Journal: Cancer Discovery

Article Title: MARK2/MARK3 Kinases Are Catalytic Codependencies of YAP/TAZ in Human Cancer

doi: 10.1158/2159-8290.CD-23-1529

Figure Lengend Snippet: MARK2/3 dependency in cancer is linked to the maintenance of YAP/TAZ function. A, mRNA expression differences comparing 19 MARK2/3-dependent with 12 MARK2/3-independent human cancer cell lines. Transcriptome data were obtained from the CCLE database, KLM1 (GSE140484), and CHL1 (this article). TPM were calculated and the difference in log 2 (TPM + 1) was plotted. P values were calculated using empirical Bayes statistics (eBayes) for differential expression with Holm–Bonferroni (BH) correction. B, Heatmap of MARK2/3-dependent and MARK2/3-independent cancer cell lines showing dependence on YAP/TAZ and expression of target genes. Competition-based fitness assays in Cas9-expressing cancer cells after lentiviral knockout of indicated genes (expression of dgRNAs was linked with GFP). Heatmap color indicates the LFC of %GFP + (normalized to day 3 or 6 after infection). n = 3. C, Crystal violet stain of indicated cells following lentiviral knockout of indicated genes. Data shown are representative of three independent biological replicates. D, Flow cytometry histogram of YAP/TAZ:TEAD reporter assay in MDA-MB-231 cells, on day 9 after infection. Data are representative of three independent experiments. E, Gene set enrichment analysis (GSEA) of Cas9 + MDA-MB-231 cancer cells after MARK2+3 dKO , including normalized enrichment score (NES) and P value. F, Heatmap showing the GSEA NES for the YAP/TAZ gene signature following MARK2+3 dKO in dependent and independent cell lines. G, Heatmap of mRNA expression [log 2 (normalized count)] z -scores in Cas9 + MDA-MB-231 cells of genes significantly downregulated or upregulated upon MARK2+3 dKO . Expression values of downregulated genes ( n = 188) and upregulated genes ( n = 91) of two replicate samples after gene knockout were grouped based on unsupervised clustering. Significant differentially expressed genes were defined as adjusted P value < 10 −4 and LFC >2 or <−1. P values from Wald test (DEseq2) adjusted using BH. H, CUT&RUN density profile of YAP:TEAD4−bound, YAP/TAZ dKO -sensitive H3K27ac marked enhancer loci ( n = 7,896) after MARK2+3 dKO . Profiles shown are an average of 50 bp bins around the summit of the enhancers. I, Occupancy profiles of public chromatin immunoprecipitation sequencing (ChIP-seq; TEAD4, YAP; GSE66083) and CUT&RUN (H3K27ac) upon indicated gene knockout at YAP/TAZ target gene loci.

Article Snippet: The HPAF-II, AsPC1, PANC1, MIA PaCa2, NCI-H1299, A549, NCI-H23, RD, MDA-MB-231, NCI-H1048, NCI-H211, NCI-H209, NCI-H1836, NCI-H1436, NCI-H2023, NCI-H1975, NCI-H1703, CHL1, OCI-AML3, THP1, HEK293T, and K562 were purchased from the ATCC.

Techniques: Expressing, Quantitative Proteomics, Knock-Out, Infection, Staining, Flow Cytometry, Reporter Assay, Gene Knockout, ChIP-sequencing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Up-regulation of miR-10a-5p expression inhibits the proliferation and differentiation of neural stem cells by targeting Chl1

doi: 10.3724/abbs.2024078

Figure Lengend Snippet: Chl1 is a direct target of miR-10a-5p (A) The public miRNA databases (TargetScan) predict that Chl1 may be a target for miR-10a-5p and that the 3′-UTR of Chl1 mRNA contains a highly conserved and complementary sequence between positions 2815 and 2821 for the binding of the seed sequence of miR-10a-5p. (B) Cells were transfected with pMIR-Chl1-WT, pMIR-Chl1-MUT or miR-10a-5p positive control (PC) reporter plasmids together with miR-10a-5p mimics or negative controls. The levels of firefly and Renilla luciferase activities were assayed 24 and 48 h later. (C) Relative expression of Chl1 mRNA was measured by RT-qPCR using β-actin as an internal control in NTD tissues and normal tissues (NCs) at E8.5, E9.5 and E10.5. (D) The expression of the Chl1 protein in NTD and NC tissues at E9.5 and E10.5 was measured by western blot analysis. (E) Representative micrographs of immunofluorescence staining for Chl1 (red) with nuclei stained blue with DAPI. Areas of Chl1-positive cells were quantified by using ImageJ. (F) The expression of Chl1 mRNA was measured by RT-qPCR using β-actin as an internal control in NSCs after transfection with LV-miR-10a or LV-NC for 48 h (left). Relative Chl1 protein expression in NSCs after transfection with LV-miR-10a or LV-NC for 72 h was measured by western blot analysis using GAPDH as an internal control (right). Scale bar: 50 μm. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: NSCs were injected with either 40 moi LV-miR-10a or LV-NC and AV-Chl1 or control viruses (AV-NC). siRNA against Chl1 (siR-Chl1) (siRNA-1: 5′-CCACATCTCTCACTTTCAA-3′; siRNA-2: 5′-GCTACAAAGCTCAGAGTTT-3′; siRNA-3: 5′-GGTCCAAGCCATCAATCAA-3′) and siR-NC (Cat No. siN0000001-1-5; https://www.ribobio.com/product/siN0000001-1-5/ ) were synthesized by RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Sequencing, Binding Assay, Transfection, Positive Control, Luciferase, Expressing, Quantitative RT-PCR, Control, Western Blot, Immunofluorescence, Staining

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Up-regulation of miR-10a-5p expression inhibits the proliferation and differentiation of neural stem cells by targeting Chl1

doi: 10.3724/abbs.2024078

Figure Lengend Snippet: Effects of Chl1 knockdown on cell proliferation and differentiation (A) The expressions of Chl1 mRNA and protein in NSCs were measured by RT-qPCR (left) and western blot (right) analysis after transfection with siR-NC, siR1-Chl1, siR2-Chl1 or siR3-Chl1 for 72 h. (B) Representative micrographs of immunofluorescence staining for BrdU (red) with nuclei stained blue with DAPI. Arrows indicate BrdU+ cells. The percentage of BrdU+ cells was significantly lower in the siR1-Chl1 group than in the blank or siR-NC groups. (C) FCM results of the cell cycle analysis are shown. The percentage of cells in the G1 phase was significantly greater and the percentage of cells in the S and G2 phases was significantly lower in the siR1-Chl1 group than in the blank or siR-NC groups. (D) Representative micrographs of immunofluorescence staining for GFAP (red) with nuclei stained blue with DAPI. The percentage of GFAP+ cells was significantly lower in the siR1-CHL1 group than in the blank or siR-NC groups. Scale bar: 50 μm. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: NSCs were injected with either 40 moi LV-miR-10a or LV-NC and AV-Chl1 or control viruses (AV-NC). siRNA against Chl1 (siR-Chl1) (siRNA-1: 5′-CCACATCTCTCACTTTCAA-3′; siRNA-2: 5′-GCTACAAAGCTCAGAGTTT-3′; siRNA-3: 5′-GGTCCAAGCCATCAATCAA-3′) and siR-NC (Cat No. siN0000001-1-5; https://www.ribobio.com/product/siN0000001-1-5/ ) were synthesized by RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Transfection, Immunofluorescence, Staining, Cell Cycle Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Up-regulation of miR-10a-5p expression inhibits the proliferation and differentiation of neural stem cells by targeting Chl1

doi: 10.3724/abbs.2024078

Figure Lengend Snippet: Re-expression of Chl1 reverses the effect of miR-10a-5p on proliferation and differentiation of NSCs (A) Representative micrographs of GFP expression in NSCs after transfection with AV-NC or AV-Chl1 for 72 h. (B) The expression of Chl1 protein in NSCs after transfection (AV-NC or AV-Chl1) or cotransfection (LV-miR-10a or LV-NC) for 72 h was measured by western blot analysis. (C) Representative micrographs of immunofluorescence staining for BrdU (red) with nuclei stained blue with DAPI. Arrows indicate BrdU+ cells. The percentage of BrdU+ cells was significantly greater in the LV-miR-10a+AV-Chl1 group than in the LV-miR-10a+AV-NC group. The miR-10a-5p-induced proliferation of NSCs was restored via Chl1 re-expression. (D) Representative micrographs of immunofluorescence staining for GFAP (red) with nuclei stained blue with DAPI. The percentage of GFAP+ cells was significantly greater in the LV-miR-10a+AV-Chl1 group than in the LV-miR-10a+AV-NC group. The miR-10a-5p-induced differentiation of NSCs was restored by Chl1 re-expression. Scale bar: 50 μm. *P<0.05, **P<0.01; ***P<0.001.

Article Snippet: NSCs were injected with either 40 moi LV-miR-10a or LV-NC and AV-Chl1 or control viruses (AV-NC). siRNA against Chl1 (siR-Chl1) (siRNA-1: 5′-CCACATCTCTCACTTTCAA-3′; siRNA-2: 5′-GCTACAAAGCTCAGAGTTT-3′; siRNA-3: 5′-GGTCCAAGCCATCAATCAA-3′) and siR-NC (Cat No. siN0000001-1-5; https://www.ribobio.com/product/siN0000001-1-5/ ) were synthesized by RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Expressing, Transfection, Cotransfection, Western Blot, Immunofluorescence, Staining

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Up-regulation of miR-10a-5p expression inhibits the proliferation and differentiation of neural stem cells by targeting Chl1

doi: 10.3724/abbs.2024078

Figure Lengend Snippet: The ERK agonist Honokiol reverses the effects of miR-10a-5p on proliferation and differentiation of NSCs (A) The protein expression levels of p-SRC, SRC, p-PI3K, PI3K, p-ERK and ERK were determined by western blot analysis in NSCs after transfection with siR-NC or siR1-Chl1 for 72 h. (B) The expression levels of p-ERK and ERK proteins were measured by western blot analysis in NSCs after treatment with DMSO or Honokiol and cotransfection with LV-miR-10a or LV-NC for 72 h. (C) Representative micrographs of immunofluorescence staining for BrdU (red) with nuclei stained blue with DAPI. Arrows indicate BrdU+ cells. The percentage of BrdU+ cells was significantly greater in the LV-miR-10a+Honokiol group than in the LV-miR-10a+DMSO group. The miR-10a-5p-induced proliferation of NSCs was restored by Honokiol. (D) Representative micrographs of immunofluorescence staining for GFAP (red) with nuclei stained blue with DAPI. The percentage of GFAP+ cells was significantly greater in the LV-miR-10a+Honokiol group than in the LV-miR-10a+DMSO group. The miR-10a-5p-induced differentiation of NSCs was reversed by Honokiol. Scale bar: 50 μm. **P<0.01; ***P<0.001.

Article Snippet: NSCs were injected with either 40 moi LV-miR-10a or LV-NC and AV-Chl1 or control viruses (AV-NC). siRNA against Chl1 (siR-Chl1) (siRNA-1: 5′-CCACATCTCTCACTTTCAA-3′; siRNA-2: 5′-GCTACAAAGCTCAGAGTTT-3′; siRNA-3: 5′-GGTCCAAGCCATCAATCAA-3′) and siR-NC (Cat No. siN0000001-1-5; https://www.ribobio.com/product/siN0000001-1-5/ ) were synthesized by RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Expressing, Western Blot, Transfection, Cotransfection, Immunofluorescence, Staining