Fig 2: THBS1 is degraded by Staufen1 mRNA decay. (A) THBS1 promoter activity is not affected by Stau1-KD in H460 cells. (B,C) THBS1 transcript half-life is increased by Stau1 knock-down. (D) THBS1 transcript half-life is increased by UPF1 knock-down. (E) Stau1 expression is rescued in sh1 and sh2 H460 cell lines by a plasmid encoding a Stau1 mRNA resistant to sh1 and sh2 mRNA degradation (pStau155Res) when compared to empty vector (pcDNA3). (F) THBS1 mRNA levels are strongly reduced when Stau1 is re-expressed in sh1 and sh2 cells. (Asterisks: * p < 0.05; ** p < 0.01 and *** p < 0.001 in two-tailed Student’s t-test).

Fig 3: Stau1 binds to THBS1 mRNA. (A) RNA immunoprecipitation of THBS1 mRNA by Stau1, but not CDH2 mRNA (left). ANKRD57 and HPRT were used as a positive and a negative control, respectively. Western blot analysis was performed to control immunoprecipitation (right). (B) Deletion constructs of THBS1 3′UTR mRNA used to map the Staufen Binding Site (SBS). (C) Stau1 pull-down by the indicated portions of THBS1 3′UTR mRNA. (D) Prediction of the 547 nt sequence containing the SBS. (E) Precise mapping of Stau1 SBS in the “b” portion on the 547 nt fragment by RNA pull-down. (F) Mutagenesis confirmed that the “b” portion corresponds to the SBS as Stau1 did not bind to the 1-Mut construct, but 1-Res restored Stau1 binding.

Fig 4: Effect of Stau1 depletion on tumor formation. (A) Representative pictures of sh0, sh1 and sh2 cell lines in 2D culture. Stau1 depletion has no visible impact. (B) Representative pictures of sh0, sh1 and sh2 cell lines on tumor spheroid formation at days 4, 8 and 20. Stau1 depletion prevents the formation of compact spheroid tumors in sh1 and sh2 cell lines. (C) Cross-sections were performed on spheroids. Actin cytoskeleton was evaluated and nuclei stained with DAPI. (D) Colony assay formation. Colonies were stained with MTT (left). Quantification did not reveal any significant difference between all three cell lines (right). Stau1 does not interfere with H460 cells’ capacities to form colonies.

Fig 5: Stau1 depletion favors tumor progression and metastasis properties. (A) Tumor volumes measured at the indicated time points after subcutaneous injection of Stau1-deficient or control H460 cells into nude mice (statistic test: ANOVA + Turkey correction. sh0 vs. sh1 p = 0.0106; sh0 vs. sh2 p = 0.0244; sh1 vs. sh2 p = 0.965. * p < 0.05). Error bars show SEM. (B) Tumors were collected after 35 days and Stau1 mRNA level was controlled by RT-PCR after RNA extraction. (C,D) Tumor metastasis was evaluated by ddPCR by measuring human GAPDH mRNA levels in lung (C) and in liver (D). (Asterisks: * p < 0.05; ** p < 0.01 and *** p < 0.001 in two-tailed Student’s t-test).