Fig 2: The changes of miRNA expression profiles caused by SP1 knockdown. This study knocked down SP1 in MG63 and 143B cells and then performed Microarray assay to find the expression of miRNAs that were influenced by SP1. A, Heatmap of Microarray assay. B, SP1 expression level in MG63 and 143B cells was assessed by PCR assay after transfecting SP1‐siRNA. C, Venn diagram shows the 18 up‐regulated miRNAs and 20 down‐regulated miRNAs in both MG63 and 143B cells after SP1 knockdown. D, Volcano plots show the significantly changed miRNAs in MG63 and 143B cells after SP1 knockdown. The expression of miR‐326 in osteosarcoma tissues and the matched adjacent normal tissues (E) as well as in OS cell lines and a normal osteoblastic cell line (F). U6 and GAPDH were employed as miRNA and mRNA internal control, respectively. KD means knockdown; T means tumour tissues; N means matched adjacent normal tissues. *P < .05, **P < .01 vs hFOB1.19 cells

Fig 3: WWP1 localizes to the Crumbs polarity complex via AMOTL2.(A) MCF10A cells transfected with GFP-WWP1 WT or ΔWW were immunostained for GFP and PALS1, and serial z-stack images along the x-plane were overlaid by orthogonal projection. Green, GFP; red, PALS1; blue, Hoescht. Scale bar, 50 μm. (B) 293T cells were transfected with the indicated combination of DNAs, then subjected to SBP pull-down assays using streptavidin agarose beads followed by Western blot analysis. (C) 293T cells were transfected with the indicated combination of DNAs, then subjected to SBP pull-down assays using streptavidin agarose beads followed by Western blot analysis. (D) 293T cells were transfected with the indicated combination of DNAs, then subjected to SBP pull-down assays using streptavidin agarose beads followed by Western blot analysis. (E) 293T cells were stably transduced with either control or Crumbs3 shRNAs, then transfected with the indicated DNAs. The resulting extracts were subjected to in vivo ubiquitination assays, followed by Western blot analysis. (F) mRNAs isolated from MCF10A cells stably transduced with control or Crumbs3 shRNAs, then re-seeded at a sparse or confluent density were analyzed for the indicated genes by qRT-PCR, and expression levels were normalized to those of GAPDH mRNA (n = 4). Data are expressed as means ± SEM (error bars; *P < 0.05, ***P < 0.001, n.s. not significant; unpaired t test).

Fig 4: A miR-29c-3p inhibitor abrogates the protective effects of sh-circ against MPTP-induced cell apoptosis. (A) Expression levels of circSAMD4A and miR-29c-3p in sh-circ, shNC, miR-29c-3p NC or miR-29c-3p inhibitor-injected MPTP-induced mice were measured via reverse transcription-quantitative PCR. (B) Western blotting was used to determine protein expression levels of caspase 3, Bcl2 and Bax in transfected mice. GAPDH or U6 were used as an internal control. (C) Apoptotic cells in treated mice were analyzed using the TUNEL assay. *P<0.05, **P<0.01, ***P<0.001. sh-circ, sh-circSAMD4A; miR, microRNA; circSAMD4A, circular RNA sterile α motif domain containing 4A; sh, short hairpin RNA; NC, negative control; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig 5: Promethazine reduces DPR protein accumulation and toxicity Left panel, immunoblotting against the HA‐tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 h with a construct expressing 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA in the GA frame and treated with 10 μM of the indicated compound for 15 h. Right panel, quantification of polyGA expression relative to the GAPDH.Left panel, immunoblotting against the HA‐tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 h with a construct expressing 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA‐tag in the PG frame and treated with 10 μM of the indicated compound for 15 h. Right panel, quantification of polyPG expression relative to the GAPDH.Cell viability (TO‐PRO‐3 FACS staining) of GT1‐7 neuronal cells treated with 1, 3, or 10 μM of promethazine and co‐transfected for 24 h with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame.Data information: Error bars indicate s.e.m. Student's t‐test, **P < 0.01, and ***P < 0.001. n = 5 independent transfection.