Journal: Bioengineered

Article Title: LINC01315 accelerates the growth and epithelial-mesenchymal transition of colorectal cancer cells via activating the Wnt/β-catenin signal

doi: 10.1080/21655979.2022.2044275

Figure Lengend Snippet: Deletion of LINC01315 inhibits CRC cells growth, invasion, and EMT. a. LINC01315 knockdown by shRNA (shLINC01315) was confirmed using qRT-PCR. b. CCK-8 assay showing proliferation of HCT116 and SW480 cancer cells following LINC01315 knockdown. c. Colony formation assays were performed to determine the proliferation of shLINC01315-transfected or shNC- transfected CRC cells. d. Wound-healing assay of LINC01315 knockdown HCT116 and SW480 cells. e. Transwell invasion assay analyzed the effect of LINC01315 knockdown on CRC cells invasion. f. Western blot assay was performed to test the expression of EMT markers (E-cadherin and N-cadherin) in the LINC01315 knockdown cells. ** P < 0.05 compared with shNC.

Article Snippet: An shRNA plasmid targeting LINC01315 (abbreviated as shLINC01315), and nonspecific shRNA (shNC) were synthesized by RiboBio (Guangzhou, China).

Techniques: Knockdown, shRNA, Quantitative RT-PCR, CCK-8 Assay, Transfection, Wound Healing Assay, Transwell Invasion Assay, Western Blot, Expressing

Journal: Bioengineered

Article Title: LINC01315 accelerates the growth and epithelial-mesenchymal transition of colorectal cancer cells via activating the Wnt/β-catenin signal

doi: 10.1080/21655979.2022.2044275

Figure Lengend Snippet: Overexpression of β-catenin reverses the effects of LINC01315 on CRC cells EMT. a. HCT116 and SW480 cells were co-transfected with shLINC01315 and pcDNA3.1-β-catenin. The expression of β-catenin was determined by Western blot. b. CCK-8 assay showing proliferation of HCT116 and SW480 cells. C . Colony formation assays were performed to determine the proliferation of shLINC01315 and pcDNA3.1-β-catenin co-transfected CRC cells. d. Wound-healing assay of HCT116 and SW480 cells. e. Transwell invasion assay analyzed CRC cells invasion. f. Western blot assay was performed to test the expression of EMT markers (E-cadherin and N-cadherin) in HCT116 and SW480 cells. ** P < 0.05 compared with shNC, ## P < 0.05 compared with shLINC01315.

Article Snippet: An shRNA plasmid targeting LINC01315 (abbreviated as shLINC01315), and nonspecific shRNA (shNC) were synthesized by RiboBio (Guangzhou, China).

Techniques: Over Expression, Transfection, Expressing, Western Blot, CCK-8 Assay, Wound Healing Assay, Transwell Invasion Assay

Journal: Scientific Reports

Article Title: The lncRNA BORG Drives Breast Cancer Metastasis and Disease Recurrence

doi: 10.1038/s41598-017-12716-6

Figure Lengend Snippet: BORG-deficiency inhibits the growth of metastatic D2.A1 cells. ( a ) qRT-PCR analysis of BORG expression in D2.A1 cells engineered to express either of two independent shRNAs against BORG. ( b ) shRNA-mediated knockdown of BORG inhibited growth of D2.A1 cells in 3D-cultures, as quantified by bioluminescent readings. Data are the mean (±SEM; * P < 0.05). ( c ) Representative photomicrographs of organoids formed by D2.A1 derivatives taken 5 days post plating. ( d ) shBORG2 significantly impaired the metastatic activity of D2.A1 cells in the lungs of BALB/c mice (mean ± SEM; * P < 0.05). ( e and f ) BORG-deficiency positively impacted the overall survival of mice inoculated with D2.A1 cells (log-rank test p values annotated ( e ); * P < 0.05; ** P < 0.0001 ( f )). ( g ) BORG expression levels are elevated in the resultant D2.A1 populations after their isolation from lung metastases and subsequent ex vivo culture (mean ± SEM; n.s. ≈ not significant). Notably, BORG expression levels in the ex vivo culture of D2.A1 shBORG2 metastases were significantly higher than expression levels in pre-inoculated D2.A1 shBORG2 cells ( a ).

Article Snippet: Cellular depletion of BORG in D2.A1 cells was achieved by VSVG lentiviral transduction of pGeneClip vectors containing a nonspecific shRNA sequence or those specific for BORG (Promega; Table ).

Techniques: Quantitative RT-PCR, Expressing, shRNA, Activity Assay, Isolation, Ex Vivo

Journal: Nature Communications

Article Title: Loss of heterozygosity of essential genes represents a widespread class of potential cancer vulnerabilities

doi: 10.1038/s41467-020-16399-y

Figure Lengend Snippet: a Schematic indicating allele-specific CRISPR approach. “Preexisting genome” represents individuals heterozygous for a germline SNP in a S. pyogenes Cas9 protospacer adjacent motif (PAM) site. A “G” allele (blue) in the PAM retains Cas9 activity at the target site, making this allele CRISPR-sensitive (S). An allele other than “G,” represented by “X” (red) abrogates Cas9 activity at the target site, making this allele CRISPR-resistant (R). Expression of an allele-specific (AS) CRISPR sgRNA targeting the polymorphic PAM site leads to specific inactivation of the S allele. b Schematic of PRIM1 SNP rs2277339 locus showing target sites for positive control, non-allele specific (NA) sgRNA and experimental, allele-specific (AS) sgRNA. Alleles appear in bold. c Crystal structure of PRIM1 gene product shows the amino acid encoded by rs2277339 (teal) lies on the surface of the primase catalytic subunit (gray) near a potentially small-molecule accessible location. d Immunoblot of PRIM1 protein levels in indicated patient-derived cell lines expressing LacZ, PRIM1 NA, or PRIM1 AS sgRNA ( n = 1 biological replicate). e Representative growth curves of indicated patient-derived cell lines expressing LacZ (black), PRIM1 NA (red), or PRIM1 AS (blue) sgRNA, as measured by CellTiter-Glo luminescence, relative to day of assay plating. n = 5 technical replicates. Data are presented as mean values ± s.d. See Supplementary Fig. for additional biological replicates. f Representative growth curves of indicated isogenic cell lines expressing LacZ (black), PRIM1 NA (red), or PRIM1 AS (blue) sgRNA, as measured by CellTiter-Glo luminescence, relative to day of assay plating. n = 5 technical replicates. Data are presented as mean values ± s.d. See Supplementary Fig. for additional biological replicates. g Disruption of PRIM1 in isogenic hemizygous PRIM1 resistant (PRIM1 R ) or PRIM1 sensitive (PRIM1 S ) cells expressing PRIM1 NA or AS sgRNA. Unaltered alleles (black), alleles with in-frame insertions or deletions (gray), and alleles with frameshift alterations (yellow) were assessed by deep sequencing of PRIM1 four days post-infection with sgRNA. Source data for Fig. 2d–g are provided as a Source Data file.

Article Snippet: Lentiviral expression constructs for non-allele specific shRNA-mediated suppression of PRIM1 were obtained through the Broad Institute of MIT and Harvard Genomic Perturbation Platform ( https://portals.broadinstitute.org/gpp/public/ ).

Techniques: CRISPR, Activity Assay, Expressing, Positive Control, Western Blot, Derivative Assay, Disruption, Sequencing, Infection

Journal: Nature Communications

Article Title: Loss of heterozygosity of essential genes represents a widespread class of potential cancer vulnerabilities

doi: 10.1038/s41467-020-16399-y

Figure Lengend Snippet: a Number of GEMINI variants (vertical axis) plotted against the number of patients per year in the US whose tumors might respond to therapeutics targeting those variants (i.e., have lost the resistant allele from a heterozygous germline; horizontal axis). Bin width = 1000 patients. b Growth of heterozygous (red circles) and hemizygous cells (pink circles) expressing positive control, non-allele specific PRIM1 -targeting shRNAs versus PRIM1 mRNA expression. Cell growth measured by CellTiter-Glo luminescence relative to day 2 post-infection and shGFP ( n = 5 technical replicates). PRIM1 mRNA expression assessed by qRT-PCR ( n = 3 technical replicates). Data are presented as mean values ± s.d. Dashed gray line indicates PRIM1 expression threshold below which substantial decreases in cell viability are observed. c Summary table representing challenges to developing allele-specific small molecules that target GEMINI vulnerabilities and associated analyses to prioritize targets. Source data for Fig. 4b is provided as a Source Data file.

Article Snippet: Lentiviral expression constructs for non-allele specific shRNA-mediated suppression of PRIM1 were obtained through the Broad Institute of MIT and Harvard Genomic Perturbation Platform ( https://portals.broadinstitute.org/gpp/public/ ).

Techniques: Expressing, Positive Control, Infection, Quantitative RT-PCR