Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer

doi: 10.1186/s13046-023-02689-y

Figure Lengend Snippet: Engineered p16 epimutation recapitulates key features of age-associated epigenetic silencing. A p16 DNA methylation profiles in cis -MEF following serial passaging (p). A schematic of the p16 promoter of the cis -element knock-in allele with the CpG maps is shown. B Kinetics of p16 promoter methylation and mRNA expression in ctr-MEF and cis -MEF. Methylation levels were averaged from CpGs from − 814 bp to − 589 bp relative to the TSS. Note that cis -MEF cells grew for more than 25 passages; in contrast, the controls (ctr-MEF) entered growth arrest at passage 8 (p8) via p16 up-regulation. The p16 gene expression is relative to β–actin. C UCSC Genome Browser tracks showing the DNA methylation status of p16 , p19 , and p15 in the INK4/ARF locus on chromosome 4. The chromosomal coordinates are annotated on the top. The WGBS tracks show DNA methylation profiling in ctr-MEF (p7) and cis-MEF (p10). The height of each bar represents the methylation level of an individual CpG between 0 and 1 (100%). Promoters are indicated by angled arrows, and the knock-in location is indicated by an asterisk. D Comparisons of p16 promoter methylation in cis -MEF before and after Ad-Cre mediated cis -element excision with increasing doses of DAC. The culture passage number of cis -MEF is at p23. E Side-by-side comparisons of p16 expression in cis -MEF under the same conditions as ( D ). For ( D) and ( E ), data are shown as mean ± SEM with individual values from three independent experiments. P values were determined by a one-way ANOVA test. F dCas9-SunTagTET1-mediated targeted demethylation of p16 in cis -MEF. p16 -gRNA indicates GFP-positive cells treated with a p16 -specific gRNA that binds the promoter region from − 792 bp to − 769 bp relative to the TSS. Ctr indicates GFP-negative cells without targeted demethylation. Data are shown from three independent experiments. G Targeted promoter demethylation resulted in p16 gene reactivation. The culture passage number of cis -MEF is at p23. P values were determined by a two-tailed Student’s t -test

Article Snippet: Plasmids containing p16 -specific gRNA or non-targeting control gRNA were constructed using an all-in-one vector from Addgene (#82,559; Watertown, MA, USA) and Gibson Assembly (NEB, Ipswich, MA, USA). gRNA sequences are listed in Supplementary Table .

Techniques: DNA Methylation Assay, Passaging, Knock-In, Methylation, Expressing, Gene Expression, Two Tailed Test

Journal: iScience

Article Title: Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase beta

doi: 10.1016/j.isci.2024.109921

Figure Lengend Snippet: Levels of PIK3CB/PI3Kβ are the highest among the four PI3K kinases in GBM regardless of MGMT status RNA-seq data were retrieved from DepMap, TCGA, CGGA, and THPA. (A) Comparison of PI3K mRNAs in DepMap glioma cell lines. Cell lines from DepMap were reannotated (see Table S1 ). mRNA levels of PI3K kinases were compared between DepMap grade II/III (GII/III) cell lines and GBM cell lines or between MGMT-deficient and MGMT-positive cells. (B) Comparison of PI3K mRNAs between TCGA GII/III and GBM tumors or between MGMT-deficient and MGMT-positive tumors. (C) Comparison of PI3K mRNAs in CGGA gliomas. (D) Immunohistochemical (IHC) staining of PI3Kα, PI3Kβ, PI3Kδ, PI3Kγ, or MGMT in a THPA MGMT-deficient HGG (Patient ID: 1587). Details of IHC staining are shown in the inset figures. Staining levels determined by the THPA are shown in each image. (E) Quantification of PI3Kα, PI3Kβ, PI3Kδ, or PI3Kγ based on images shown in D using ImageJ. Four persons performed ImageJ quantification independently. Each data point stands for an intensity reading from one individual. (F) Quantification of PI3Kα, PI3Kβ, PI3Kδ, or PI3Kγ in THPA HGG or LGG tumors. Four persons performed quantification using ImageJ. Averages of intensity readings from four persons consist of individual data points. Case numbers (N) are shown. Error bars are standard errors. p values were obtained by Student’s t test or One-way ANOVA. ns: not significant; ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001.

Article Snippet: PIK3CB gRNA , Addgene , Cat#76192.

Techniques: RNA Sequencing, Comparison, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: iScience

Article Title: Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase beta

doi: 10.1016/j.isci.2024.109921

Figure Lengend Snippet: mRNA Levels of PIK3CB associate levels of PI3K activation and chemosensitivity in MGMT-deficient GBMs RNA-seq and RPPA data were retrieved from DepMap and TCGA. The Pearson correlation assay was used to determine associations between two subjects. (A) Pearson correlations between pAKTS473/T308 and levels of PIK3CA/B/D/G mRNAs in DepMap MGMT-deficient or MGMT-positive GII/III glioma cell lines or GBM cell lines. (B) Pearson correlations between pAKTS473/T308 and levels of PIK3CA/B/D/G mRNAs in TCGA MGMT-deficient or MGMT-positive GII/III gliomas or GBMs. (C) Pearson correlations between TMZ IC50s and levels of PIK3CA/B/D/G mRNAs in DepMap MGMT-deficient or MGMT-positive GII/III glioma cell lines or GBM cell lines. (D) Pearson correlations between TMZ IC50s and levels of pAKTs or PTEN in DepMap MGMT-deficient or MGMT-positive GII/III glioma cell lines or GBM cell lines. Pearson coefficients (r) with 95% confidence intervals (95% CI) are shown. Representative Pearson correlation results in MGMT-deficient GBMs are also shown. Original results of Pearson correlation in other gliomas are shown in Figures S7 , , , and . ∗: p < 0.05; ∗∗: p < 0.01. Statistically not significant Pearson coefficients are not labeled.

Article Snippet: PIK3CB gRNA , Addgene , Cat#76192.

Techniques: Activation Assay, RNA Sequencing, Two-Photon Excitation Fluorescence Cross-Correlation Assay, Labeling

Journal: iScience

Article Title: Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase beta

doi: 10.1016/j.isci.2024.109921

Figure Lengend Snippet: PIK3CB predicts clinical outcomes of MGMT-deficient GBMs RNA-seq/RPPA data and associated clinical data were retrieved from TCGA, CGGA, and other GBM datasets downloaded from the GlioVis online program. Results from primary MGMT-deficient/IDH-wt gliomas were pooled together for Kaplan-Meier survival analyses using JMP and Prism 10. Plotted are the resulting HRs with 95% CIs (HR > 1: more chance of death; HR < 1: more chance of survival). (A) Comparison of high and low levels of PI3K kinases in primary MGMT-deficient/IDH-wt or MGMT-positive/IDH-wt GII/III or GBM patients. (B) Comparison of high or low levels of pAKTs in primary MGMT-deficient/IDH-wt or MGMT-positive/IDH-wt GII/III or GBM patients. (C) Comparison of untreated (Chemo–) and treated (Chemo+) cases in primary MGMT-deficient/IDH-wt or MGMT-positive/IDH-wt GII/III or GBM patients expressing various levels of PI3K kinases. Representative Kaplan-Meir survival curves in MGMT-deficient GBMs are shown. Other Kaplan-Meir survival curves can be found in Figures S12–S14 . HRs, Log Rank Ps (long-term survival), and Wilcoxon Ps (short-term survival) with 95% CIs are shown. Only statistically significant HRs are marked with ∗. ∗: p < 0.05.

Article Snippet: PIK3CB gRNA , Addgene , Cat#76192.

Techniques: RNA Sequencing, Comparison, Expressing

Journal: iScience

Article Title: Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase beta

doi: 10.1016/j.isci.2024.109921

Figure Lengend Snippet: PIK3CB/PI3Kβ, but not other PI3K kinases, activates PI3K signaling and regulates TMZ sensitivity in MGMT-deficient GBMs (A) Combinations of TMZ and knockdown of individual PI3K kinases. PI3Kβ-high SF295 or PI3Kβ-low LN229 cells were transfected with viruses harboring non-silencing (NS) shRNA or shRNA of PIK3CA, PIK3CB, or PIK3CD. Cells were then treated with DMSO or 200 μM TMZ. 4 days after treatment, cell viability was measured using the MTS assay. Cells treated with shNS and DMSO serve as controls (100% of viability). (B) Knockout of PI3Kα or PI3Kβ. PI3Kα/β-high U87MG cells were transduced with viruses harboring gRNA of non-targeting (NT) or PIK3CA. PI3Kβ-high SF295 cells were transduced with viruses harboring gRNA of NT or PIK3CB. Levels of PI3Kα, PI3Kβ, or PI3K signaling were monitored using immunoblotting. β-actin (ACTB) was the loading control. (C) Combinations of TMZ and knockout of PI3Kα or PI3Kβ. PI3Kα/β-high U87MG or PI3Kβ-high SF295 cells were transduced with viruses having gRNAs of NT, PIK3CA, or PIK3CB. Cells were then treated with DMSO or 200 μM TMZ. 4 days after treatments, cell viability was measured using the MTS viability assay. Cells treated with NT gRNA and DMSO serve as controls (100% of viability). (D) Overexpression of active AKT1, AKT2, or AKT3. PI3Kα/β-high U87MG cells were transduced with viruses having pBABE (vector control) or plasmids with active myristoylated AKT isoforms (pBABE-Myr-AKT1, pBABE-Myr-AKT2, or pBABE-Myr-AKT3). Cells were then treated with 20 μM of TGX-221 (PI3Kβ-selective inhibitor) and 200 μM TMZ. Cell viability was measured using the MTS assay. Cells treated with DMSO and pBABE serve as controls (100% of viability). Error bars are standard deviations derived from three to four independent replicates. Student’s t test and One-Way ANOVA were used to determine p values. ns: not significant.

Article Snippet: PIK3CB gRNA , Addgene , Cat#76192.

Techniques: Knockdown, Transfection, shRNA, MTS Assay, Knock-Out, Transduction, Western Blot, Control, Viability Assay, Over Expression, Plasmid Preparation, Derivative Assay

Journal: iScience

Article Title: Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase beta

doi: 10.1016/j.isci.2024.109921

Figure Lengend Snippet:

Article Snippet: PIK3CB gRNA , Addgene , Cat#76192.

Techniques: Recombinant, Transfection, Proliferation Assay, Concentration Assay, Software, Protein Array, Expressing, shRNA, Modification

Journal: Cell Reports Medicine

Article Title: A kinome-wide CRISPR screen identifies CK1α as a target to overcome enzalutamide resistance of prostate cancer

doi: 10.1016/j.xcrm.2023.101015

Figure Lengend Snippet:

Article Snippet: ATM gRNA2 , Doench et al. , Addgene Cat# 77531.

Techniques: Magnetic Beads, Virus, Recombinant, Mutagenesis, Plasmid Preparation, CRISPR, Knock-Out, Software

Journal: Light, Science & Applications

Article Title: Near-infrared light activatable chemically induced CRISPR system

doi: 10.1038/s41377-025-01917-8

Figure Lengend Snippet: a Custom LED illumination setup. b Fluorescence of eGFP-expressing HEK293FT cells treated with rapamycin dimer, transfected with full-length dCas9. At the top of the setup there is a holder for a 35 mm glass-bottom dish. The 3D-printed double slit screen is located in the focal plane of the illumination setup. c Fluorescence of eGFP-expressing HEK293FT cells treated with rapamycin dimer, transfected with split dCas9 without NIR illumination. d Fluorescence of eGFP-expressing HEK293FT cells treated with rapamycin dimer, transfected with split dCas9 under 30 seconds of NIR illumination. e 3D-printed double-slit screen with each slit being 500 μm wide and 2 mm long. f NIR illumination using double slit screen. g Corresponding pattern of the fluorescence of eGFP-expressing cells

Article Snippet: The plasmids encoding Cas9 (PX851, #62883 and PX852, #62884), split dCas9 (PX855, #62887 and PX856, #62888), dCas9-VP64 (#61425), an eGFP-expressing surrogate reporter (#60718), and a sgRNA targeting the eGFP reporter (#60719) were obtained from Addgene.

Techniques: Fluorescence, Expressing, Transfection