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1.
Fig 9

Fig 9. From: MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death.

MYC regulation by HIF under hypoxic conditions. MYC is posttranslationally degraded via proteasomal and nonproteasomal pathways, resulting in loss of MYC protein and global suppression of MYC transcriptional activity under low O2 tension. See the text for details.

Waihay J. Wong, et al. Mol Cell Biol. 2013 September;33(17):3494-3504.
2.
Fig 6

Fig 6. From: MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death.

HIF-dependent regulation of MYC protein under low O2 tension. (A) HCT116 cells were transfected with the scrambled control (Scr) or siRNA targeting HIF1A, HIF2A, or both, and lysates were immunoblotted for HIF and MYC proteins. Densitometric quantifications are presented as the mean of three independent experiments. N.S., not significant. *, P < 0.05. (B) HCT116 cells were transfected with siRNAs against scrambled sequence, HIF1A, or HIF2A and grown at 21% or 0.5% O2 for 24 to 30 h, and lysates were immunoblotted for HIF and MYC proteins. Densitometric measurements are presented as the means of two independent experiments.

Waihay J. Wong, et al. Mol Cell Biol. 2013 September;33(17):3494-3504.
3.
Fig 4

Fig 4. From: MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death.

Hypoxia promotes cathepsin gene expression. (A) HCT116 cells were grown at 21% or 0.5% O2 for 24 h, and expression of cathepsin genes (CTSS, CTSK, etc.) was determined using qRT-PCR. Mean results from four independent experiments are shown. *, P < 0.05. (B) Confocal microscopy was used to determine cellular localization of cathepsins D (CTSD) and S (CTSS) in HCT116 cells at 21% and 0.5% O2. LysoTracker (red) and DAPI (4′,6-diamidino-2-phenylindole) (blue) were used to visualize acidic lysosomes and cell nuclei, respectively. Scale bars, 10 μm.

Waihay J. Wong, et al. Mol Cell Biol. 2013 September;33(17):3494-3504.
4.
Fig 8

Fig 8. From: MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death.

Overexpression of stabilized MYC promotes hypoxia-induced cell death. (A) The viability of HCT116-HA-MYC and HCT116-MYCT58A cells after 48 h of growth at 21% or 0.5% O2 was determined by annexin V (AnnV) and propidium iodide (PI) staining; data from one representative experiment are shown. (B) Mean results from three independent experiments are quantified. (C and D) HCT116-HA-MYC and HCT116-MYCT58A cells were grown at 21% or 0.5% O2, and expression of PUMA (C) and NOXA (D) was determined by qRT-PCR. (E) HCT116-HA-MYC and HCT116-MYCT58A cells were transfected with scrambled control (Scr) or shRNA targeting NOXA, PUMA, or both, and viability was determined by annexin V and propidium iodide staining after 48 h of growth at 21% or 0.5% O2. KD, knockdown. Mean results from three independent experiments are shown. *, P < 0.05.

Waihay J. Wong, et al. Mol Cell Biol. 2013 September;33(17):3494-3504.
5.
Fig 5

Fig 5. From: MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death.

MYC downregulation under hypoxia is dependent on FBXW7 and DDB1. (A) HCT116 cells were transfected with the scrambled control (Scr) or siRNAs targeting various MYC E3 ligases and grown at 21% or 0.5% O2 for 24 h, and lysates were immunoblotted for MYC protein. Densitometric measurements were obtained from two independent experiments. (B) Knockdown (KD) efficiency of siRNAs targeting various MYC E3 ligases was assessed using qRT-PCR. (C) HCT116 FBXW7−/− cells were transfected with the scrambled control (CONT) or siRNA targeting DDB1, and DDB1 expression was determined using qRT-PCR. (D) HCT116 FBXW7−/− cells transfected with the scrambled control or siRNA targeting DDB1 were grown at 21% or 0.5% O2 for 24 h, and lysates were immunoblotted for MYC protein. Gene expression data are shown as the mean of two independent experiments. *, P < 0.05. Western blot densitometry was calculated using ImageJ and normalized to the β-actin control.

Waihay J. Wong, et al. Mol Cell Biol. 2013 September;33(17):3494-3504.
6.
Fig 2

Fig 2. From: MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death.

MYC downregulation under hypoxia occurs posttranslationally. (A) MYC was coimmunoprecipitated with MAX from HCT116 cells grown at 21% or 0.5% O2 for 6 or 24 h. (B) HCT116 cells were grown at 0.5% O2 for the indicated times, and lysates were immunoblotted (IB) for MYC protein. (C) HCT116 cells were grown at 21% or 0.5% O2 for 24 h, and MYC transcript levels were measured using qRT-PCR. Mean results from three independent experiments are shown. N.S., not significant. (D) HCT116 cells were grown at 21% or 0.5% O2 for 24 h and pulsed with [35S]Cys/Met for 1 h. Newly translated MYC protein was immunoprecipitated and visualized by autoradiography. (E) HCT116 cells grown at 21% or 0.5% O2 for 24 h were pulsed with [35S]Cys/Met for 15 min, and lysates were obtained during subsequent chase periods as indicated. MYC protein was immunoprecipitated and visualized by autoradiography. Densitometric and half-life (t1/2) measurements are shown.

Waihay J. Wong, et al. Mol Cell Biol. 2013 September;33(17):3494-3504.
7.
Fig 3

Fig 3. From: MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death.

Hypoxia promotes MYC degradation via proteasome- and protease-dependent events. (A) HCT116 cells grown at 21% or 0.5% O2 for 44 h were treated with various chemical inhibitors for 2 h, and lysates were immunoblotted for MYC protein and quantified (top panel). Densitometric quantifications are presented in graphical form as the mean of two independent experiments (bottom panel). DMSO, dimethyl sulfoxide. *, P < 0.05. (B) HCT116 cells grown at 21% or 0.5% O2 for 24 h were pulsed with [35S]Cys/Met in the presence of cathepsin inhibitor III, and lysates were obtained in the presence of inhibitor during subsequent chase periods as indicated. MYC protein was immunoprecipitated and visualized by autoradiography. Densitometric and half-life measurements are shown.

Waihay J. Wong, et al. Mol Cell Biol. 2013 September;33(17):3494-3504.
8.
Fig 7

Fig 7. From: MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death.

MYCT58A mutation opposes hypoxia-induced MYC suppression. (A and B) REF52 cells were transduced with retroviral vectors encoding HA-MYC (A) or MYCT58A (B), grown at 21% or 0.5% O2 for 24 h, and pulsed with [35S]Cys/Met, and lysates were obtained at the indicated times during the subsequent chase period. MYC protein was immunoprecipitated and visualized by autoradiography. Average densitometric values and half-life measurements from two independent experiments are shown. (C) MYC immunoblot of lysates from HCT116 and HCT116-MYCT58A cells. (D) HCT116-HA-MYC and HCT116-MYCT58A cells were seeded on day 0 and counted at the indicated times. Mean results from two independent experiments are shown. (E) HCT116-MYCHA-MYC and HCT116-MYCT58A cells were grown at 21% or 0.5% O2 for 24 h, and the percentages of cells in the G1 (left), S (middle), and G2 (right) phases of the cell cycle were determined. Mean results from two independent experiments are shown. *, P < 0.05 at 0.5% O2; **, P < 0.05 at 21% O2.

Waihay J. Wong, et al. Mol Cell Biol. 2013 September;33(17):3494-3504.
9.
Fig 1

Fig 1. From: MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death.

Hypoxia opposes MYC-dependent transcription. (A) HCT116 cells were grown at 21% or 0.5% O2 for 24 h, and expression of MYC-induced (CCND2, MCM5, MCM7, TRNAR, TRNAL, TRNAY, and RN5S) or suppressed (CDKN1A) genes was determined using qRT-PCR. HIF target genes (PGK1, MXD1, and MXI1) and RPLP0 serve as positive and negative controls, respectively. While RN5S expression was unchanged under hypoxia, lower levels of TRNAR, TRNAL, and TRANY were statistically significant. Mean results from three independent experiments are shown. *, P < 0.05. (B) MYC occupancy at target gene promoters was determined by chromatin immunoprecipitation after 24 h of growth at 21% or 0.5% O2. The filaggrin (FLG) promoter does not contain MYC binding sites and was used as a negative control. Mean results from two independent experiments are shown. *, P < 0.05.

Waihay J. Wong, et al. Mol Cell Biol. 2013 September;33(17):3494-3504.

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