Journal: Biomedicines

Article Title: Whole Exome Sequencing Identifies PHF14 Mutations in Neurocytoma and Predicts Responsivity to the PDGFR Inhibitor Sunitinib

doi: 10.3390/biomedicines10112842

Figure Lengend Snippet: PHF14 Depletion Enhances Cell Proliferation and Anchorage Independent Cell Growth. ( A ) A conserved 19-bp region in the human, mouse and rat PHF14 gene in Exon 10 (CGC ATG ATT CAA ATT CAG GAA) was selected as shRNA target to knockdown PHF14 expression. Five cell lines originating from human, mouse and rat were used to evaluate the biological effect of PHF14 depletion. PHF14 knockdown stable transfectants were established by puromycin selection, and the knockdown efficiency was determined by Western Blot using anti-PHF14 antibody. The densitometric analyses of the protein bands vs. the individual loading controls were shown under individual blot using the ImageQuant 5.2 software (GE Healthcare, Pittsburgh, PA). ( B ) Cell proliferation rate was enhanced in shRNA PHF4 knockdown transfectants compared to Nonsense controls as measured by CellTiter-Glo ® luminescent cell viability assay in a variety of cell lines. ( C ) Soft agar assay demonstrated that PHF14 knockdown induced an increase in colony size in human neuroblastoma SHSY-5Y cells. ( D ) A single guiding RNA (sgRNA) targeting a 20-bp region (TGG ATC GCA GCT CCA AGA GG) in PHF14 Exon 1 was designed for CRISPR-Cas9 mediated genetic editing. The knockout efficiency was confirmed by Western Blot. PHF14 knockout in human neuroblastoma SHSY-5Y cells promoted cell proliferation as measured by CellTiter-Glo ® luminescent cell viability assay. PHF14 knockout increased colony formation in soft agar assay. The results shown were representative of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: ShRNA PHF14 (TRCN0000312505) was purchased from Sigma-Aldrich Corp. sgRNA/Cas9 all-in-one expression clone targeting PHF14 (HCP223067-CG01-3-B) and scrambled sgRNA control were from GeneCopoeia, Inc. (Rockville, MD, USA).

Techniques: shRNA, Knockdown, Expressing, Selection, Western Blot, Software, Cell Viability Assay, Soft Agar Assay, CRISPR, Knock-Out

Journal: Cancer discovery

Article Title: The AMPK-related kinases SIK1 and SIK3 mediate key tumor suppressive effects of LKB1 in NSCLC.

doi: 10.1158/2159-8290.CD-18-1261

Figure Lengend Snippet: (A) Schematic of the experimental design using pSECC lentivirus to deliver Cre-recombinase, Cas9 and a Sik1 or Sik3 sgRNA as a single payload to the lungs of KrasG12D-mice (K) and Kras-Sik1 floxed mice (KSik1) after intratracheal virus delivery.

Article Snippet: As a cohort, KPSik1 mice showed an increase from 10% to 28% in tumor burden over KP mice, (a 2.8-fold increase) compared to a tumor burden of 52% in the KPL mice (5.25-fold increase over KP) , and this was consistent with the results observed in KP mice treated with SIK1 sgRNA guides ( Figure S3B , 2.5 fold tumor burden increase of KP+sgSik1 over KP+sgControls).

Techniques: Virus

Journal: Cancer discovery

Article Title: The AMPK-related kinases SIK1 and SIK3 mediate key tumor suppressive effects of LKB1 in NSCLC.

doi: 10.1158/2159-8290.CD-18-1261

Figure Lengend Snippet: (A) Schematic of the experimental design using pSECC lentivirus to deliver Cre-recombinase, Cas9 and a sgRNA of choice as a single payload to the lungs of KrasG12D-mice (K) and Kras-p53 floxed mice (KP) after intratracheal virus delivery.

Article Snippet: As a cohort, KPSik1 mice showed an increase from 10% to 28% in tumor burden over KP mice, (a 2.8-fold increase) compared to a tumor burden of 52% in the KPL mice (5.25-fold increase over KP) , and this was consistent with the results observed in KP mice treated with SIK1 sgRNA guides ( Figure S3B , 2.5 fold tumor burden increase of KP+sgSik1 over KP+sgControls).

Techniques: Virus

Journal: Cancer discovery

Article Title: The AMPK-related kinases SIK1 and SIK3 mediate key tumor suppressive effects of LKB1 in NSCLC.

doi: 10.1158/2159-8290.CD-18-1261

Figure Lengend Snippet: (A) ELISA analysis of IL-6 secretion in mouse KP lines. KP cells lines were transduced with a sgRNA guide targeting Lkb1 or with two independent sets of guides to target Sik1 and Sik3 simultaneously. 500,000 control or KO cells were seeded in 6 well plates and supernatant was collected at 48hrs to quantitate the amount of IL6 released by the cells into the media.

Article Snippet: As a cohort, KPSik1 mice showed an increase from 10% to 28% in tumor burden over KP mice, (a 2.8-fold increase) compared to a tumor burden of 52% in the KPL mice (5.25-fold increase over KP) , and this was consistent with the results observed in KP mice treated with SIK1 sgRNA guides ( Figure S3B , 2.5 fold tumor burden increase of KP+sgSik1 over KP+sgControls).

Techniques: Enzyme-linked Immunosorbent Assay, Transduction, Control

Journal: Nature Communications

Article Title: PTPN23-dependent ESCRT machinery functions as a cell death checkpoint

doi: 10.1038/s41467-024-54749-2

Figure Lengend Snippet: a Gene essentiality of mouse PTPs in RN2 cells and NIH 3T3 cells. sgRNA frequencies at Day 2 and Day 14 were determined by next-generation sequencing, with average logarithmic frequency changes calculated. Yellow dots represent pan-essential genes spiked into the screen library. b Histogram of PTPN23 gene knockout effect in 1078 cancer cell lines (DepMap 22Q2). The dashed line indicates the median score. c GFP competition growth assays performed in RN2 cells ( n = 3 independent experiments). d Left, Human PTPN23 and ALIX full-length constructs and PTPN23 truncations used in rescue experiments. Right, Heatmap visualizing the complementation effects aligned with the corresponding numbers on D14 in ( e ). His: His domain; PRR: Proline-rich region, located at the N-terminus of the His domain. e GFP competition growth assays performed in RN2 stable cell lines expressing indicated cDNAs. Top, control sgRNAs; bottom, PTPN23 sgRNAs ( n = 3 independent experiments). f Left, immunoblot of NOMO-1 cells overexpressing empty vector (EV), sgRNA-sensitive (PTPN23-WT) or sgRNA-resistant (PTPN23-r2) PTPN23 cDNA ( n = 3 independent experiments). Right, GFP competition growth assays performed using NOMO-1 stable cell lines expressing either PTPN23-WT ( n = 3 independent experiments) or PTPN23-r2 ( n = 4 independent experiments). g Top, acute depletion of PTPN23 with dTAG system. NOMO-1 cells stably expressing sgRNA-resistant PTPN23 fused with dTAG and endogenous PTPN23 were depleted with two sgRNAs. Bottom, immunoblotting analysis of PTPN23 examined after 4-day treatment of dTAG-13 (100 nM). h CellTiter-Glo (CTG) luminescence cell viability assay measured on cells treated with either DMSO or dTAG-13. DMSO or 500 nM dTAG-13 were added to three PTPN23-dTAG NOMO-1 cell lines established in ( g ) ( n = 3 independent experiments). i Cell death indicated by Sytox Green positivity. PTPN23-dTAG NOMO-1 cells were treated with 200 nM dTAG-13 for 4 days, and stained with Sytox Green ( n = 60 fields captured, 3 independent experiments). Scale bar: 30 μm. Data are presented as mean ± SEM, statistical analysis for ( h ) by Two-way ANOVA, Sidak’s multiple comparisons test; ( i ) by One-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: The pooled library containing phosphatase domain targeting and control sgRNAs (Supplementary Data ) was synthesized in duplicates or triplicates on an array platform (Twist Bioscience) and then cloned into the LRG2.1 vector with Gibson Assembly kit (NEB).

Techniques: Next-Generation Sequencing, Gene Knockout, Construct, Stable Transfection, Expressing, Control, Western Blot, Plasmid Preparation, Viability Assay, Staining