA or p27 siRNA (left panel). Graph shows means ± SEM of the quantification of ENO1 levels normalized to actin (n = 4) (right panel). *: p < 0.05. ( g ) Immunoblots for Aldolase A (ALDOA), p27 and actin in hTert-RPE1 transfected with control siRNA or p27 siRNA (left panel). Graph shows means ± SEM of the quantification of ALDOA levels normalized to actin (n = 4) (right panel). *: p < 0.05. ( h ) Immunoblots for Lactate Dehydrogenase B (LDHB), p27 and actin in hTert-RPE1 transfected with control siRNA or p27 siRNA (left panel). Graph shows means ± SEM of the quantification of LDHB levels normalized to actin (n = 3) (right panel). *: p < 0.05. ( i ) Immunoblots for Lactate Dehydrogenase A (LDHA), p27 and actin in hTert-RPE1 transfected with control siRNA or p27 siRNA (left panel). Graph shows means ± SEM of the quantification of LDHA levels normalized to actin (n = 3) (right panel). *: p < 0.05.

Article Snippet: Mouse anti-p27 (SX53G8.5, sc-53871; Immunofluorescence (IF) 1/100, Western Blot (WB) 1/1000), Aldolase A (sc-377058; WB 1/1000), Enolase 1 (sc-271384; WB 1/1000) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques: Transfection, Control, Western Blot