PMC

Uch37 interacts with Tcf7 and removes polyubiquitin chain from Tcf7 protein. As a result, Tcf7 stably binds target DNA for gene transcription. However, absence of Uch37 promotes polyubiquitination on Tcf7 protein, causing transcriptional silence.

A, B Western blot results of TCF7 and c-Myc protein expression by lncRNA02Rik overexpression. C, D mRNA levels of TCF7 and c-Myc by lncRNA02Rik overexpression. E, F Western blot results of TCF7 and c-Myc protein expression by siA02Rik. G, H mRNA levels of TCF7 and c-Myc by siA02Rik. I, J Western blot results of TCF7 and c-Myc protein expression by shA02Rik in vitro. K, L mRNA levels of TCF7 and c-Myc by shA02Rik in vitro. M, N Western blot results of TCF7 and c-Myc protein expression by shA02Rik in vivo. O, P mRNA levels of TCF7 and c-Myc by shA02Rik in vivo. *P < 0.05 vs. Sham/Control group, ^#P < 0.05 vs. TAC + N.C/Ang-II/lncRNAA02Rik group, ^@P < 0.05 vs. Ang-II + siA02Rik/Ang-II + shA02Rik/TAC + shA02Rik group, n = 6.

(A) Nanog interacts with Tcf7 through its N terminal. Different Myc-tagged Nanog were constructed and co-transfection with HA-Tcf7 in HEK293T cells. Among all the mutated types of Nanog, only the N-terminal truncated Nanog (Nanog-ΔN) could not coprecipitate with Tcf7, indicating that Nanog physically interacts with Tcf7 through its N terminal. (B) Tcf7 interacts with Nanog through its GroBD. Different Myc-tagged Tcf7 were constructed and co-transfection with HA-Nanog in HEK293T cells. Among all the mutated types of Tcf7, only the GroBD deleted Tcf7 (Tcf7_ΔGroBD) could not coprecipitate with Nanog, indicating that Tcf7 binds with Nanog through GroBD. (C) Nanog and β-catenin competitively binds with Tcf7. Co-transfection of increasing amount of Nanog decreases the interaction between β-catenin and Tcf7 in a dose-dependent manner. When increased amount of Nanog was transfected into Tcf7 and β-catenin co-transfected cells, decreased amount of β-catenin could be coprecipitated. The molecular weight of HA-Nanog is around 55 Kda, and HA-β-catenin is around 100 KDa, so we could distinguish the 2 anti-HA bands by different protein sizes. (D) Co-transfection of Ctnnbip1 increased the binding affinity between Nanog and Tcf7. Two different amounts of HA-ctnnbip1 were co-transfected with Tcf7, Nanog and β-catenin in HEK293T cells; because Ctnnbip1 was overexpressed, an increased amount of Nanog was coprecipitated by myc-Tcf7. The molecular weight of HA-ctnnbip1 is around 10 KDa. Note that HA-β-catenin level was reduced when Ctnnbip1 was overexpressed. (E) Co-immunoprecipitation assay showed that increased amount of endogenous β-catenin could interact with Tcf7 in MZnanog mutants compared with WT embryos. Embryos were collected at 4 hpf. (F) The intensity ratio of β-catenin to Tcf7 in panel E. Error bars, mean ± SD, *P < 0.05. The P values in this figure were calculated by Student t test. The underlying data in this figure can be found in . GroBD, Groucho binding domain; HEK293T cells, human embryonic kidney 293T cells; MZnanog, maternal -zygotic mutant of nanog; hpf, hours post fertilization; WT, wild type.

Requirement of TCF7 for MSCs. (A) Silencing or rescue of TCF7 in MSCs. MSCs were transfected with TCF7-shRNA to knock down the expression of the internal TCF7. As a control, shRNA-scrambled was included in the transfection. To rescue the expression of TCF7 in the TCF7-silenced cells, the cells were co-transfected with TCF7-shRNA and the plasmid expressing TCF7 (TCF7-rescue). The endogenous TCF7 was silenced by TCF7-shRNA in MSCs, while the plasmid expressed the recombinant TCF7 protein. At 48 h after transfection, TCF7 was examined using quantitative real-time PCR and Western blot (**, p < 0.01). β-tubulin was used as a control. (B) Impact of TCF7 knockdown and rescue on the proliferation of MSCs. The cell viability was examined at different times after transfection. ShRNA-scrambled was used as a control (*, p < 0.05; **, p < 0.01). (C) Role of TCF7 in cell cycle. The TCF7-silenced or rescued MSCs were subjected to the examination of cell cycle at 48 h after treatment (**, p < 0.01). (D) Influence of TCF7 knockdown and rescue on apoptosis of MSCs. The TCF7-silenced or rescued MSCs were subjected to caspase 3/7 activity assay at 48 h after transfection (**, p < 0.01). (E) Determination of apoptosis using Annexin V assays. Apoptosis of MSCs was detected by flow cytometry at 48 h after transfection with TCF7-shRNA, TCF7-rescue or shRNA-scrambled (**, p < 0.01). (F) Tumorsphere formation assay of TCF7-silenced or rescued MSCs. At 10 days after transfection, the percentage of tumorsphere formation of MSCs was evaluated (**, p < 0.01). Scale bar, 20 μm. (G) Effects of TCF7 on the expressions of stemness genes. The expressions of stemness genes in MSCs were examined using quantitative real-time PCR and Western blot at 48 h after the transfection (*, p < 0.05; **, p < 0.01). (H) Expression level of TCF7 in cancerous tissues of melanoma patients. According to the GEPIA database (http://gepia.cancer-pku.cn/index.html), the expression of TCF7 from melanoma patients in the cancerous tissues and normal tissues was estimated (*, p < 0.05). (I) Kaplan-Meier survival analysis. The Kaplan-Meier curve showed the survival curves of melanoma patients with low vs. high expression level of TCF7. (J) Expression level of LINC006987, miR-3132 and TCF7 in MSCs after transfection of vector alone, LINC00698 or LINC00698 and miR-3132 mimic. Quantitative real-time PCR was used to examine the expression levels of LINC006987, miR-3132 and TCF7 at 48 h after transfection (*, p < 0.05; **, p < 0.01). (K) Expression level of TCF7 detected by Western blot analysis. The TCF7 protein level in MSCs was detected at 48 h after transfection with vector alone, LINC00698 or LINC00698 and miR-3132 mimic (*, p < 0.05; **, p < 0.01)