Fig 2: Re-expression of PSAT1 restores the nuclear localization of PKM2 and cell migration in silenced PC9 cells. (A) Immunoblot analysis for PKM2 and PSAT1 localization in PSAT1-silenced PC9 cells stably expressing an empty vector (EV) or FLAG-PSAT1. Cytoplasmic and nuclear fractions from the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PSAT1 PC9 cells were analyzed using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three independent experiments. (B) Wound healing assay of the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PSAT1 PC9 cells. Shown are representative images at 0 h and 24 h. The migrating cells are demarcated by continuous white lines. Data are presented as a mean ± SE migrated area after 24 h from three independent experiments. A statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. ** p < 0.005 and * p < 0.05. A.U.: arbitrary unit.

Fig 3: Re-expression of nuclear-localized acetyl-mimetic (K433Q) PKM2, but not wild-type PKM2, partially rescues the migration defect due to the loss of PSAT1. (A) Immunoblot analysis for PKM2 localization in PSAT1-suppressed PC9 cells stably expressing the nuclear-targeted wild-type PKM2 (FLAG-PKM2NLS-WT). Cytoplasmic and nuclear fractions from the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PKM2NLS-WT-expressing cells were analyzed using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three independent experiments. (B) Wound healing assay of serum-starved PC9 cells expressing the control-EV, shPSAT1-EV, or shPSAT1-FLAG-PKM2NLS-WT. Shown are representative images at 0 h and 24 h with migrating cells demarcated by continuous white lines. Data are presented as a mean ± SE migrated area after 24 h from three independent experiments. A statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. * p < 0.0001 and N.S.: not significant. (C) Immunoblot analysis for PKM2 localization in PSAT1-suppressed PC9 cells stably expressing nuclear-targeted acetyl-mimetic (K433Q) PKM2. Cytoplasmic and nuclear fractions from the control-EV, shPSAT1-EV, and shPSAT1-FLAG-PKM2NLS-K433Q-expressing cells were analyzed using anti-PKM2 and anti-PSAT1 antibodies. Oct-1 and α-tubulin served as loading controls for the nuclear and cytoplasmic compartments, respectively. Shown are representative images from three independent experiments. (D) Wound healing assay of serum-starved PC9 cells expressing the control-EV or shPSAT1-EV and shPSAT1-FLAG-PKM2NLS-K433Q. Shown are representative images at 0 h and 24 h with migrating cells demarcated by continuous white lines. Data are presented as a mean ± SE migrated area after 24 h from three independent experiments. A statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparison test. ** p < 0.0001 and * p < 0.05. EV: empty vector; A.U.: arbitrary unit; NLS: nuclear localization signal.

Fig 4: The CSN6–TRIM21–OCT1 axis is involved in ALDH1A1 expression.a HCT116 cells were transfected with the indicated plasmids. Cell lysates were immunoblotted with anti-Flag and anti-OCT1 antibodies. b TRIM21 knockout cells were transfected with the indicated plasmid, and cell lysates were immunoblotted with anti-Flag and anti-OCT1 antibodies. c HEK293T cells were transfected with HA-Ubi and the indicated plasmids. Cell lysates were subjected to IP with anti-Flag and immunoblotted with anti-HA antibodies. d HEK293T cells were transfected with His-Ubi and the indicated plasmids. Cell lysates were subjected to PD with Ni-NTA beads and immunoblotted with anti-Flag and anti-CSN6 antibodies. e HEK293T cells were transfected with the indicated plasmids. Cell lysates were subjected to PD with Ni-NTA beads and immunoblotted with anti-Flag antibodies. f HEK293T cells were transfected with HA-Ubi and Flag-OCT1 and subsequently treated with shCSN6 and siTRIM21. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. g DLD-1 cells were engineered to stably express scrambled or CSN6-specific shRNA and were then rescued with Flag-OCT1 plasmids. Sphere formation was measured. Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1. *P < 0.05 and **P < 0.01.

Fig 5: CSN6 facilitates ubiquitination-mediated degradation of TRIM21.a HEK293T cells were transfected with His-Ubi together with Flag-TRIM21 and HA-CSN6. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with Ni-NTA beads and immunoblotted with anti-Flag and anti-HA antibodies. b HEK293T cells were transfected with HA-Ubi and Flag-TRIM21 and subsequently treated with shCSN6. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with the anti-Flag and anti-HA antibodies. c In DLD-1 cells expressing scrambled or CSN6-specific shRNA, CSN6 knockdown reduced the turnover rate of the TRIM21 protein and increased the turnover rate of OCT1. CHX cycloheximide. d DLD-1 cells were seeded into six-well plates. Forty-eight hours later, the cells were treated with MG132 at the indicated time points. e HEK293T cells were co-transfected with HA-Ubi and Flag-TRIM21. Forty-eight hours later, MG132 or NH4Cl was added to the cells at the indicated time points. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. f HCT116 cells were co-transfected with HA-CSN6 and Flag-TRIM21-WT or Flag-TRIM21-LD (C16A, C31A and H33W) mutant. Cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. g The knockout efficiency of TRIM21 in HEK293T cells by TRIM21 sgRNAs was evaluated by western blotting. h HEK293T cells were co-transfected with the HA-Ubi-WT, Ubi-K48 only or Ubi-K63 only construct and the Flag-TRIM21-WT or Flag-TRIM21-LD construct. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. i HEK293T KO cells were co-transfected with the indicated HA-Ubi constructs and Flag-TRIM21 or the Flag-TRIM21 ΔRING mutant. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-HA antibodies. j Prediction of TRIM21 ubiquitination sites by UbiSite (http://140.138.144.145/~ubinet/index.php) and UbPred (http://www.ubpred.org/index.html). k HEK293T cells were co-transfected with HA-Ubi and Flag-TRIM21-WT or its mutants. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. l HCT116 cells were co-transfected with HA-CSN6 and Flag-TRIM21-WT or the Flag-TRIM21 K214R/K217R mutant. Cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. m HCT116 cells were co-transfected with HA-CSN6 and Flag-TRIM21 or the Flag-TRIM21 K214R/K217R mutant and were then treated with cycloheximide. Cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. n HEK293T KO cells were co-transfected with the indicated HA-Ubi constructs and Flag-TRIM21-WT or the Flag-TRIM21 K214R/K217R mutant. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-HA antibodies.