Colocalization of endogenous p62 with PHD3. (A) Immunofluorescence analysis of endogenous p62 (red) and PHD3–EGFP or PHD2–EGFP (green) under normoxia. (B) Quantification of p62 colocalization with PHD3 and PHD2. Values are means and s.d. of three visual fields. (C) HeLa cells grown on slides were stained for both endogenous PHD3 and p62 and their expression pattern studied by confocal microscopy. Images from both PHD3 and p62 were studied separately as well as from overlay images (merge). A histogram showing colocalization of the two proteins along the line of the red arrow was generated from the image using LSM510 software (lower panel). Arrows indicate colocalization in aggregates. (D) Consecutive HNSCC tissue sections stained for p62 and PHD3 show aggregates for both proteins in same regions. Examples of aggregates are indicated by arrows.

p62 interacts with PHD3. (A) Immunoprecipitation analysis of PHD3 and p62 interaction under normoxia. HeLa cells grown under normoxia were transfected with either EGFP-p62 alone or together with FLAG-PHD2 or FLAG-PHD3. Anti-FLAG antibody was used for co-immunoprecipitation (+) or was omitted (−) and captured p62 was analyzed by western blotting. (B) HeLa cells were transfected with FLAG-PHD3 and myc-tagged wild-type p62 (myc-p62). Co-immunoprecipitation analysis using FLAG antibody was performed. (C) HeLa cells were transfected with either FLAG-PHD2 or FLAG-PHD3. Endogenous p62 co-immunoprecipitated by FLAG antibody was detected by western blot analysis. PHD3, but not PHD2, captured endogenous p62. (D) HeLa cells were transfected with either wild-type p62 or p62 with LIR domain deletion [p62(delLIR)] together with FLAG-PHD3. p62 co-immunoprecipitated by FLAG antibody was detected by western blot analysis. PHD3 captured wild-type p62 but not p62(delLIR). (E) The proximity of endogenous PHD3 and p62 proteins were examined on HeLa cells using a commercial in situ PLA kit. As a control p62 depletion with siRNA was used. (F) Quantification of p62–PHD3 interactions detected as in E in control and p62 siRNA-treated cells. Means and s.d. of five optical fields are shown.

p62 is required for PHD3 aggregation in normoxia. (A) HeLa cells grown under normoxia were transfected with PHD3-EGFP (green) followed by transfection with either control siRNA (Scr siRNA) or with p62 siRNA and imaged for colocalization with endogenous p62 (red). (B) Quantification of p62 colocalization with PHD3 after transfection with the indicated siRNAs. Values are means and s.d. of five visual fields. (C) HeLa cells transfected with PHD3-EGFP (green) and with either wild-type p62 (myc-p62), a point mutated PB1 domain [myc-p62(D69A)] or UBA-domain deletion [myc-p62(1–358)] plasmids, were immunostained for myc tag (red). Both the UBA domain and PB1 domain of p62 were required for aggregation of PHD3. (D) Quantification of p62 colocalization with PHD3 after treatment as in C. Values are means and s.d. of five optical fields.

p62 expression in hypoxia forces aggregation of PHD3. (A) p62 expression is lost in hypoxia. HeLa cells were grown either under normoxia (21% O₂) or hypoxia (1% O₂) for 24 hours and analyzed for p62 expression by western blotting. β-actin was used as loading control. (B) Forcing p62 expression under hypoxia restores the aggregation of PHD3. HeLa cells were transfected with PHD3-EGFP alone (top image) or together with wild-type myc-p62 (red; lower panels) and grown in hypoxic conditions (1% O₂) for 24 hours. (C) p62 expression was restored 6 h after reoxygenation, simultaneously with the disappearance of PHD3. HeLa cells were grown either under normoxia (21% O₂) or hypoxia (1% O₂) for 24 hours followed by 1 h or 6 h reoxygenation of hypoxia-treated cells. Western blot analysis was performed to determine the levels of p62 and PHD3. β-actin was used as a loading control.

p62 enhances the degradation of PHD3. (A) p62 depletion upregulates the PHD3 protein level. HeLa cells were left untreated or treated with either control siRNA (scr) or p62 siRNA. Cells were grown in normoxic conditions (21% O₂) for 24 hours or 48 hours and subsequently analyzed for PHD3 and p62 expression with western blotting. (B) Western blot intensities were analyzed with the MCID™ imaging software system and the fold expression relative to control was calculated. Values are means and s.d. of three experiments. (C) HeLa cells were grown on coverslips in normoxia and either left untreated or transfected with sip62. Endogenous PHD3 was detected by immunostaining and imaged using confocal microscopy. sip62 exposure increased PHD3 expression. (D) Cells were transfected with control or wild-type p62 (myc-p62) plasmid and grown either under normoxia (−) or hypoxia (+) for 24 hours, followed by western blot analysis of myc, PHD3, PHD2 and HIF-1α. (E) HeLa cells were transfected with control or PHD2, PHD3 and p62 siRNAs. RNA was collected and the mRNA levels determined by quantitative RT-PCR. p62 siRNA did not induce PHD3 mRNA expression. (F) Western blot analysis of PHD3, p62 and HIF-1α under hypoxia and hypoxia with forced p62 expression. Cells were exposed to autophagosome inhibitors 3-MA (4 and 10 hours), bafilomycin A1, chloroquine or the proteasome inhibitor MG-132 (all 10 hours). MG-132 was the only inhibitor that increased PHD3 levels.

Re-expression of p62 under reoxygenation restores the normoxic expression pattern of PHD3. (A) Western blot analysis of the effects of p62 siRNA treatment on the expression levels of PHD3, PHD2, p62 and HIF-1α in normoxia, hypoxia and reoxygenation (Rox) at three different time points (1–6 hours). p62 siRNA markedly slowed the disappearance of PHD3 expression. (B) Cells were imaged with confocal microscopy for the pattern and level of expression of PHD3 and p62 after normoxia, hypoxia and at two different time points after reoxygenation. (C) Quantification of p62–PHD3 interaction under the indicated conditions shown as colocalizing aggregates/cell. Values are means and s.d. of five cells.