Fig 2: LSM12 knockdown repressed the growth of CRC xenograft tumor. (A) The effect of LSM12 knockdown in SW480-Tet-On-shLSM12 cells on the xenograft tumor growth was monitored with the tumor volume under indicating time points. (B) The tumor weights at the end time point (35 days) was compared between LSM12 knockdown and control tumors. (C) LSM12 knockdown and Ki67 positive cell population were observed by immunohistochemical staining on xenograft tumor tissues. (D) The cleaved levels of Caspase-3 and -9 by LSM12 knockdown in xenograft tumors were assessed by Western blot. All experiments were independently conducted in triplicate technical repeats. Data shown are means ± SEM. *p < 0.05, **p < 0.01 or ***p < 0.001.

Fig 3: LSM12-mediated CTNNB1/TCF transcription is involved in the apoptosis of CRC cells. (A) Cell apoptosis by LSM12 knockdown in SW480 and HCT116 cells was determined by flow cytometry using Annexin V antibody and PI. (B) Cell apoptosis by LSM12 knockdown in SW480 and HCT116 cells was confirmed by TUNEL assay. The right graph indicates the quantification of TENEL-positive cells. (C) The cleaved levels of Caspase-3 and -9 by LSM12 knockdown in CRC cells were assessed by Western blot. (D) Cell apoptosis by ICG001 (10 µM) treatment in SW480 and HCT116 cells was determined by flow cytometry using Annexin V antibody and PI. (E) The cleaved levels of Caspase-3 and -9 by ICG001 treatment in CRC cells was assessed by Western blot. All experiments were independently conducted in triplicate technical repeats. Data shown are means ± SEM. *p < 0.05, **p < 0.01 or ***p < 0.001.

Fig 4: LSM12 expression in CRC specimens and cells. (A) The expression level of LSM12 mRNA in tumor (n = 275) and adjacent normal (n = 41) specimens from TCGA-COAD database, as well as in tumor (n = 92) and adjacent normal (n = 10) specimens from TCGA-READ database. *p < 0.05. (B) The expression level of LSM12 mRNA in CRC (n = 30) and adjacent normal (n = 30) specimens was revealed by qRT-PCR. ***p < 0.001. (C) The expression level of LSM12 protein in CRC (n = 3) and adjacent normal (n = 3) specimens was detected by Western blot. ***p < 0.001. Note: Wilcox test focused on the differences in medians, while the t-test focused on the differences in means. For the data in (C), three tissue samples were included for each group, so the mean was more representative than the median. Thus, the paired t-test was used for data comparison in (C). (D) Expression of LSM12 mRNA in CRC (SW620, SW480, HCT116, Caco-2, LS174T, HT-29) and normal (FHC) cell lines was determined by qRT-PCR. **p < 0.01. (E) The expression of LSM12 protein in CRC cell lines and normal cell lines was demonstrated by Western blot. **p < 0.01 or ***p < 0.001. All cell line-based experiments were independently conducted in triplicate technical repeats. Data shown are means ± SEM.

Fig 5: LSM12/CTNNB1 interaction activated WNT signaling. (A) Protein-Protein-Interaction (PPI) network construction of interacting proteins with LSM12 using GPS-Prot platform. (B) Interaction of LSM12 with CTNNB1 was confirmed by co-IP in Flag-LSM12 and Myc-CTNNB1 overexpressed 293T cells. (C) Endogenous interaction between LSM12 and CTNNB1 was validated by co-IP using LSM12 antibody in SW480 cells. (D) CTNNB1 protein degradation was detected by Western blot in SW480-Tet-On-shLSM12 and HCT116-Tet-On-shLSM12 cells with or without DOX (Doxycycline, 100 ng/mL) treatment following exposure to Cycloheximide (CHX,50 µg/mL) for the indicated times. The right graph indicates CTNNB1 protein degradation pattern and half-life time (t1/2). (E) Effect of LSM12 knockdown on the interaction between CTNNB1 and LEF1 was determined by co-IP in 293T-Tet-On-shLSM12 cells. (F) Effect of LSM12 knockdown on the interaction between CTNNB1 and TCF1 was validated by co-IP in 293T-Tet-On-shLSM12 cells. (G) Expression changes of WNT downstream mRNAs (cMYC, c-JUN, WISP1, PPARD) by LSM12 knockdown in SW480 and HCT116 cells were assessed by qPCR. (H) Expression changes of WNT downstream proteins (cMYC, c-JUN, WISP1, PPARD) by LSM12 knockdown in SW480 and HCT116 cells were confirmed by Western blot. All experiments were independently conducted in triplicate technical repeats. Data shown are means ± SEM. *p < 0.05 or **p < 0.01.