Fig 2: MKRN1 knockdown induces cell growth retardation and cellular senescence through p14ARF regulation in gastric cancer cells. A) Treatment of MKRN1 siRNAs suppressed the expression of MKRN1. After 96-h transfection with control, MKRN1 #5, or MKRN1 #6 siRNAs, cell lysates were subjected to immunoblotting using anti-MKRN1 and actin antibodies. B) The suppression of cell proliferation in p53 wt AGS cells and p53 mutant SNU601 cells under MKRN1 knockdown. After treatment with siRNAs as described above, the cells were stained with crystal violet. C) Treatment of MKRN1 siRNAs induced cellular senescence. Cells transfected with MKRN1 siRNAs as above were stained for ß-galactosidase activities. The scale bar indicates 80.26 µm. D) Knockdown of MKRN1 induces stabilization of p14ARF without changes in mRNA levels of p14ARF. Cells transfected with control or MKRN1 #6 were analyzed by RT-PCR using primers specific for p14ARF, MKRN1, and GAPDH. Cell lysates were analyzed using the antibodies indicated. E) MKRN1 siRNA #6 and p14ARF siRNAs treated in AGS and SNU601 cells suppressed the expression of MKRN1, p14ARF, or both. (F) p14ARF depletion blocks the inhibition of gastric cancer cell growth induced by MKRN1 ablation. G) Cellular senescence induced by MKRN1 depletion is reversed by p14ARF ablation. The scale bar indicates 80.26 µm. All experiments from E) to G) were carried out as described above. H) Ablation of p14ARF rescued the cell growth retardation induced by MKRN1 depletion. AGS and SNU601 cells were transfected with siRNA as indicated. After 24, 48, 72, and 96h, cell viability was assessed using CellTiter-Glo Reagent. The error bars indicate 95% confidence intervals. *, P = .004; **, P = .005 using a two-sided t test. Three independent experiments were performed. MKRN1 = Makorin ring finger protein 1; p14ARF = p14 alternative reading frame.

Fig 3: The tumor-suppressive effects of MKRN1 knockdown via stabilization of p14ARF were observed in the p53 mutated gastric cancer cell line SNU601. A–C) Mice (N = 4) were inoculated subcutaneously in both ?anks with 106 cells of each SNU601 cell line. The stable SNU601 cell lines were prepared in the same ways as mentioned in Figure 7. A) At 23 days after inoculation, mice were killed in 7.5% CO2 chamber, and mice and excised tumors from mice were photographed. B) Quantification of tumor formation was performed by measurement of tumor size 4, 6, 8, 11, and 13 days after inoculation (N = 4 tumors for four mice). The error bars indicate 95% confidence intervals. *, P = .02; **, P = .01, two-sided t test. All statistical tests were two-sided. C) Immunohistochemical analysis and hematoxylin and eosin (H&E) staining were performed for each group of tumors as described in Materials and Methods (magnification ×200). SA-ß-galactosidase-stained slides were viewed under ×200 magnification. The scale bar indicates 100 µm. MKRN1 = Makorin ring finger protein 1; p14ARF = p14 alternative reading frame.

Fig 4: MKRN1 interacts with p14ARF. A, B) MKRN1 and p14ARF proteins interact with each other. Plasmids expressing HA/MKRN1 alone or with p14ARF/FLAG were transfected into 293 cells. WCL were immunoprecipitated with anti-FLAG or anti-HA antibodies, and immunoprecipitates and WCLs were immunoblotted using the antibodies against MKRN1 and p14ARF. C, D) Endogenous MKRN1 and p14ARF bind to each other. HeLa cell lysates were immunoprecipitated with anti-p14ARF or MKRN1 antibodies, and immunoprecipitated proteins were analyzed using the same antibodies. Arrow indicates p14ARF. E) Recombinant p14ARF and MKRN1 proteins bind to each other directly. Purified GST/p14ARF and in vitro translated HA-MKRN1 were incubated at 37°C (input) followed by pull-down (PD) using glutathione-sepharose. The precipitated samples were detected using anti-MKRN1 and p14ARF antibodies. F) MKRN1 induces p14ARF translocation to nucleo/cytoplasm. H1299 cells were transfected with plasmids expressing HA/MKRN1 and p14ARF/FLAG. Fixed cells were detected by monoclonal anti-HA mouse and polyclonal anti-FLAG rabbit antibodies and stained by Alexa Fluor 594-conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-mouse antibodies, analyzed using N-SIM superresolution microscopy (×150 magnification). The percentages on panels 6 and 10 and graph indicate the fractions of counted cells (200) that displayed such p14ARF/FLAG localization. G) The Pearson correlation coefficient for the colocalization of p14ARF and MKRN1. The error bars indicate 95% confidence intervals using two-sided t test. Thirty cells were tested for the analyses. The tan bars show the Pearson correlation for p14ARF and MKRN1 colocalization. MKRN1 = Makorin ring finger protein 1; p14ARF = p14 alternative reading frame.

Fig 5: MKRN1 functions as an E3 ligase by inducing ubiquitination and subsequent proteasomal degradation of p14ARF. A) MKRN1 induces proteasomal degradation of p14ARF. H1299 cells were transfected with mixtures of plasmids expressing p14ARF/FLAG, HA/MKRN1, or GFP followed by 3-h treatment with 20 µM MG132 or LLnL. The lysates were immunoblotted with anti-FLAG, HA, and GFP antibodies. B) E3 ligase activity of MKRN1 is required for p14ARF degradation. H1299 cells were transfected with mixtures of plasmids expressing p14ARF/FLAG, HA/MKRN1, HA/H307E (E3 ligase activity–deficient mutant), or GFP. The cell lysates were immunoblotted using anti-FLAG, HA, and GFP antibodies. C) Both MKRN1 WT and H307E interact with p14ARF. Mixtures of plasmids expressing p14ARF/FLAG, HA/MKRN1, or HA/H307E were transfected into 293T cells followed by immunoprecipitation using anti-HA antibodies and detection of the immunoprecipitated proteins with anti-FLAG and HA antibodies. D) MKRN1 ubiquitinates p14ARF. H1299 cells were transfected with mixtures of plasmids expressing His/Ub, p14ARF/FLAG, HA/MKRN1, or mock as indicated, with or without MG132. The cell lysates were pulled down with Ni2+-NTA resin and detected using anti-HA and FLAG antibodies. E) MKRN1 H307E is defective in inducing p14ARF ubiquitination. H1299 cells were transfected with mixtures of plasmids expressing His/Ub, p14ARF/FLAG, HA/MKRN1, or mock as indicated. The cell lysates were pulled down with Ni2+-NTA resin and detected with anti-FLAG and HA antibodies. F) MKRN1 depletion suppresses ubiquitination of endogenous p14ARF. HeLa cells were transfected with control or MKRN1 siRNAs followed by MG132 treatment. The cell lysates were immunoprecipitated with anti-p14ARF antibodies, and the immunoprecipitates were detected with anti-p14ARF and HA ubiquitin antibodies. All of the ubiquitination analyses were performed under conditions of denaturation. MKRN1 = Makorin ring finger protein 1; p14ARF = p14 alternative reading frame; Ni2+-NTA = Ni2+-nitrilotriacetic acid.