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) 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