Fig 2: MKRN1 induces the degradation of p14ARF. A) MKRN1 induces the degradation of p14ARF in a dose-dependent manner. H1299 cells were transfected with mixtures of plasmids expressing p14ARF/FLAG and HA/MKRN1 with increasing concentrations, or with GFP. The cell lysates were detected with anti-FLAG, HA, and GFP antibodies. B) MKRN1 decreases the half-life of p14ARF. H1299 cells were transfected with mixtures of plasmids expressing p14ARF/FLAG, HA/MKRN1, or GFP followed by cycloheximide (CHX) treatment before harvest. The lysates were immunoblotted using antibodies against FLAG, HA, and GFP. C) Exogenous MKRN1 induces degradation of endogenous p14ARF. HeLa cells were transfected with increasing amounts of HA/MKRN1 plasmids. The cell lysates were immunoblotted with anti-p14ARF, HA, and actin antibodies. D) Exogenous MKRN1 reduces the stability of endogenous p14ARF. HeLa cells were transfected with mock vector or plasmid expressing HA/MKRN1 followed by CHX treatment before harvest. The cell lysates were immunoblotted with anti-p14ARF, HA, and actin antibodies. E) MKRN1 depletion stabilizes endogenous p14ARF. HeLa cells were transfected with control, MKRN1 #5, or MKRN1 #6 siRNAs and lysed after 48h. WCL were immunoblotted with anti-p14ARF, MKRN1, and actin antibodies. F) MKRN1 ablation does not affect p14ARF mRNA levels. HeLa cells were treated as above and analyzed by RT-PCR using primers specific for p14ARF, MKRN1, and GAPDH. G) MKRN1 depletion increases stability of endogenous p14ARF. HeLa cells were transfected with control and MKRN1 siRNAs followed by treatment with CHX and were then lysed and immunoblotted with anti-p14ARF, MKRN1, and actin antibodies. The graphs indicate the relative amounts of p14ARF protein compared with the levels of actin or GFP in the western blot. The expression of each protein was analyzed using the Image J program. MKRN1 = Makorin ring finger protein 1; p14ARF = p14 alternative reading frame; CHX = cycloheximide; WCL = whole cell lysates.

Fig 3: Characterization of retroviral vector for one-step immortalization. a Schematics of the MSCV-pic2 vector design. For one-step immortalization, a hTERT cDNA and a shRNA targeting p16INK4A and p14ARF were introduced into this vector. b Western blot verifying the knockdown of p16INK4A and p14ARF in stably transduced SAECs. c Telomerase activities in SAECs stably transduced with hTERT cDNA (error bars represent SD for all bar charts unless otherwise stated)

Fig 4: iSAECs are genomically stable. a Cell cycle distribution of early and late passage iSAECs in log phase culture (p values were determined using Student’s t test for all bar charts). b Chromosomal count of early and late passages (P14 and P79, respectively) iSAECs using metaphase spread of colcemid arrested mitotic cells. Early passage (P18) BJ fibroblasts were used as control. Numbers in each column indicates the number of metaphase spreads analyzed. c Telomere fluorescence in situ hybridization of late passage iSAECs. A representative metaphase spread image was shown

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.