Fig 2: NLK phosphorylation of c-Myb results in ubiquitination and proteasome degradation.a CD34+ CB HSPCs transduced with shRNA against control (shLuc) or RPS19 (shRPS19) were differentiated for 10 days and sorted into CD71+ and CD71– fractions. Three independent experiments were pooled and populations were probed by Western blot analysis examining phospho-Ser863-Raptor and total Raptor. This experiment was performed in conjunction with Fig. 3c. b Cord blood CD34+ progenitors were transduced with shRNA against luciferase (shLuc) or RPS19 (shRPS19) and siRNA against NLK (siNLK) or a non-targeting (NT) sequence and differentiated for 8 days. Cells were fixed, permeablized and incubated with Cy3-labeled antibody against Raptor (pseudo-colored red) and lysosomes were visualized by incubation with FITC-labeled antibody recognizing LAMP1 (pseudo-colored green). Areas of co-localization (merge) are indicated in yellow and yellow dotted boxes are magnified in inserts to the far right. Scale bar, 15 µm c Fetal liver CD34+ progenitors were transduced with shRNA against luciferase (shLuc) or RPS19 (shRPS19) in conjunction with siRNA against NLK (siNLK) or a non-targeting sequence (NT). Cells were differentiated for 5 days and split into two aliquots. One aliquot was lysed and probed for c-Myb protein expression by Western blot, while the second aliquot was subjected to qRT-PCR to examine c-Myb mRNA expression. d Kp53A1 cells were treated with vehicle alone, lactacystin or chloroquine for 30 min, prior to switching cells from 37 to 32 °C for the indicated times. Cells were lysed, normalized for protein and split into two. C-Myb was immunoprecipitated from one sample before Western blot analysis for phosphorylated serine, while the other sample was subjected to Western blot for c-Myb and GAPDH. Bars represent means ± SD with individual data points overlaid. n = 3 independent experiments performed in triplicate. Also see Supplementary Fig. 7. Source data are provided as a Source Data file.

Fig 3: The function of non-dissociable homodimer of 14-3-3ζ. (A) Two copies of the 14-3-3ζ gene via a 10-amino acid Gly-rich linker to get a non-dissociable dimer of 14-3-3ζ (14-3-3ζ-Fusion) with lentiviral vector. Expression of endogenous 14-3-3ζ and 14-3-3ζ-Fusion was detected by western blot analysis in A549 cells transfected with the vector and 14-3-3ζ-Fusion lentivirus (left: Under denaturing condition, right: Under non-denaturing condition). Densitometric analysis of the relative expression level of dimer and monomer of 14-3-3ζ. *P<0.005 compared to the Vector group. Lower images: The cells exhibited different morphologies with the transfection of the vector, 14-3-3ζ-Fusion or 14-3-3ζ-Fusion plus untagged NLK lentivirus (scale bar, 100 µm). (B) Expression of E-cadherin and β-actin was detected by western blot analysis in A549 cells transfected with the vector lentivirus, 14-3-3ζ-Fusion or 14-3-3ζ-Fusion plus untagged NLK lentivirus. Densitometric analysis of the relative expression level of E-cadherin. *P<0.001 compared to the Vector group. (C) Left: A549 cells were lysed and subjected to IP with NLK antibodies, controlled by corresponding IgG. Immunoprecipitated protein were then analyzed by western blot analysis under non-denaturing condition. Right: Immunoprecipitated 14-3-3ζ-Fusion-FLAG-associated protein in A549 cells was subjected to western blot analysis. (D) Wound healing analysis of A549 cells transfected with the vector, 14-3-3ζ, 14-3-3ζ-Fusion, 14-3-3ζ plus NLK or 14-3-3ζ-Fusion plus NLK lentivirus. *P<0.005 compared to the Vector or 14-3-3ζ+NLK groups (scale bar, 200 µm). NLK, nemo-like kinase; IP, immunoprecipitation.

Fig 4: NLK inhibits the dimerization of 14-3-3ζ. (A) A549 cells stably sole-expressing or co-expressing FLAG-14-3-3ζ and HA-14-3-3ζ. Cell lysates was immunoprecipitated with HA Affinity Gel and subjected to western blot analysis as detected by the FLAG or HA antibody. (B) A549 cells co-expressing FLAG-14-3-3ζ and HA-14-3-3ζ were transfected with the vector lentivirus or untagged NLK lentivirus. Cell lysates was immunoprecipitated with HA Affinity Gel and subjected to western blot analysis. (C) Densitometric analysis of the relative expression level of untagged NLK and FLAG-tagged 14-3-3ζ associated with equal amounts of HA-tagged 14-3-3ζ. *P<0.001 compared to Vector group. NLK, nemo-like kinase.

Fig 5: NLK inhibits the cell growth and migration of A549 cells. (A) Western blot analysis of NLK protein expression in A549 cells transfected with the NLK lentivirus or vector lentivirus; Densitometric analysis of the relative expression level of NLK. *P<0.001 compared to the Vector group. (B) Colony formation assay (CFA): Representative images and quantification of the colony formation of A549 cells transfected with NLK or vector lentivirus. *P<0.01 compared to the Vector group. Sphere formation assay (SFA): The spheres were derived from A549 cells transfected with NLK lentivirus or vector lentivirus. **P<0.001 compared to the Vector group (scale bar, 200 µm). (C) Wound healing assay indicated that NLK overexpression suppressed A549 cell migration. *P<0.01 compared to the Vector group (scale bar, 200 µm). NLK, nemo-like kinase.