Fig 1: LZTFL1 destabilizes AKT through ZNRF1-mediated ubiquitin proteosome degradation pathway.a GSEA of genes in TGCA cohorts that express high and low LZTFL1, respectively. b Western blot of proteins indicated in ACHN-NC, ACHN-LZTFL1, A498-NC, A498-sh1, andA498-sh2 cells. c Western blot of LZTFL1 in ACHN-NC and ACHN-LZTFL1 cells at various time points after addition of translational inhibitor cycloheximide (CHX) (left panel). The expression levels of LZTFL1 in Western blots were quantified by densitometry and normalized against loading control GAPDH (right panel). Mean ± SD of three independent experiments. *, P < 0.05, two-way ANOVA. d HEK293T cells transfected with myc-LZTFL1 and Flag-ZNRF1 were immunoprecipitated with control IgG or anti-myc antibody and western blotted with anti-flag or anti-myc antibody. 10% of input was loaded on the gel. e HEK293T cells were immunoprecipitated with control IgG or anti-LZTFL1 antibody and western blotted with anti-AKT or anti-LZTFL1 antibody. 10% of input was loaded on the gel. f–h HEK293T cells (f) and A498 cells (g, h) transfected with plasmids expressing indicated proteins were treated with MG132 for 4 h before harvested. His-ubiquitin (His-ub) conjugated proteins were pulled down with Ni-NTA agarose beads and subjected to Western blot analysis with anti-HA antibody.
Fig 2: LZTFL1 suppresses cell proliferation in vitro and in vivo.a Western blots of the endogenous LZTFL1 in various ccRCC cell lines. GAPDH was used as loading control. b Western blots of LZTFL1 in ACHN and Caki1 cell lines transduced with lentiviruses expressing control vector (ACHN-NC, caki1-NC) or LZTFL1 (ACHN-LZTFL1, Caki1-LZTFL1), and in A498 cell line transduced with lentiviruses expressing control (A498-NC) or two different LZTFL1 shRNAs (A498-sh1 & A498-sh2). c Relative cell growth of ccRCC cell lines with control, LZTFL1-overexpressed or knocked down as indicated. Mean ± SD, ****, P < 0.0001, two-way ANOVA. d Colony forming ability of ccRCC cells with control, LZTFL1-overexpressed or knocked down as indicated. N = 3, mean ± SD, **, P < 0.01, ****, P < 0.0001, Student’s t test. e, f 5 × 106 cells with LZTFL1 overexpressed (e), knockdown (f), or corresponding control vectors were inoculated subcutaneously into the mice. Tumor volume was recorded weekly (left panel). Data are shown as mean ± SD, *, P < 0.05, ***, P < 0.001, two-way ANOVA. Tumor weight (middle panel) and tumor micrographs (right panel) at time of sacrifice were shown. mean ± SD, *, P < 0.05, **, P < 0.01, Student’s t test.
Fig 3: LZTFL1 induces cell cycle arrest at G1/S transition.a GO terms of differentially expressed genes (DEGs) between lower and upper quartile expression of LZTFL1 in TCGA-ccRCC cohort. Count indicates the number of DEGs enriched in the pathway. GeneRatio indicates the ratio of enriched DEGs to background genes. b Flow cytometry cell cycle analysis of ACHN and A498 cells stably overexpressing LZTFL1 and knockdown, respectively, and their respective control cells (left panels). Percentages (%) of cell populations at different cell cycle phases (right panels). mean ± SD of three independent experiments, *, P < 0.05, **, P < 0.01, unpaired student t test. c Representative immunofluorescent staining of Edu (red) and DAPI (blue) of cells indicated (left panel) and % of Edu-positive cells (right panel). Mean ± SD of three independent experiments, ***, P < 0.001, ****, P < 0.0001, unpaired student t test. scale bar is 60 µm.
Fig 4: ZNRF1 knockdown partially rescued cell proliferation inhibited by LZTFL1.a Relative cell growth of ACHN-NC, ACHN-LZTFL1, and ACHN-LZTFL1-siZNRF1 cells. Mean ± SD of three independent experiments, *, P < 0.05, ***, P < 0.001, two-way ANOVA. b Colony forming ability of ACHN-NC, ACHN-LZTFL1 and ACHN-LZTFL1-siZNRF1. a, b Mean ± SD of three independent experiments, *, P < 0.05, ****, P < 0.0001, student’s t test. c Western blots of proteins indicated in ACHN-NC, ACHN-LZTFL1 and ACHN-LZTFL1-siZNRF1 cells. d FACS of ACHN-NC, ACHN-LZTFL1 and ACHN-LZTFL1-siZNRF1 (top panel) and % cell populations at different stages of cell cycles (bottom panel). Mean ± SD of three independent experiments, **, P < 0.01, unpaired student t test. e Representative immunofluorescence micrographs stained with Edu (red) and DAPI (blue) of ACHN-NC, ACHN-LZTFL1 and ACHN-LZTFL1-siZNRF1 cells (left panel). % Edu-positive cells from the left panel were quantified (right panel). Mean ± SD of three independent experiments, ****, P < 0.0001, unpaired student t test.
Fig 5: LZTFL1 is significantly down regulated in ccRCC compared to normal tissues.a The expression level of LZTFL1 in tumor and normal tissues based on data from the TCGA database. b Relative mRNA level as a function of the relative copy number of LZTFL1 in the TCGA database. c Correlation of LZTFL1 mRNA and CNV segment means from ccRCC patients using TCGA level 3 data. d Kaplan–Meier analysis of survival and the COX proportional hazards model for the hazard ratio of LZTFL1 mRNA levels as a prognostic marker in ccRCC patients. e LZTFL1 protein level in ccRCC patient’s tissue samples was compared with those in normal renal tissues in the CPTAC database. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, NS not significant, unpaired t-test.
Supplier Page from Proteintech Group Inc for LZTFL1 antibody