Table S1. Alteration of ZNF322A and FBXW7 protein expression levels in relation to clinicopathological parameters in 135 lung cancer patients.

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1 SUPPLEMENTARY TABLES Table S1. Alteration of ZNF322A and FBXW7 protein expression levels in relation to clinicopathological parameters in 135 lung cancer patients. ZNF322A Normal Overexpression expression FBXW7 Preserved Low expression expression Characteristics N (%) N (%) 38 (28.1%) 97 (71.9%) Age Sex a The data was analyzed by Pearson χ 2 test. b ADC, adenocarcinoma; SCC, squamous cell carcinoma. c T describes the size of the tumor. d N describes regional lymph nodes. e M describes distant metastasis. P- value a 72 (53.3%) 63 (46.7%) N (%) N (%) P- value a <65 15 (20.3) 59 (79.7) (50.0) 37 (50.0) >65 23 (37.7) 38 (62.3) 35 (57.4) 26 (42.6) Male 20 (29.4) 48 (70.6) (45.6) 37 (54.4) Female 18 (26.9) 49 (73.1) 41 (61.2) 26 (38.8) Smoking No 22 (27.2) 59 (72.8) (59.3) 33 (40.7) Yes 13 (31.0) 29 (69.0) 18 (42.9) 24 (57.1) Tumor type b ADC 28 (23.7) 90 (76.3) (54.2) 54 (45.8) SCC 9 (56.2) 7 (43.8) 7 (43.8) 9 (56.2) Tumor stage I & II 29 (34.5) 55 (65.5) (58.3) 35 (41.7) III & IV 9 (17.6) 42 (82.4) 23 (45.1) 28 (54.9) T status c I & II 31 (27.7) 81 (72.3) (56.3) 49 (43.8) III & IV 7 (31.8) 15 (68.2) 8 (36.4) 14 (63.6) N status d N0 26 (37.7) 43 (62.3) (59.4) 28 (40.6) >N1 12 (18.2) 54 (81.8) 31 (47.0) 35 (53.0) M status e M0 37 (31.1) 82 (68.9) (52.1) 57 (47.9) >M1 1 (6.70) 14 (93.3) 9 (60.0) 6 (40.0) 1

2 Table S2. Antibodies and their reaction conditions used in the study. Target K.D. Raised In Application a Dilution Source Catalog No. CK1 47 Rabbit IB 1:1000 GeneTex GTX Flag -- b Mouse IB 1:1000 IP 1:500 Sigma Aldrich F1804 FBXW7 110 Rabbit IB 1:1000 Bethyl Laboratories Inc. A A-T FBXW7 110 Rabbit IHC 1:100 Bethyl Laboratories Inc. IHC T GSK3 46 Rabbit IB 1:1000 Cell signaling #9315S GAPDH 37 Mouse IB 1:1000 Santa Cruz sc GFP tag -- b Rabbit IB 1:1000 GeneTex GTX26556 GST tag -- b Rabbit IB 1:1000 GeneTex GTX p-h2ax 17 Mouse IB 1:1000 Millipore HA tag -- b Rabbit IB 1:1000 IP 1:500 GeneTex GTX

3 HA tag -- b Mouse IB 1:1000 Bioman HAT001M Myc tag -- b Rabbit IB 1:1000 IP 1:500 GeneTex GTX ZNF322A 47 Rabbit IHC 1:100 IB 1:1000 Kelowna International Scientific Inc. Homemade c ZNF322A ZNF322A p Rabbit Rabbit IHC 1:100 IB 1:1000 ZNF322A p Rabbit IB 1:1000 Kelowna International Scientific Inc. Kelowna International Scientific Inc. Homemade c Homemade c a. IB: immunoblotting, IP: immunoprecipitation, IHC: immunohistochemistry. b. --, Molecular weight is variable. c. Homemade Anti-ZNF322A, Anti-ZNF322A p-ser-391 and Anti-ZNF322A p-ser-396 were generated by Kelowna International Scientific Inc, Taiwan using synthetic peptides ZNF322A (CNVSEKGLELSPPHASE), ZNF322A p-ser-391 (CNVSEKGLEL-pS-PPHASE), ZNF322A p-ser- 396 (CNVSEKGLELSPPHA-pS-E). 3

4 Table S3. The plasmids and their characteristics used in the study. Plasmid Target Function Source pcmv-ha None Vector control Homemade a HA-ZNF322A Wild type ZNF322A Overexpression Homemade a HA-ZNF322A S391A ZNF322A S391A Overexpression Homemade a HA-ZNF322A S396A ZNF322A S396A Overexpression Homemade a HA-ZNF322A S396E ZNF322A S396E Overexpression Homemade a HA-ZNF322A S399A ZNF322A S399A Overexpression Homemade a HA-ZNF322A S391/396A ZNF322A S391/396A Overexpression Homemade a EGFP-C1 None Vector control Homemade a EGFP-ZNF322A Wild type ZNF322A Overexpression Homemade a EGFP-ZNF322A S391/396A ZNF322A S391/396A Overexpression Homemade a pgex4t-1-znf322a Wild type ZNF322A Recombinant protein purification Homemade a 4

5 pgex4t-1-znf322a S391A ZNF322A S391A Recombinant protein Homemade a pgex4t-1-znf322a S396A ZNF322A S396A Recombinant protein Homemade a pgex4t-1-znf322a S399A ZNF322A S399A Recombinant protein Homemade a pcdna3.1 None Vector control From Dr. C-W Chiang b pcdna3.1-flag-ck1δ CK1δ Overexpression From Dr. C-W Chiang b pcdna3.1-flag-ck1α CK1α Overexpression From Dr. C-W Chiang b pcdna3.1-flag-ck1ε CK1ε Overexpression From Dr. C-W Chiang b PCMV5 None Vector control From Dr. M-H Lee c PCMV5-Flag-FBXW7 FBXW7 Overexpression From Dr. M-H Lee c HA-FBXW7 FBXW7 Overexpression From Dr. Wenyi Wei d EGFP-GSK3 -CA Constitutively active: S9A GSK3 Overexpression From Dr. C-F Lin e EGFP-GSK3 -KD Kinase dead: K85A GSK3 Overexpression From Dr. C-F Lin e pcmv-tag 5A None Vector control Addgene Tag5A myc-gsk3 CA Constitutively active: S9A GSK3 Overexpression Addgene 5

6 pgl3-vector None Vector control Promega pgl3-add1 ADD1 promoter Promoter activity assay Homemade a HA-WT-ub Ubiquitin Overexpression From Dr. H-K Lin f HA-K48R-ub Ubiquitin K48R Overexpression From Dr. H-K Lin f HA-K63R-ub Ubiquitin K63R Overexpression From Dr. H-K Lin f a Human ZNF322A cdna was PCR-amplified and cloned into pcmv-ha and EGFP-C1 expression vector to generate HA-tagged and GFP-tagged ZNF322A expression vector. For recombinant protein purification, human ZNF322A cdna was PCR-amplified and cloned into pgex4t-1 expression vector to generate GST-tagged ZNF322A expression vector. b Plasmid was kindly provided by Dr. Chi-Wu Chiang from Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan. c Plasmid was kindly provided by Dr. Mong-Hong Lee from Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. d Plasmid was kindly provided by Dr. Wenyi Wei from Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. e Plasmid was kindly provided by Dr. Chiou-Feng Lin from Department of Microbiology and Immunology, College of Medicine, 6

7 Taipei Medical University, Taipei, Taiwan. f Plasmid was kindly provided by Dr. Hui-Kuan Lin from Department of Cancer Biology, Wake Forest School of Medicine, Winston- Salem, NC, USA. 7

8 Table S4. The sh-rna plasmids used for generating stable knockdown cell lines. sh-rna a sh-ck1δ#1 Oligo sequences (5 3 ) CCGGCGAAAGGATTAGCGAGAAGAACTCGAGTTCTTCTCGCTAATCCTTTCGTTTTT sh-ck1δ#2 CCGGCCGATGAGAACTCTCCTTATTCTCGAGAATAAGGAGAGTTCTCATCGGTTTTTG sh-gsk3 #1 CCGGCACTGGTCACGTTTGGAAAGACTCGAGTCTTTCCAAACGTGACCAGTGTTTTT sh-gsk3 #2 CCGGAGCAAATCAGAGAAATGAACCTCGAGGTTCATTTCTCTGATTTGCTCTTTTTG sh-fbxw7 #1 CCGGGGCAACAACGACGCCGAATTACTCGAGTAATTCGGCGTCGTTGTTGCCTTTTTG sh-fbxw7 #2 CCGGTCAAACCAGGTGCAATTATTTCTCGAGAAATAATTGCACCTGGTTTGATTTTTG a sh-rna plasmids were purchased from RNAi core, Academia Sinica, Taiwan. 8

9 Table S5. The primer used in site-directed mutagenesis and RT-qPCR. Gene Primer Sequences (5 3 ) Application a GAPDH mrna ZNF322A mrna GSK3β mrna CK1 mrna FBW7 mrna ZNF322A S391A ZNF322A S396A ZNF322A S396E ZNF322A S399A Forward GAG TCA ACG GAT TTG GTC GT Reverse TTG ATT TTG GAG GGA TCT CG qrt-pcr Forward GTG GTC TGC GTG TGA GAG TGG C Reverse TTC TGA CGC ATG GGG AGG GCT qrt-pcr Forward GGT CTT CCG ACC CCG AAC T Reverse GGA TGG TAG CCA GAG GTG GAT qrt-pcr Forward ACC CCC ATC GAA GTG TTG TG Reverse TCC AGT CGA ACA CGT AGT CAT AGG qrt-pcr Forward TGAACCATTGCACATCAAGAGAA Reverse TCATCATGTCCTTTCAGCACCTT qrt-pcr Forward TCTTGAGCTTGCTCCTCCCCATG Site-direct Reverse CATGGGGAGGAGCAAGCTCAAGA mutagenesis Forward AATGTCGACAATGTACACTTCAGAAGAGAAATGTAATC Site-direct Reverse ATATGCGGCCGCTCAAGACATCTGTGAGGCTTCAGCCGC mutagenesis Forward AATGTCGACAATGTACACTTCAGAAGAGAAATGTAATC Site-direct Reverse ATATGCGGCCGC TCAAGACATCTGTGAGGCTTCCTCCGC mutagenesis Forward AATGTCGACAATGTACACTTCAGAAGAGAAATGTAATC Site-direct Reverse ATATGCGGCCGCTCAAGACATCTGAGCGGCTTCTGACGC mutagenesis 9

10 a For construction of ZNF322A S391/396A or E mutant, we used S391A/E as PCR template and the primers of S396A/E in site-direct mutagenesis. 10