gene specific sgrna  (Synthego Inc)

Journal: Epigenetics & Chromatin

Article Title: SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes

doi: 10.1186/s13072-026-00661-6

Figure Lengend Snippet: Schematic of the CRISPR-mediated whole genome screen (see also Fig. and Supplemental Figures S2, S3). A The top of the schematic shows the Brunello sgRNA library, which encompasses 76,441 sgRNAs for 19,114 endogenous genes (green rectangle). The sgRNAs (blue rounded rectangle and hairpin) were constitutively expressed using a U6 promoter (blue arrow). In addition, productively-infected cells also constitutively express the puromycin resistance gene ( Puro ; orange rounded rectangle) using an EF1α promoter (orange arrow). The middle part of the schematic shows a transgene cassette that inducibly expresses Cas9. From left to right the cassette contains a tetracycline response element (TRE; blue and yellow hatched rectangle), a normally inactive promoter (black arrow with red X), the Cas9 gene (blue rectangle), a constitutive EF1α promoter (black arrow) driving expression of rtTA (the gene = red rectangle and the expressed protein is a red PacMan™) and the neomycin resistance gene ( Neo ; yellow rectangle). The bottom part of the schematic shows the dual-reporter cassette stably integrated at the AAVS1 locus (black line). Gene expression of the cassette is constitutively driven by a CMV promoter (green arrow). The reporter contains two drug resistance genes, one for BSD (red rectangle) and one for mycophenolic acid ( IMPDH ; red rectangle) which can be co-expressed due to the presence of the ribosome-skipping peptide, P2A (orange rectangle). In the indicated configuration, neither BSD nor IMPDH are expressed due to the presence of engineered tandem stop codons (2X stop signs and green rectangles) in their respective open reading frames (see also Supplemental Figures S2, S3). This panel corresponds to T(-3) > T0 in Fig. . B In the presence of the inducer, Dox (+ Dox, yellow triangle; also referred to as Track A), or Cas9 mRNA (green squiggly line) transfections (see text and Fig. ; also referred to as Track B), Cas9 protein (blue circles) is expressed, which facilitates the functional inactivation of the 19,114 target genes (red X over green rectangle). This panel corresponds to T0 > T3 in Fig. . C After 3 days, Dox was washed out (Track A) or the Cas9 mRNA was given time to decay (Track B). This panel corresponds to T3 > T7 of Fig. . D nCas9 (light blue circles) protein along with a sgRNA targeting the dual stop codon region in the BSD gene was introduced into the cells along with an ssDI donor ODN that encoded a portion of the wild type BSD open reading frame to correct the double stop codon. Over the course of the next 11 days, corrected cells expressing BSD resistance (green rectangle) were selected for. This panel corresponds to T11 > T18 of Fig. . E nCas9 protein was again introduced into the cells, along with a sgRNA targeting the dual stop codon region in the IMPDH gene. This time, an ssDI donor ODN that encoded a portion of the wild type IMPDH open reading frame to correct the double stop codon was utilized. Over the course of the next 15 days, corrected cells expressing IMPDH resistance (green rectangle) were selected for. This panel corresponds to T18 > T33 of Fig.

Article Snippet: For individual gene KOs in HCT116 cells, 1 × 10 6 cells in 10 μL Neon buffer R were combined with 1 μL CleanCap Cas9 mRNA (TriLink) along with 1 μL of 100 mM gene specific sgRNA (Synthego) and electroporated with 1 pulse at 1530 volts for 30 msec in a 10 μL Neon tip.

Techniques: CRISPR, Infection, Expressing, Stable Transfection, Gene Expression, Transfection, Functional Assay

Journal: Epigenetics & Chromatin

Article Title: SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes

doi: 10.1186/s13072-026-00661-6

Figure Lengend Snippet: Flow diagram of the CRISPR-mediated whole genome KO strategy. From left to right: a Brunello sgRNA library, which encompasses 76,441 sgRNAs for 19,114 genes was infected into HAP1 cells at (arbitrarily) day − 3 [T(-3)]. The cells were treated for 3 days with puromycin ( Puro ) to select for the cells which had productively taken up a sgRNA lentivirus. After 3 days, doxycycline (+ Dox, blue rectangle) induction was started at (T0) on half of the population. At day (T1) the other half of the population was transfected with Cas9 (+ Cas9) mRNA. Henceforth the + Dox-only sample was referred to as the “A Track” and the + Cas9 mRNA sample as the “B track”. At day 7 (T7) a ssDI experiment was performed on both populations of cells (approximately 70 million cells each) to functionally correct an imbedded, but inactive BSD gene (see also Fig. and Supplemental Figures S2, S3). After 48 h (i.e., T9) a blasticidin selection was started for both populations of cells. After an additional 24 h (T10) a sample of cells was saved from both tracks for subsequent bioinformatics analysis. After a week (T18) a second ssDI experiment was performed on both populations of cells (again, approximately 70 million cells each) to functionally correct an imbedded, but inactive IMPDH gene (see also Fig. and Supplemental Figures S2, S3). After 72 h (T21) a mycophenolic acid selection was started. 12 days later (T33) the experiment was terminated for the A and B tracks of cells, which were now resistant to both BSD and mycophenolic acid (BSD + :IMPDH + ). In addition, a sample of cells (T33) was saved from both tracks for subsequent bioinformatics analysis (see also Fig. ).

Article Snippet: For individual gene KOs in HCT116 cells, 1 × 10 6 cells in 10 μL Neon buffer R were combined with 1 μL CleanCap Cas9 mRNA (TriLink) along with 1 μL of 100 mM gene specific sgRNA (Synthego) and electroporated with 1 pulse at 1530 volts for 30 msec in a 10 μL Neon tip.

Techniques: CRISPR, Infection, Transfection, Selection

Journal: bioRxiv

Article Title: Identification of host factors for Rift Valley Fever Phlebovirus

doi: 10.1101/2023.09.28.559935

Figure Lengend Snippet: A549 cells were transfected with 50 nM of siRNAs. At 48 hours post-transfection, the cells were infected with RVFV MP-12 virus at 0.1 MOI. At 24 hours post-infection, the supernatant was collected and titered by plaque assay. NTC-non-target control siRNA, si46N-anti-RVFV siRNA, WDR7-, SLC35B2-, EXOC4-, LRP1-, EMC3-, CTL47A1-gene specific siRNAs. Each bar represents the average virus titer (pfu/ml) along with the corresponding standard deviation. Statistical analysis was done on two independent experiments with four replicates for each, using Mann-Whitney U independent Student’s t-test (** p-value < 0.005, *** p-value <0.001).

Article Snippet: LentiCRISPRv2 plasmids 1 and 5 containing sgRNAs specific for WDR7 gene were purchased from Genescript, USA.

Techniques: Transfection, Infection, Virus, Plaque Assay, Control, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Identification of host factors for Rift Valley Fever Phlebovirus

doi: 10.1101/2023.09.28.559935

Figure Lengend Snippet: ( A ) A549 cells, CT (non-knock-out control) cells, and WDR7 gene KO cell lines #1 and #2 were analyzed for WDR7 protein expression by western blot using a WDR7-specific polyclonal antibody. ( B, C, D & E ) CT cells and WDR7 KO A549 cells were infected with RVFV MP-12 vaccine strain, ( B ) with the wild-type RVFV Kenya 128B-15 strain, ( C ) or with La Crosse encephalitis virus ( D, E ) at 0.1 MOI. Supernatant was collected at 6, 12 or 24 h post infection (h pi) and titered by plaque assay (RVFV) or TCID 50 -CPE assay (LACV). RVFV MP-12 testing on CT A549 cells, WDR7 KO lines #1 or #2, and NTC-non-target control cells involved three to five independent experiments with three to four technical replicates each. RVFV Kenya 128B-15 testing involved independent experiments with three technical replicates each. LACV testing was performed in two independent experiments with eight technical replicates each. Statistical analysis was done using Mann-Whitney U independent Student’s t-test (* p-value < 0.05, ** p-value < 0.005, *** p-value <0.001).

Article Snippet: LentiCRISPRv2 plasmids 1 and 5 containing sgRNAs specific for WDR7 gene were purchased from Genescript, USA.

Techniques: Knock-Out, Control, Expressing, Western Blot, Infection, Virus, Plaque Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Identification of host factors for Rift Valley Fever Phlebovirus

doi: 10.1101/2023.09.28.559935

Figure Lengend Snippet: CT and WDR7 KO #1 cells were infected with the ( A ) RVFV MP-12 vaccine strain or ( B ) the LAC virus at 0.1 MOI. Total cellular RNA was harvested at various hour(s) post-infection (h pi). One-step RT-qPCR was performed to detect the level of viral RNA using the PGK1 gene as an internal control. CT and WDR7 KO #1 cells were utilized. Each bar graph represents the average fold change in viral RNA expression, along with the corresponding standard deviation. Statistical analysis was done on three independent experiments with two to three technical replicates for each, using Mann-Whitney U independent Student’s t-test (* p-value <0.05, *** p-value <0.001, ns, non-significant).

Article Snippet: LentiCRISPRv2 plasmids 1 and 5 containing sgRNAs specific for WDR7 gene were purchased from Genescript, USA.

Techniques: Infection, Virus, Quantitative RT-PCR, Control, RNA Expression, Standard Deviation, MANN-WHITNEY

Journal: Cancer Research

Article Title: Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis

doi: 10.1158/0008-5472.CAN-22-1622

Figure Lengend Snippet: Clinical relevance of CaMKK2 in metastatic TNBC. A, Kaplan–Meier plots showing the association between CaMKK2 level and the overall survival of patients with basal ( n = 431), luminal A ( n = 592), luminal B ( n = 439), or Her2 + ( n = 361) tumor subtypes. Patients were grouped into CaMMK2-high or -low by upper quartile (lower or higher than 75th percentile). The x -axis shows time in months, and the y -axis shows the overall survival probability. Plots were generated using KM-plotter ( https://kmplot.com ), B–E, Violin plots depicting the score for the gene signatures published in the indicated articles . Samples were grouped into CaMMK2-high or -low by quartiles (equal or less than 25th percentile and equal or greater than 75th percentile). The x -axis shows breast cancer subtypes, and the y -axis shows the score between −1 and 1. Data, mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ns, nonsignificant; by Student t test ( B – E ) and log-rank test ( A ).

Article Snippet: sgRNAs targeting specific sequences at the N-terminal end of the CaMKK2 gene were purchased from Synthego Corp. For each sgRNA, a ribonucleoprotein (RNP) complex was formed with the Cas9 2NLS nuclease (Synthego), at a sgRNA: Cas9 ratio of 6:1.

Techniques: Generated

Journal: Cancer Research

Article Title: Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis

doi: 10.1158/0008-5472.CAN-22-1622

Figure Lengend Snippet: Ablation of CaMKK2 impairs migration in invasive breast cancer cells. A and B, Representative blots showing knockdown of CaMKK2 in MDA-MB-231-4175 ( A ) and BT-20 cells ( B ). MDA-MB-231-4175 and BT-20 cells were transfected with either control siRNAs (siCtrl #1, siCtrl #2) or siRNAs targeting CaMKK2 (siKK2 #1, siKK2 #2). Forty-eight hours posttransfection, cells were harvested and immunoblots were used to analyze CaMKK2 and phospho-AMPKα (T172) expression. C and D, CaMKK2 knockdown impaired migration of MDA-MB-231-4175 cells ( C ) and BT-20 cells ( D ) in vitro. Data plotted as mean ± SEM; n = 6 random fields measurements from four individual experiments. E and F, Treatment with STO-609 (10 μmol/L; 48 hours) and GSKi (1 μmol/L; 48 hours) reduced migration of MDA-MB-231–4175 cells ( E ) and BT20 cells ( F ). Data plotted as mean ± SEM; n = 9 random fields measurements from two individual experiments done in triplicate. G and H, STO-609 (10 μmol/L; 48 hours) and GSKi (1 μmol/L; 48 hours) treatment reduced invasiveness in MDA-MB-231-4175 cells ( G ) and BT20 cells ( H ) in vitro . Data plotted as mean ± SEM; n = 4–5 random fields measurements from three individual experiments done in duplicate, I , Representative blots confirming CRISPR-mediated knockout of CaMKK2 in MDA-MB-231-4175 cells (see also Supplementary Fig. S1H), J, CRISPR-mediated ablation of CaMKK2 reduced migration in CaMKK2-KO clones in vitro , Data plotted as mean ± SEM; n = 4–5 random fields measurements from three individual experiments done in duplicate. K, Depletion of CaMKK2 reduced cellular movement in MDA-MB-231-4175 cells cultured with fresh human breast tissue. Fluorescence microscopy and brightfield images were captured every 5 minutes over 6 hours to track cellular movement (see also Supplementary Videos S1–S4). L, Representative blots confirming addback of CaMKK2 expression in CaMKK2-KO clones #1 and #2. M and N, Overexpression of CaMKK2 rescues migratory capability in both CaMKK2 KO clones. Data are plotted as mean ± SEM; n = 4–5 random fields measurements from three individual experiments done in duplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. P values were calculated using unpaired Student t test.

Article Snippet: sgRNAs targeting specific sequences at the N-terminal end of the CaMKK2 gene were purchased from Synthego Corp. For each sgRNA, a ribonucleoprotein (RNP) complex was formed with the Cas9 2NLS nuclease (Synthego), at a sgRNA: Cas9 ratio of 6:1.

Techniques: Migration, Knockdown, Transfection, Control, Western Blot, Expressing, In Vitro, CRISPR, Knock-Out, Clone Assay, Cell Culture, Fluorescence, Microscopy, Over Expression

Journal: Cancer Research

Article Title: Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis

doi: 10.1158/0008-5472.CAN-22-1622

Figure Lengend Snippet: Genetic ablation of CaMKK2 impairs metastasis from the primary tumor in vivo. A, Six-week-old female nude mice were orthotopically injected in the mammary fat pad with two control cell clones, Ctrl #1 ( n = 9), Ctrl #3 ( n = 8); two CaMKK2-KO cell clones, KK2-KO #1 ( n = 9), KK2-KO #2 ( n = 6); and the CaMKK2 KO cell clones with reexpression of CaMKK2, KO #1-KK2 ( n = 8) and KO #2-KK2 ( n = 7). All clones were derived from the parental MDA-MB-231–4175 cell line. The mice were sacrificed after tumors reached approximately 2,500 mm 3 , and the lung and liver metastatic burden was analyzed ex vivo. Representative BLI images showing lung metastatic foci were visualized. B–D, Quantitation of the number of metastatic foci in the lung ( B ) and liver ( C ), and total metastatic burden (includes metastatic foci in the liver and lung taken together for each animal; D ). Data, mean ± SEM. *, P < 0.05; **, P < 0.01; ns, nonsignificant; by Student t test.

Article Snippet: sgRNAs targeting specific sequences at the N-terminal end of the CaMKK2 gene were purchased from Synthego Corp. For each sgRNA, a ribonucleoprotein (RNP) complex was formed with the Cas9 2NLS nuclease (Synthego), at a sgRNA: Cas9 ratio of 6:1.

Techniques: In Vivo, Injection, Control, Clone Assay, Derivative Assay, Ex Vivo, Quantitation Assay

Journal: Cancer Research

Article Title: Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis

doi: 10.1158/0008-5472.CAN-22-1622

Figure Lengend Snippet: Pharmacologic inhibition of CaMKK2 impairs metastasis from the primary tumor in vivo. A, MDA-MB-231-4175 cells were orthotopically injected into the mammary fat pad of 6-week-old female nude mice. Five days postinjection, the animals were dosed with vehicle ( n = 10), STO-609 (30 mg/kg; n = 8), or GSKi (10 mg/kg; n = 9) by intraperitoneal injections every third day. Tumor growth rate was monitored until the tumors reached a volume of 2,000 mm 3 . B–D, Quantitative analysis of metastatic foci in the lung ( B ), liver ( C ), and the total metastatic burden (includes metastatic foci in the liver and lung taken together; D ). The mice were sacrificed after the primary tumor reached approximately 2000 mm 3 , and the metastatic foci were analyzed ex vivo . E, Subcutaneous tumor growth in 6-week-old female nude mice orthotopically injected with murine 4T1 cells. The mice were randomized (five days after tumor cell injection in the mammary fat pad) and dosed with vehicle ( n = 7) or STO-609 (30 mg/kg; n = 9) by intraperitoneal injections every third day. Tumor growth rate was monitored until the tumors reached a volume of 2,000 mm 3 . F, Quantitative analysis of metastatic nodules in the lung. Data, mean ± SEM. *, P < 0.05; **, P < 0.01; by two-way ANOVA followed by Bonferroni multiple-correction test ( A and E ) or unpaired Student t test ( B–D and F ).

Article Snippet: sgRNAs targeting specific sequences at the N-terminal end of the CaMKK2 gene were purchased from Synthego Corp. For each sgRNA, a ribonucleoprotein (RNP) complex was formed with the Cas9 2NLS nuclease (Synthego), at a sgRNA: Cas9 ratio of 6:1.

Techniques: Inhibition, In Vivo, Injection, Ex Vivo

Journal: Cancer Research

Article Title: Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis

doi: 10.1158/0008-5472.CAN-22-1622

Figure Lengend Snippet: Depletion of CaMKK2 leads to increased phosphorylation of VASP at the Serine 239 residue, which leads to impaired cytoskeletal assembly and cell motility. A, Representative images showing impaired cytoskeletal assembly in CaMKK2-KO cells. Visualization of F-actin by phalloidin staining in CaMKK2-WT (Ctrl #1) and CaMKK2-KO (KK2-KO #1) cells revealed almost complete loss of ventral stress fibers in cells lacking CaMKK2 expression. Yellow arrows, dorsal stress fibers; white arrows, ventral stress fibers; yellow bracket, transverse arcs; green arrowhead, leading edge; yellow arrowhead, contractile rear of the cell. Scale bar, 10 μm. B, Representative blot showing increased phosphorylation of VASP at Serine 239 in CaMMK2 KO cell clones. C, Representative blot showing VASP phosphorylation at Serine 239 is enhanced in CaMKK2-KO cells in response to ionomycin (1 μmol). Cells were treated with either DMSO or ionomycin (1 μmol/L) with CaCL 2 (1 mmol/L) for varying time periods (as indicated). D and E, Inhibition of CaMKK2 (STO-609, 10 μmol) increases phosphorylation of VASP [especially with ionomycin (1 μmol; 2 hours)] in metastatic MDA-MB-231-4175 cells ( D ) and BT-20 cells ( E ). DMSO or STO-609–treated cells were exposed to either DMSO or ionomycin (1 μmol/L) with CaCL 2 (1 mmol/L) for 2 hours before immunoblotting. F and G, CaMKK2 inhibition decreases migration of MDA-MB-231-4175 cells ( F ) and BT20 cells ( G ) in vitro. DMSO or STO-609–treated cells were exposed to either DMSO or ionomycin with CaCL 2 (see D and E ) before migration assays were performed. Data are plotted as mean ± SEM; n = 4–5 random fields measurements from four individual experiments done in duplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values were calculated using unpaired Student t test.

Article Snippet: sgRNAs targeting specific sequences at the N-terminal end of the CaMKK2 gene were purchased from Synthego Corp. For each sgRNA, a ribonucleoprotein (RNP) complex was formed with the Cas9 2NLS nuclease (Synthego), at a sgRNA: Cas9 ratio of 6:1.

Techniques: Phospho-proteomics, Residue, Staining, Expressing, Clone Assay, Inhibition, Western Blot, Migration, In Vitro

Journal: Cancer Research

Article Title: Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis

doi: 10.1158/0008-5472.CAN-22-1622

Figure Lengend Snippet: Inhibition of PKG1 blocks phosphorylation of VASP in CaMKK2-depleted cells and restores cellular migration in vitro . A, Schematic representation of working hypothesis of the mechanism by which CaMKK2 drives cell migration and metastasis B and C , RKRARKE treatment (100 μmol; 2.5 hours) decreased VASP phosphorylation in CaMKK2-inhibited MDA-MB-231-4175 cells ( B ) and BT-20 cells ( C ). Vehicle or STO-609–treated cells were dosed with RKRARKE for 30 minutes before exposure to DMSO or ionomycin (1 μmol/L) with CaCL 2 (1 mmol/L) for 2 hours. Representative immunoblots from three independent experiments ( B ) and one experiment ( C ) are shown. D and E, Inhibition of PKG1 with RKRARKE blocks VASP phosphorylation at Serine 239 in CaMKK2-KO cells. CaMKK2-WT (Ctrl #1) and CaMKK2-KO (KK2-KO #1) cells were pretreated with RKRARKE (100 μmol) for 30 minutes before exposure to DMSO or ionomycin (1 μmol/L) with CaCL 2 (1 mmol/L) for 2 hours. Representative blots from two independent experiments ( D ) and quantitative results for the band intensities of phospho-VASP versus total VASP ( E ) are shown. F and G, RKRARKE (100 μmol; 2.5 hours) pretreatment restores the migratory phenotype in CaMKK2-KO cells ( F ) and STO-609-treated MDA-MB-231-4175 cells ( G ). Cells were pretreated with RKRARKE before exposure to ionomycin (1 μmol/L) with CaCL 2 (1 mmol/L) for 2 hours prior to migration assays. Data plotted as mean ± SEM; n = 4–5 random fields measurements from two individual experiments. H, Pretreatment with KT5823 (5 μmol; 16 hours) in CaMKK2-inhibited cells decreased VASP phosphorylation. MDA-MB-231-4175 cells were pretreated with either vehicle or KT5823 overnight before exposure to DMSO or ionomycin. Representative blots (left) and quantitative analysis (right) of band intensities from two independent experiments are shown. I, KT5823 pretreatment (5 μmol; 16 hours) of CaMKK2-inhibited cells restores the migratory phenotype of STO-609–treated MDA-MB-231-4175 cells. Data are plotted as mean ± SEM; n = 4–5 random fields measurements from four individual transwells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values were calculated using unpaired Student t test.

Article Snippet: sgRNAs targeting specific sequences at the N-terminal end of the CaMKK2 gene were purchased from Synthego Corp. For each sgRNA, a ribonucleoprotein (RNP) complex was formed with the Cas9 2NLS nuclease (Synthego), at a sgRNA: Cas9 ratio of 6:1.

Techniques: Inhibition, Phospho-proteomics, Migration, In Vitro, Western Blot

Journal: Cancer Research

Article Title: Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis

doi: 10.1158/0008-5472.CAN-22-1622

Figure Lengend Snippet: Depletion of CaMKK2 causes reduced expression of PDE1A, an upstream negative regulator of PKG1. A, CaMKK2 depletion decreased PDE1A protein levels in MDA-MB-231-4175 cells. B and C, Inhibition of CaMKK2 (STO-609, 10 μmol/L) reduced PDE1A expression in MDA-MB-231-4175 cells ( B ) and BT20 cells ( C ). D, PDE1 inhibition with vinpocetine increased VASP phosphorylation at Serine 239 in MDA-MB-231-4175 cells. Cells were pretreated with vinpocetine (50 μg/mL; 16 hours), IBMx (100 μmol/L; 16 hours), or sildenafil (10 μmol/L; 1 hour) before exposure to either DMSO or ionomycin (1 μmol/L) with CaCL 2 (1 mmol/L) for 2 hours E, Vinpocetine pretreatment enhances phosphorylation of VASP at Serine 239 in CaMKK2-WT cells, phenocopying CaMKK2-KO cells. F, PDE1 inhibition impairs migration of MDA-MB-231-4175 cells in a dose-dependent manner. Following pretreatment with vinpocetine, MDA-MB-231-4175 cells were exposed to ionomycin (1 μmol/L) with CaCL 2 (1 mmol/L) for 2 hours before assaying for migration. Data plotted as mean ± SEM; n = 4–5 random fields measurements from two individual experiments. G and H, Treatment with L-NNA blocked VASP phosphorylation in ionomycin-treated CaMKK2-KO cells. CaMKK2-WT and CaMKK2-KO cells were cultured with NOS inhibitors (16 hours) or vinpocetine, as indicated, before exposure to either DMSO or ionomycin (1 μmol/L) with CaCL 2 (1 mmol/L) for 2 hours. Representative blots ( G ) and quantitative results ( H ) for the band intensities of phospho-VASP versus total VASP are shown. Data plotted as mean ± SEM from two independent experiments. I, Pretreatment of CaMKK2-KO cell lines with L-NNA rescued migratory ability in a dose-dependent manner. CaMKK2-KO cells were cultured with or without L-NNA (1 mmol/L; 16 hours) before exposure to ionomycin (1 μmol/L) with CaCL 2 (1 mmol/L) for 2 hours and assayed for migration. Data plotted as mean ± SEM; n = 4–5 random fields measurements from two individual experiments. Significance calculated relative to Ctrl#1. J, Schematic model of the mechanism by which CaMKK2 drives tumor cell migration and metastasis. **, P < 0.01; ***, P < 0.005; ns, nonsignificant. P values were calculated using unpaired Student t test.

Article Snippet: sgRNAs targeting specific sequences at the N-terminal end of the CaMKK2 gene were purchased from Synthego Corp. For each sgRNA, a ribonucleoprotein (RNP) complex was formed with the Cas9 2NLS nuclease (Synthego), at a sgRNA: Cas9 ratio of 6:1.

Techniques: Expressing, Inhibition, Phospho-proteomics, Migration, Cell Culture

Journal: Cancer Research

Article Title: Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis

doi: 10.1158/0008-5472.CAN-22-1622

Figure Lengend Snippet: CaMKK2 expression is associated with lower survival rates in patients with HGSOC. A, Representative photomicrographs of tumor microarray sections stained for CaMKK2 by immunohistochemistry, with corresponding intensity scores (×10 magnification). B and C, PFS ( B ) and OS ( C ) of patients with stage III HGSOC were evaluated and demonstrated that low CaMKK2 expression (intensity score < 3) correlated with longer median PFS (19.1 months vs. 6.2 months, P = 0.0242) and OS (60.6 vs. 17.1 months, P = 0.0180) than patients with high expression. D, PFS of patients with stage II to IV HGSOC were evaluated, which demonstrated that low CaMKK2 expression correlated with longer median PFS than patients with high expression (14.7 months vs. 14.5 months, P = 0.0453). E, OS of patients with stage II to IV were evaluated and showed a trend toward higher median OS in patients with low expression (58.0 months vs. 32 months, P = 0.2745). F, Eight-week-old female nude mice were inoculated with SKOV3ip1 cells (intraperitoneally) and received vehicle (control) or STO-609. Representative images ( F ), weight of metastatic tumor burden ( G ), and weight of non-pelvic metastasis ( H ) are shown ( n = 15 per group). Data, mean ± SEM. *, P < 0.05; **, P < 0.01; by Student t test ( G and H ) and log-rank test ( B – E ).

Article Snippet: sgRNAs targeting specific sequences at the N-terminal end of the CaMKK2 gene were purchased from Synthego Corp. For each sgRNA, a ribonucleoprotein (RNP) complex was formed with the Cas9 2NLS nuclease (Synthego), at a sgRNA: Cas9 ratio of 6:1.

Techniques: Expressing, Microarray, Staining, Immunohistochemistry, Control