Journal: Cell Death & Disease
Article Title: RB1 controls differentiation through positive regulation of phosphoglycerate mutases
doi: 10.1038/s41419-025-07850-3
Figure Lengend Snippet: A OCR, an index of oxygen consumption in mitochondria indicating oxidative phosphorylation coupled with glycolytic activity, was measured in 53KOLS cells expressing scramble or Rb shRNA. Cells were sequentially treated with 10.5 mM glucose and 100 mM 2-deoxy glucose. B ECAR, an index of the rate of lactate production indicating glycolytic activity, was measured in 53KOLS cells expressing scramble or Rb shRNA. Cells were sequentially treated with 10.5 mM glucose and 100 mM 2-deoxy glucose (2-DG). C Relative 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) fluorescence intensity showing glucose uptake in 53KOLS cells expressing scramble or Rb shRNA. Paired student’s t-test was performed. * P < 0.05 and n.s., not significant. D ECAR was measured in 53KOLS cells expressing scramble or Rb shRNA following the treatment with 1 mM pyruvate. E ECAR was measured in 53KOLS. cells expressing scramble or Rb shRNA following the treatment with 4 mM glutamine. F OCR in 53KOLS cells expressing scramble or Rb shRNA was measured in the presence or absence of a glutaminase inhibitor, BPTES (5 μM). Tukey HSD test was performed. ** P < 0.01 and n.s., not significant. G Relative incorporation of [U- 14 C]-glucose into lipid. 53KOLS cells were cultured in the presence of [U- 14 C]-glucose (6.25 μCi/ml) for 24 h. 14 C activity was measured using liquid scintillation counter. Paired student’s t-test was performed. *** P < 0.001. H Relative incorporation of [U- 14 C]-glutamine into lipid. 53KOLS cells were cultured in the presence of [U- 14 C]-glutamine (62.5 μCi/ml) for 24 h. Paired student’s t-test was performed. n.s., not significant. I Relative lipid incorporation of 3 H 2 O. 53KOLS cells were cultured in the presence of 3 H 2 O (20 v/v%) for 24 h. 3 H activity was measured using liquid scintillation counter. Paired student’s t-test was performed. * P < 0.05. J IB of the indicated proteins in 53KOLS cells overexpressing Pgam2. K Relative lipid incorporation of [U- 14 C]-glucose in 53KOLS cells overexpressing Pgam2. Tukey’s HSD test was performed. * P < 0.05 and n.s., not significant. L ECAR was measured in AGS cells expressing scramble or RB1 shRNA. Cells were sequentially treated with 10.5 mM glucose and 100 mM 2-DG. M Relative 2-NBDG fluorescence intensity in AGS cells expressing scramble or RB1 shRNA. Tukey HSD test was performed. *** P < 0.001 and ** P < 0.01. N Relative 2-NBDG fluorescence intensity in SNU-638 cells expressing scramble or RB1 shRNA. Tukey HSD test was performed. ** P < 0.01 and ** P < 0.05. O Absolute glucose consumption rate within 24 h in SNU-638 cells expressing scramble or RB1 shRNA. Paired student’s t-test was performed. * P < 0.05. P Absolute lactate secretion rate within 24 h in SNU-638 cells expressing scramble or RB1 shRNA. Paired student’s t-test was performed. * P < 0.05. Q Relative incorporation of [U- 14 C]-glucose into lipid in AGS cells expressing scramble or RB1 shRNA those were cultured in the presence of [U- 14 C]-glucose (6.25 μCi/ml) for 24 h. 14 C activity was measured using liquid scintillation counter. Tukey HSD test was performed. ** P < 0.01 and * P < 0.05. R Effect of PGAM1 overexpression on glycolytic flow in RB1-depleted AGS cells. S Effect of PGAM1 overexpression on glucose consumption for 24 h in RB1-depleted AGS cells. Glucose concentration in medium was determined using colorimetric assay. Tukey HSD test was performed. * P < 0.05. All data are shown as the mean ± SEM.
Article Snippet: Taqman probes used are as follow: mouse Actb (Mm00607939_s1), mouse Rb (Mm00485586_m1), mouse Hprt (Mm00446968_m1), mouse Nanog (Mm02019550_s1), mouse Pgam2 (Mm01187768_m1), mouse Myh7 (Mm00600555_m1), mouse Myog (Mm00446194_m1), mouse Myod1 (Mm00440387_m1) human ACTB (Hs99999903_m1), human RB (Hs01078066_m1), human PGAM1 (Hs01652468_g1) and human HPRT (Hs02800695_m1).
Techniques: Phospho-proteomics, Activity Assay, Expressing, shRNA, Fluorescence, Cell Culture, Over Expression, Concentration Assay, Colorimetric Assay
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![A OCR, an index of oxygen consumption in mitochondria indicating oxidative phosphorylation coupled with glycolytic activity, was measured in 53KOLS cells expressing scramble or Rb shRNA. Cells were sequentially treated with 10.5 mM glucose and 100 mM 2-deoxy glucose. B ECAR, an index of the rate of lactate production indicating glycolytic activity, was measured in 53KOLS cells expressing scramble or Rb shRNA. Cells were sequentially treated with 10.5 mM glucose and 100 mM 2-deoxy glucose (2-DG). C Relative 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) fluorescence intensity showing glucose uptake in 53KOLS cells expressing scramble or Rb shRNA. Paired student’s t-test was performed. * P < 0.05 and n.s., not significant. D ECAR was measured in 53KOLS cells expressing scramble or Rb shRNA following the treatment with 1 mM pyruvate. E ECAR was measured in 53KOLS. cells expressing scramble or Rb shRNA following the treatment with 4 mM glutamine. F OCR in 53KOLS cells expressing scramble or Rb shRNA was measured in the presence or absence of a glutaminase inhibitor, BPTES (5 μM). Tukey HSD test was performed. ** P < 0.01 and n.s., not significant. G Relative incorporation of [U- 14 C]-glucose into lipid. 53KOLS cells were cultured in the presence of [U- 14 C]-glucose (6.25 μCi/ml) for 24 h. 14 C activity was measured using liquid scintillation counter. Paired student’s t-test was performed. *** P < 0.001. H Relative incorporation of [U- 14 C]-glutamine into lipid. 53KOLS cells were cultured in the presence of [U- 14 C]-glutamine (62.5 μCi/ml) for 24 h. Paired student’s t-test was performed. n.s., not significant. I Relative lipid incorporation of 3 H 2 O. 53KOLS cells were cultured in the presence of 3 H 2 O (20 v/v%) for 24 h. 3 H activity was measured using liquid scintillation counter. Paired student’s t-test was performed. * P < 0.05. J IB of the indicated proteins in 53KOLS cells overexpressing <t>Pgam2.</t> K Relative lipid incorporation of [U- 14 C]-glucose in 53KOLS cells overexpressing Pgam2. Tukey’s HSD test was performed. * P < 0.05 and n.s., not significant. L ECAR was measured in AGS cells expressing scramble or RB1 shRNA. Cells were sequentially treated with 10.5 mM glucose and 100 mM 2-DG. M Relative 2-NBDG fluorescence intensity in AGS cells expressing scramble or RB1 shRNA. Tukey HSD test was performed. *** P < 0.001 and ** P < 0.01. N Relative 2-NBDG fluorescence intensity in SNU-638 cells expressing scramble or RB1 shRNA. Tukey HSD test was performed. ** P < 0.01 and ** P < 0.05. O Absolute glucose consumption rate within 24 h in SNU-638 cells expressing scramble or RB1 shRNA. Paired student’s t-test was performed. * P < 0.05. P Absolute lactate secretion rate within 24 h in SNU-638 cells expressing scramble or RB1 shRNA. Paired student’s t-test was performed. * P < 0.05. Q Relative incorporation of [U- 14 C]-glucose into lipid in AGS cells expressing scramble or RB1 shRNA those were cultured in the presence of [U- 14 C]-glucose (6.25 μCi/ml) for 24 h. 14 C activity was measured using liquid scintillation counter. Tukey HSD test was performed. ** P < 0.01 and * P < 0.05. R Effect of PGAM1 overexpression on glycolytic flow in RB1-depleted AGS cells. S Effect of PGAM1 overexpression on glucose consumption for 24 h in RB1-depleted AGS cells. Glucose concentration in medium was determined using colorimetric assay. Tukey HSD test was performed. * P < 0.05. All data are shown as the mean ± SEM.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0115/pmc12290115/pmc12290115__41419_2025_7850_Fig2_HTML.jpg)
