Journal: Molecular Therapy. Nucleic Acids

Article Title: Protection of animals against devastating RNA viruses using CRISPR-Cas13s

doi: 10.1016/j.omtn.2024.102235

Figure Lengend Snippet: Schematic workflow of the used approach in this study to inhibit FMDV infection using the CRISPR-Cas system (1) Transmission and circulation of FMD disease between wildlife-livestock ecosystems due to animal interactions. (2) Finding conserved regions among different FMDV serotypes by considering multiple criteria and designing efficient and specific crRNAs by the CaSilico tool. (3) Designed crRNAs of type VI-A/B/D were synthesized as DNA oligos, and then, each crRNA was cloned into separate crRNA cloning backbones. In addition to crRNA designing, several shRNAs were designed to compare virus knockdown by RNAi and CRISPR-Cas systems. (4) Virus propagation and titration of two FMDV serotypes (O and A) were performed on BHK-21 cells to be used for viral challenge. (5) The sgRNA screening was performed to validate potent crRNAs. (6) Two methods, RT-qPCR and TCID50 assay were used to measure viral RNA and FMDV infectivity, respectively. (7) CRISPR-Cas systems can potentially be leveraged to aid antiviral development for viral diseases in animals. Figure was created with BioRender.com .

Article Snippet: To generate crRNAs and shRNAs, spacers, shRNAs, and their reverse complementary DNA oligomers were synthesized as single-stranded DNA (ssDNA) by GenScript Biotech.

Techniques: Infection, CRISPR, Transmission Assay, Synthesized, Clone Assay, Cloning, Virus, Knockdown, Titration, Quantitative RT-PCR, TCID50 Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Protection of animals against devastating RNA viruses using CRISPR-Cas13s

doi: 10.1016/j.omtn.2024.102235

Figure Lengend Snippet: Effect of Cas13 and RNAi on FMDV inhibition, serotype O (A) Experimental procedure for evaluating Cas13s and RNAi antiviral activity in BHK-21 cells. BHK-21 cells were transfected with plasmids containing targeting and non-targeting shRNAs or Cas13 and crRNAs that target or do not target FMDV 24 h before infection. Then the supernatants were collected 24 hpi to evaluate the antiviral activity. Silencing efficiency of Lwa Cas13a (B), Psp Cas13b (C), Rfx Cas13d (D), and RNAi system (E) on FMDV virus according to RT-qPCR results. In (B) to (E), data points in the graph are five independent biological experiments performed in technical replicates, lines indicate means; error bars represent SD; t test was performed to compare each treatment to non-targeting crRNA or shRNA (green and red color of the boxes indicate p < 0.05 and p < 0.01, respectively).

Article Snippet: To generate crRNAs and shRNAs, spacers, shRNAs, and their reverse complementary DNA oligomers were synthesized as single-stranded DNA (ssDNA) by GenScript Biotech.

Techniques: Inhibition, Activity Assay, Transfection, Infection, Virus, Quantitative RT-PCR, shRNA

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A. Expression of ERG, ESE1 and ESE3 determined by qRT-PCR in normal prostate and prostate tumors. Tumors are grouped according to the predominantly expressed ETS factor. B. Principal component analysis. Dots represent individual samples with their location determined by the principal components of the transcriptome. C. Number of differentially expressed genes with Q≤0.1 in each ETS subgroup. D-E. Venn diagrams showing shared and distinct differentially expressed genes among the indicated tumor subgroups.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A . Number of unique, similar and common features among the differentially expressed genes compared to normal prostate in ERG high , ESE3 low , ESE1 high and NoETS tumors according to Metacore. B. Commonly affected GeneGo Pathway Maps in the tumor subgroups. C. Differentially affected GeneGo Pathway Maps in ERG high , ESE3 low , ESE1 high and NoETS tumors.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques:

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A. EZH2 level in normal prostate, ESE3 low and NoETS tumors. B. EZH2 level in NoETS and ESE3 low tumors in Wallace et al. microarray dataset. C. EZH2 level in control (sh-) and stable ESE3 knock-down (sh 4, 6, 7) LNCaP clones determined by RT-PCR. D. EZH2 level in control (sh-) and ESE3 knock-down (sh17, 28) LHS clones determined by RT-PCR. E. Nkx3.1 level in normal prostate, ESE3 low and NoETS tumors. F. Nkx3.1 level in control (sh-) and ESE3-knock-down LNCaP and LHS cells determined by RT-PCR and Western blot ( right bottom ). G. Nkx3.1 promoter activity in PC3 cells transfected with human NKx3.1 promoter reporter along with the indicated expression vectors. Luciferase reporter activity was measured after 24 h. *p<0.01; **, p<0.001.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Microarray, Control, Knockdown, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Transfection, Expressing, Luciferase

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A. RNA was extracted from control (sh-) and ESE3 knock-down (sh7) LNCaP cells 48 h post-transfection with siEZH2 and control siRNA and analyzed by RT-PCR. B. H3K27 methylation was assessed in sh- and sh7 LNCaP cells by ChIP and qPCR with primers encompassing the EBS and ARE in the Nkx3.1 gene. ETS2 was used as negative control C. ESE3 binding to the EZH2 and Nkx3.1 promoter was assessed by ChIP and qPCR in sh- and sh7 LNCaP cells. ETS2 was used as negative control . D. ESE3 binding to the EZH2 and Nkx3.1 promoter in control and ERG-expressing (ERG-18) LNCaP cells assessed by ChIP and qPCR. ETS2 was used as negative control . *, p<0.01.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Control, Knockdown, Transfection, Reverse Transcription Polymerase Chain Reaction, Methylation, Negative Control, Binding Assay, Expressing

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: A. ERG over-expression and ESE3 knock-down (B) enhance cell migration. Cells were grown until confluence and starved for 24 h when a scratch was performed on the monolayer. Pictures were taken at 0 and 72 h. Representative photographs of triplicate experiments are shown. C. Control and ERG over-expressing LNCaP cell clones were plated in polyhema coated 96-well plates and cell viability was measured using a colorimetric assay at the indicated time points. D. Control and ESE3 knock-down LNCaP cells were plated in polyhema and assayed as described above. E. VCaP cells were transfected with siRNAs against ERG or EZH2 and plated in polyhema after 24 h. Cell viability was measured as described above. F. Parental and ERG-expressing (ERG-18) LNCaP cells were transfected with siRNAs against EZH2 and assayed as indicated above. G. Parental and ERG-expressing (ERG-18) LNCaP cells were transfected with full length Nkx3.1 expression vector and assayed as indicated above. H. Parental (sh-) and ESE3 knock-down (sh-7) LNCaP cells were transfected with full length Nkx3.1 expression vector and assayed as indicated above. Data are presented as mean ± SD of triplicate experiments. *, p<0.01; **, p<0.001.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Over Expression, Knockdown, Migration, Control, Expressing, Clone Assay, Colorimetric Assay, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

doi: 10.1371/journal.pone.0010547

Figure Lengend Snippet: ESE3 controls expression of EZH2 and Nkx3.1 in normal prostate epithelial cells. ERG over-expression or loss of ESE3 leads to abnormal expression of these key genes and promotes cell transformation.

Article Snippet: To establish stable ESE3 knock-down cell lines, cells were transfected with ESE3 targeting shRNAs (Cat. no. KH14264N SuperArray, Frederick, MD, USA) using Lipofectamine 2000 and selected with G418.

Techniques: Expressing, Over Expression, Transformation Assay

Journal: The Journal of Biological Chemistry

Article Title: The kinesin-14 family motor protein KIFC2 promotes prostate cancer progression by regulating p65

doi: 10.1016/j.jbc.2023.105253

Figure Lengend Snippet: KIFC2 expression is related to enzalutamide-resistance PCa and CRPC. A , the Venn diagram gives the overlapped genes of high expression related to enzalutamide-resistance and CRPC based on three GEO datasets. B , the expression of KIFC2 and that in comparison among enzalutamide-treated and DMSO-treated cells in GSE69249 dataset. C and D , KIFC2 expression of CRPC and primary tumor in the GSE70770 dataset and GSE70768 dataset. Data are mean ± SD (n = 3). CRPC, castration-resistance prostate cancer; DEGs, differentially expressed genes; Enza, enzalutamide; KIFC2, kinesin family member C2; PCa, prostate cancer.

Article Snippet: Two siRNAs targeting KIFC2 were designed and purchased from GenePharma Technology.

Techniques: Expressing, Comparison

Journal: The Journal of Biological Chemistry

Article Title: The kinesin-14 family motor protein KIFC2 promotes prostate cancer progression by regulating p65

doi: 10.1016/j.jbc.2023.105253

Figure Lengend Snippet: KIFC2 was highly expressed in prostate cancer and associated with a poor prognosis. A , KIFC2 mRNA expression was investigated between PCa tissues and normal prostate tissues in TCGA database. B , comparison of KIFC2 expression levels between N classifications from TCGA database . C and D , TCGA database showed KIFC2 expression in PCa and correlated with Gleason scores. E , TCGA database showed KIFC2 expression in PCa and correlated with lymph nodes metastasis. F , survival curve was analyzed based on TCGA database. G , KIFC2 protein expression in PCa and normal tissues was detected via Western blot. H , representative IHC images for KIFC2 expression levels in PCa and normal prostate tissues. Data are mean ± SD (n = 3). ∗∗ p < 0.01. IHC, immunohistochemistry; KIFC2, kinesin family member C2; PCa, prostate cancer; TCGA, The Cancer Genome Atlas.

Article Snippet: Two siRNAs targeting KIFC2 were designed and purchased from GenePharma Technology.

Techniques: Expressing, Comparison, Western Blot, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: The kinesin-14 family motor protein KIFC2 promotes prostate cancer progression by regulating p65

doi: 10.1016/j.jbc.2023.105253

Figure Lengend Snippet: Correlation between KIFC2 expression and clinicopathological characteristics of PCa

Article Snippet: Two siRNAs targeting KIFC2 were designed and purchased from GenePharma Technology.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: The kinesin-14 family motor protein KIFC2 promotes prostate cancer progression by regulating p65

doi: 10.1016/j.jbc.2023.105253

Figure Lengend Snippet: KIFC2 promotes proliferation and migration of prostate cancer cells. A , Western blot and qRT-PCR analysis of KIFC2 knockdown efficiencies in PCa cells. B – D , the proliferation capabilities of PCa cells under KIFC2 knockdown were determined via CCK8 and colony-formation assays. E and F , representative images of wound-healing assays are presented. G and H , representative images of Transwell migration assays are presented. Data are mean ± SD (n = 3). ∗∗∗ p < 0.001. CCK8, cell counting kit-8; KIFC2, kinesin family member C2; PCa, prostate cancer.

Article Snippet: Two siRNAs targeting KIFC2 were designed and purchased from GenePharma Technology.

Techniques: Migration, Western Blot, Quantitative RT-PCR, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: The kinesin-14 family motor protein KIFC2 promotes prostate cancer progression by regulating p65

doi: 10.1016/j.jbc.2023.105253

Figure Lengend Snippet: KIFC2 influences the sensitivity of PCa cells to enzalutamide. A , the protein expression of KIFC2 in LNCaP-P/EnzaR and 22Rv1-P/EnzaR cells. B and C , CCK8 assays were used to measure the viability of the PCa cells treated with different concentrations of Enza for 24 h. D and E , CCK8 assays were used to assess cell viabilities of PCa cells after Enza intervention (15 μM) and/or siKIFC2 treatment. F and G , colony-formation assays were used to assess cell growth of PCa cells after Enza intervention and/or siKIFC2 treatment. Data are mean ± SD (n = 3). ∗∗ p < 0.01, ∗∗∗ p < 0.001. C2; Enza, enzalutamide; CCK8, cell counting kit-8; PCa, prostate cancer; KIFC2, kinesin family member.

Article Snippet: Two siRNAs targeting KIFC2 were designed and purchased from GenePharma Technology.

Techniques: Expressing, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: The kinesin-14 family motor protein KIFC2 promotes prostate cancer progression by regulating p65

doi: 10.1016/j.jbc.2023.105253

Figure Lengend Snippet: KIFC2 regulates NF-κB pathway and promotes p65 protein expression. A , the differentially expressed genes from TCGA PRAD dataset was presented. B , the KEGG pathways for differentially expressed genes were presented. C , GSEA analysis of NF-κB pathway. D , the overexpression efficiencies of KIFC2 were assessed via Western blot. E , the total protein and phosphorylation levels of p65 were assessed via Western blot in KIFC2-knockdown or -overexpression PCa cells. F , correlation analysis between KIFC2 and p65 was conducted based on TCGA database. G , qRT-PCR was used to determine the target genes expression of NF-κB pathway. H , correlation analysis between KIFC2 and target genes of NF-κB pathway was conducted based on TCGA database. I , the nuclear or cytoplasmic protein levels of p65 were assessed via Western blot. J , IF analysis of the nuclear levels of the p65 in DU145 cell transfected with siNC or siKIFC2. Data are mean ± SD (n = 3). ∗∗∗ p < 0.001. KIFC2, kinesin family member C2; TCGA, The Cancer Genome Atlas.

Article Snippet: Two siRNAs targeting KIFC2 were designed and purchased from GenePharma Technology.

Techniques: Expressing, Over Expression, Western Blot, Quantitative RT-PCR, Transfection

Journal: The Journal of Biological Chemistry

Article Title: The kinesin-14 family motor protein KIFC2 promotes prostate cancer progression by regulating p65

doi: 10.1016/j.jbc.2023.105253

Figure Lengend Snippet: KIFC2 modulates PCa progression and enzalutamide resistance via NF-κB pathway. A and B , CCK8 assays showed that NF-κB pathway inhibitor GSK583 reversed KIFC2-induced promotion of proliferation in PCa cells. C – E , wound-healing and Transwell migration assays showed that GSK583 reversed KIFC2-induced promotion of migration in PCa cells. F , colony formation assays showed that GSK583 reversed KIFC2-induced enhancement of resistance to enzalutamide. Data are mean ± SD (n = 3). ∗∗ p < 0.01, ∗∗∗ p < 0.001. CCK8, cell counting kit-8; KIFC2, kinesin family member C2; PCa, prostate cancer.

Article Snippet: Two siRNAs targeting KIFC2 were designed and purchased from GenePharma Technology.

Techniques: Migration, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: The kinesin-14 family motor protein KIFC2 promotes prostate cancer progression by regulating p65

doi: 10.1016/j.jbc.2023.105253

Figure Lengend Snippet: KIFC2 depletion inhibits PCa cell growth in vivo . A , PC3 cells transfected with shKIFC2 with or without GSK583 treatment. B , the sizes of tumor xenografts were measured every 7 days. C , the tumor masses were harvested from nude mice at the experimental endpoint. D , KIFC2, p65, and Ki-67 expression levels in tumor tissues were measured via IHC analysis. Data are mean ± SD (n = 3). ∗∗ p < 0.01, ∗∗∗ p < 0.001. IHC, immunohistochemistry; KIFC2, kinesin family member C2; PCa, prostate cancer.

Article Snippet: Two siRNAs targeting KIFC2 were designed and purchased from GenePharma Technology.

Techniques: In Vivo, Transfection, Expressing, Immunohistochemistry