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

Journal: Nature Communications

Article Title: Target-dependent RNA polymerase as universal platform for gene expression control in response to intracellular molecules

doi: 10.1038/s41467-023-42802-5

Figure Lengend Snippet: a Application of TdRNAP to control genome editing in a target-dependent manner in human cells. GCN4-dRNAP induces gRNA expression when cells express GCN4-fused EGFP (EGFP-GCN4). The gRNA targets and knocks out the EGFP gene by forming a complex with Cas9. b Establishment of the 293FT cell lines carrying EGFP or EGFP-GCN4 gene in the AAVS1 locus. GCN4-dependent EGFP knockout should be triggered preferentially in the cells expressing EGFP-GCN4 than EGFP. c Flow cytometry analysis of the GCN4-dependent EGFP knockout in the established 293FT cell lines. d , e EGFP-negative cell population measured by flow cytometry in EGFP knockout experiments using T7 promoter for GCN4-dependent gRNA expression ( d ) and U6 promoter for constitutive gRNA expression ( e ) along with 200 ng of Cas9 expression plasmid. The protein structures were drawn with PDB data (PDB ID: 1QLN, 2Y0G, and 1P4B) ( a ). Values represent mean ± s.d. of n = 7 ( d ) and n = 5 ( e ) biological replicates. Statistical analysis by unpaired two-tailed t test ( d , e ), ** P <0.01, n.s., not significant ( P >0.05). Each P value is listed in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: The cells were transfected with 1 μg of SpCas9 plasmid (Addgene plasmid #41815), 1 μg of AAVS1 -targeting sgRNA plasmid (Addgene plasmid #41818), and 1 μg of the donor plasmid using 2.0 μL of Lipofectamine 2000 following the manufacturer’s instructions.

Techniques: Control, Expressing, Knock-Out, Flow Cytometry, Plasmid Preparation, Two Tailed Test

Journal: Cell Biology and Toxicology

Article Title: CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

doi: 10.1007/s10565-024-09848-7

Figure Lengend Snippet: CD36 expression is positively correlated with cell proliferation and migration in vitro . A–F A549 cells were transfected with pCMV or pCMV-CD36 plasmid, and NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid for 12 h in serum-free medium and then cultured in complete medium for another 24 h. Cells were collected for determination of cell viability ( A ), cell cycle ( B , C ), migration ( D ), and apoptosis ( E ) by MTT assay, FACS, wound healing test, and Annexin V-FITC/PI staining, respectively. Protein expression of CD36, CDH1, PCNA, vimentin, Bcl-2, and BAX was determined by Western blot with density quantitative analysis ( F ). G , H NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid for 12 h and then cultured in complete medium for 24 h, followed by FFAs (150 µM) treatment for 24 h. Cells were collected for determination of cell viability ( G ) and apoptosis ( H ). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; A , G : n = 6; B – F , H : n = 3

Article Snippet: CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase.

Techniques: Expressing, Migration, In Vitro, Transfection, Plasmid Preparation, Cell Culture, MTT Assay, Staining, Western Blot

Journal: Cell Biology and Toxicology

Article Title: CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

doi: 10.1007/s10565-024-09848-7

Figure Lengend Snippet: The effects of pitavastatin on cell proliferation, migration, and apoptosis are related to CD36 expression. A549 cells were transfected with pCMV or pCMV-CD36 plasmid, and NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid for 12 h and then cultured in complete medium for 24 h, followed by received pitavastatin (5 µM) treatment for 24 h. Cells were collected for determination of cell viability ( A ), cell cycle ( B , C ), migration ( D ), and apoptosis ( E ). Protein expression of CD36, PCNA, Bcl-2, and BAX was determined by Western blot with density quantitative analysis ( F , G ). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; A : n = 6; B – F : n = 3; Pita, pitavastatin

Article Snippet: CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase.

Techniques: Migration, Expressing, Transfection, Plasmid Preparation, Cell Culture, Western Blot

Journal: Cell Biology and Toxicology

Article Title: CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

doi: 10.1007/s10565-024-09848-7

Figure Lengend Snippet: The reduction effects of pitavastatin on tumor progression are regulated by CD36/AKT/mTOR pathway. A–E A549 ( A ) and NCI-H520 ( B ) cells were treated with 150 µM FFAs or 5 µM pitavastatin plus FFAs for 24 h. A549 cells were transfected with pCMV or pCMV-CD36 plasmid for 12 h ( C ); NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid ( D , E ) for 12 h and then cultured in complete medium for 24 h, followed by treatment with 5 µM pitavastatin ( C , D ) or 150 µM FFAs ( E ) for 24 h. Protein expression of p-AKT, AKT, p-mTOR, and mTOR was detected by Western blot. F , G Tumor paraffin sections collected from Fig. A ( F ) or Fig. A ( G ) were performed IHC staining to detect the expression of p-AKT and p-mTOR with MD quantified by ImageJ software. H–L A549 cells were transfected with pCMV or pCMV-CD36 plasmid for 12 h and then cultured in complete medium for 24 h, followed by received LY294002 (10 µM) treatment for 24 h. Cells were collected for determination of cell viability ( H ) and apoptosis ( I , J ). Protein expression of CD36, vimentin, PCNA, BAX, p-AKT, AKT, p-mTOR, and mTOR was determined by Western blot ( K , L ). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; A – E , I – L : n = 3; F , G : n = 5; H : n = 6; Pita, pitavastatin; LY, LY294002

Article Snippet: CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Expressing, Western Blot, Immunohistochemistry, Software