Journal: Targeted Oncology

Article Title: Absorption, Distribution, and Binding Profile of ME-401, a Potent and Selective Oral Small-Molecule Inhibitor of Phosphatidylinositol 3-Kinase δ (PI3Kδ) in Animal and B-Cell Lymphoma Models

doi: 10.1007/s11523-019-00668-y

Figure Lengend Snippet: Binding kinetics of ME-401 and idelalisib on PIK3CD/PIK3R1

Article Snippet: Kinase protein immobilization: An aliquot of biotinylated PIK3CD/PIK3R1 (Carna Biosciences, Inc., Kobe, Japan) was thawed, diluted to 40 µg/mL in precooled Biacore buffer (50 mM Tris pH 7.5, 0.05% Tween, 150 mM NaCl, and 5 mM MgCl 2 ), and immobilized with the biotin tag on a Series S Sensor Chip SA.

Techniques: Binding Assay

Journal: The Scientific World Journal

Article Title: Leucaena leucocephala Fruit Aqueous Extract Stimulates Adipogenesis, Lipolysis, and Glucose Uptake in Primary Rat Adipocytes

doi: 10.1155/2014/737263

Figure Lengend Snippet: List of probes used in Real-Time PCR.

Article Snippet: Phosphatidylinositol-3-kinase , Pik3r1/PI3KA , Rn00564547_m1 , NM_013005.1.

Techniques: Binding Assay

Journal: Medicine

Article Title: Characterization and Prognostic Significance of Methylthioadenosine Phosphorylase Deficiency in Nasopharyngeal Carcinoma

doi: 10.1097/MD.0000000000002271

Figure Lengend Snippet: MTAP gene dosage and methylation-specific PCR for analysis of MTAP promoter methylation in nasopharyngeal carcinoma. Representative low-stage NPC with intact MTAP expression has a preserved MTAP gene, with a Ct value similar to that of PIK3R1 , both <40 in a quantitative DNA-PCR (A). Representative high-stage and MTAP-deficient NPC has nondetectable MTAP gene, characterized by a Ct value no less than 40 (B). Methylation-specific PCR detects MTAP promoter methylation in MTAP protein-deficient NPCs without homozygous gene deletion (C). PCR products amplified from MTAP promoter with methylation-specific primers can be distinctly visualized in both NPC69 and NPC74, while the MTAP gene promoter of a representative MTAP-intact NPC, NPC22, is unmethylated. Pyrosequencing is performed to quantify the percentage of methylation in specific CpG sites of MTAP (D). The percentages of methylation in NPC69 and NPC74 are 23% and 26%, respectively. While in NPC22, the percentage is 4%, which is less than the cut-off value of methylation and regarded as unmethylated. M = methylation-specific primers, M.SssI = a healthy donor's blood DNA treated with M. SssI methyltransferase, MTAP = methylthioadenosine phosphorylase, NP = DNA from nontumor nasopharyngeal mucosal tissue without treatment of M.SssI methyltransferase, NPC = nasopharyngeal carcinoma, PCR = polymerase chain reaction, U = unmethylation-specific primers.

Article Snippet: The TaqMan assays targeting PIK3R1 (Hs06028467_cn) in 5q13.1 and the exon 8 of the MTAP gene (Hs02079487_cn) in 9p21.3 were purchased from Applied Biosystems.

Techniques: Methylation, Expressing, Amplification, Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: PIK3R1 is down-regulated at both the genomic and mRNA expression level in breast cancer. (A) The incidence of PIK3R1 copy-number loss or mutation in TCGA breast cancer cohorts. (B) PIK3R1 expression in breast cancer compared with normal breast (Oncomine). Means ± SEM are shown. *P < 0.05, **P < 0.01, ****P < 0.0001; unpaired t test.

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques: Expressing, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: Compilation of sources and data used for Oncomine analysis of PIK3R1 expression levels via microarray in breast cancer datasets

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques: Expressing, Microarray

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: PIK3R1 knockdown increases RTK-mediated PI3K/AKT signaling in and transformation of HMECs. (A) Control and shPIK3R1 HMECs were starved and then stimulated with EGF before immunoblotting. (B) Cell suspensions of control and shPIK3R1 HMECs were grown in agar. (C) Control, shPIK3R1, and rescue HMECs were treated as in A before immunoblotting. (D) Cell suspensions of control, shPIK3R1, and PIK3R1 rescue HMECs were grown in agar. (E) Control, shPIK3R1, or p110α-H1047R HMECs expressing activated HER2/neu were starved overnight before immunoblotting. (F) Control and shPIK3R1 HER2/neu HMECs were treated as in A before immunoblotting. (G) Cell suspensions of control and shPIK3R1 HER2/neu HMECs were grown in agar. Means ± SEM are shown; n = 3. *P < 0.05, **P < 0.01, ****P < 0.0001; unpaired t test.

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques: Transformation Assay, Western Blot, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: (A) HMECs of the indicated genotypes were starved for 4 h; protein lysates were collected and subjected to immunoblotting. (B) Bands from immunoblots as in A were quantified by densitometry. Means ± SEM are shown; n = 3 for each. Statistical significance was determined by unpaired t test. (C) Control, PIK3R1 knockdown, or PIK3R1 rescue HMECs were starved and then stimulated with EGF (20 ng/mL). Protein lysates were collected and subjected to immunoblotting. *P < 0.05, ****P < 0.0001; unpaired t test; ns, not significant.

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques: Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: (A) Mouse mammary epithelial cells were derived from the mammary glands of individual nulliparous females of the indicated genotypes and collected and subjected to immunoblotting. (B) Whole mounts were prepared from the fourth inguinal mammary glands of MMTV-Cre, MMTV-Cre; Pik3r1+/−, and MMTV-Cre; Pik3r1−/− female mice during puberty (6 and 12 wk nulliparous), pregnancy (14 d pregnant), and lactation (2 d postpartum). Mammary glands were stained with carmine red to visualize ducts. Representative images from each genotype and stage are shown. (C) Hematoxylin and eosin (H&E) staining of primary spontaneous mammary tumors from the indicated genotypes. (Scale bar, 50 μm.) (D) H&E staining of lung metastases of primary mammary tumors from mice with the indicated genotypes. (Scale bars, 100 μm.)

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques: Derivative Assay, Western Blot, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: Mouse tumor outcomes from the indicated genotypes

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: Tumors characterized by activated Her2/Neu and p85α loss are sensitive to pan-PI3K and p110α-selective inhibition. (A) Tumor-free survival of the indicated mouse genotypes. Median tumor-free survival: NIC, 140 d (n = 25); NIC; Pik3r1+/−, 125 d (n = 38); NIC; Pik3r1−/−, 126 d (n = 43); log-rank (Mantel–Cox) test. (B) Mammary tumor lysates of the indicated genotypes were subjected to immunoblotting. MG, mammary gland from MMTV-Cre mouse genotype. (C) Tumor volume of orthotopically transplanted NIC; Pik3r1+/− mammary tumors following once-daily treatment with GDC-0941, BYL-719, or KIN-193. End-point tumor volumes: vehicle, 942.6 ± 222.9 mm3; GDC-0941, 199.5 ± 66.8 mm3; BYL-719, 293.2 ± 85.0 mm3; KIN-193, 801.6 ± 111.1 mm3. Means ± SEM are shown; all treatment groups n ≥ 10; two-way ANOVA with Tukey’s multiple-comparisons test. (D) Transplanted NIC; Pik3r1+/− mammary tumors were treated as in C. One hour after treatment on day 4, recipients were killed and tumor tissue was subjected to immunoblotting. (E) The percentage of Ki67- or TUNEL-positive nuclei in Fig. S3G. Means ± SEM are shown; all groups n = 8. **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired t test.

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques: Inhibition, Western Blot, TUNEL Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: (A) Mammary tumor tissue from NIC, NIC; Pik3r1+/−, and NIC; Pik3r1−/− female mice was fixed in formalin and stained with H&E. (Scale bar, 50 μm.) (B) The total wet weight of mammary tumors plus associated mammary gland tissue per mouse from NIC (n = 16), NIC; Pik3r1+/− (n = 20), and NIC; Pik3r1−/− (n = 20) females was determined 5 wk after initial tumor palpation. Means ± SEM are shown. Statistical significance was determined by unpaired t test. Significance for NIC; Pik3r1+/− and NIC; Pik3r1−/− in comparison with the NIC control is shown. (C) Number of lung metastases per mouse 5 wk after initial tumor palpation. n = 8 for each genotype. (D) Number of total tumors per mouse 5 wk after initial tumor palpation. n = 8 for each genotype. (E, Top) Immunohistochemical (IHC) analysis of formalin-fixed mammary tumor tissue from mice of the indicated genotypes. (Scale bar, 50 μm.) (E, Bottom) The percentage of Ki67-positive nuclei from IHC analysis. n = 12. (Scale bar, 50 μm.) (F) Body weight of NCrNu female mice with orthotopic transplant of NIC; Pik3r1+/− mammary tumors treated daily with GDC-0941 (125 mg/kg), BYL-719 (45 mg/kg), KIN-193 (20 mg/kg), or vehicle control as in Fig. 4C. Means ± SEM are shown; n ≥ 10 for each treatment group. (G) IHC analysis of mammary tumor tissue from transplanted NIC; Pik3r1+/− mammary tumors treated as in Fig. 4D. (Scale bars, 50 μm.) **P < 0.01, ****P < 0.0001; unpaired t test.

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques: Staining, Immunohistochemical staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: (A) Control and shPIK3R1 HMECs were starved and then stimulated with EGF (20 mg/mL) for 5 min. Protein lysates were collected and subjected to immunoprecipitation (IP) for endogenous p85α; immunoprecipitates were subjected to immunoblotting. (B) Control and shPIK3R1 HMECs were starved and then stimulated with EGF (20 mg/mL) for 30 min. Protein lysates were collected and subjected to immunoprecipitation for endogenous PTEN; immunoprecipitates were subjected to immunoblotting. (C) 293T cells were transfected with wild-type PIK3R1. Protein lysates were collected and subjected to immunoprecipitation for PTEN or p85α; immunoprecipitates were subjected to immunoblotting. (D) mRNA levels of INPP4B and PTEN mRNA in control and shPIK3R1 HMECs. Means ± SD are shown for one experiment performed in triplicate. Levels were normalized to the corresponding mean for shControl. (E and F) In vitro lipid phosphatase assays for PTEN immunoprecipitated from control and shPIK3R1 HMECs. A malachite green reagent was used to detect phosphate in prepared standards (E) or released upon incubation of PtdIns(3,4,5)P3 substrate with PTEN immunoprecipitates (F). Three negative controls were used: PIP3 only, assay performed using substrate but no source of PTEN; PTEN only, assay performed using PTEN immunoprecipitated from shControl cells but with no substrate added; and beads only, assay performed using precipitates from shControl cells in which no PTEN antibody was used. The amount of phosphate released in in vitro assays was interpolated from comparison with the phosphate standards. (E) Means ± SD are shown for standards in triplicate. (F) Means ± SEM are shown for assays performed using three independent immunoprecipitation reactions per cell line. Statistical analysis was performed using unpaired t test compared with shControl. *P < 0.05; ns, not significant.

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques: Immunoprecipitation, Western Blot, Transfection, In Vitro, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α

doi: 10.1073/pnas.1704706114

Figure Lengend Snippet: PIK3R1 knockdown increases the amount of RTK-bound p110α–p85 in HMECs. (A) Control and shPIK3R1 Flag-ErbB3 HMECs were starved and then lysed. ErbB3 was immunoprecipitated using anti-Flag beads and subjected to immunoblotting. IP, immunoprecipitation. (B–G) Bands from immunoblots as in A were quantified. (B–D) Quantification of protein levels in ErbB3 IPs. (B) Protein levels of p110α were normalized to pan-p85 in IPs, then to the mean for shControl. (C and D) Protein levels were normalized to ErbB3 in the IPs, then to the mean for shControl. (E–G) Quantification of protein in inputs. (E and F) Protein was normalized to vinculin, then to the mean for shControl. (G) Protein was normalized to AKT, then to the mean for shControl. Means ± SEM are shown; n = 4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired t test. (H) Model in which free p85 competes with p85–p110 to negatively regulate RTK-mediated PI3K/AKT signaling in mammary epithelial cells. (H, Top) In cells with normal p85α levels, p85 is present in excess of p110. Both free p85 and p85–p110 can compete for binding to activated RTKs, but only p85–p110 can signal. (H, Bottom) In cells with reduced p85α, the pool of free p85 is selectively depleted. More binding sites on activated RTKs are available for p85–p110, allowing for up-regulated PI3K/AKT signaling.

Article Snippet: To generate constructs for rescue of PIK3R1 knockdown, a pCMV6 entry vector containing the cDNA ORF of wild-type human PIK3R1 with C-terminal Myc and Flag tags (OriGene; RC210544) was subcloned into the pWZL-blasticidin retroviral vector.

Techniques: Immunoprecipitation, Western Blot, Binding Assay

Journal: International Dental Journal

Article Title: High-Glucose Microenvironment Accelerates Malignant Progression Via O-GlcNAcylation in Oral Squamous Cell Carcinoma

doi: 10.1016/j.identj.2025.103897

Figure Lengend Snippet: The effects of OGT on cell proliferation, metastasis, and apoptosis partially depend upon the PI3K/AKT/mTOR signalling pathway in OSCC cells. (A-C) Levels of PI3K, AKT, mTOR, p-PI3K, p-AKT, p-mTOR, OGT, and O-GlcNAcylation proteins were assessed in OSCC cells exposed to a medium with different glucose concentrations and OGT knockdown. n = three independent experiments. Data were presented as the mean ± SD. P values were determined by one-way ANOVA. * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: Antibodies against N-cadherin (N-cad) (1:1000 for WB, HY- P80238 ), E-cadherin (E-cad) (1:1000 for WB, P80113), vimentin (1:1000 for WB, HY- P80371 ), Bcl2 (1:1000 for WB, P80566 ), Bax (1:1000 for WB, P80028 ), PI3K (1:1000 for WB, HY- P80867 ), and phosphorylated P13K (p-PI3K) (1:1000 for WB, HY- P80846 ) were purchased from MedChemExpress; antibodies against AKT (1:1000 for WB, 4691T) and p-AKT (1:1000 for WB, 13038T) were purchased from Cell Signaling Technology; antibodies against OGT (1:5000 for WB, 1:250 for IHC, 66823-1-Ig), mammalian target of rapamycin (mTOR) (1:5000, 66888-1-Ig), p-mTOR (1:5000, 67778-1-Ig), and β-Actin (1:20,000 for WB, 66009-1-Ig) were obtained from Proteintech; and the O-GlcNAc antibody (1:1000 for WB, 1:200 for IHC, MA1-072) was purchased from Invitrogen.

Techniques: Knockdown

Journal: Journal of Clinical Pathology

Article Title: Microbial infections in eight genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis

doi: 10.1136/jcp.2009.072561

Figure Lengend Snippet: CFS/ME-associated genes and transcription factors in patients with CFS/ME, Q-fever-associated CFS/ME and endogenous depression

Article Snippet: PIK3R1 , NM_181523 , Hs00236128_m1 , 4.04 , 0.005 , 2.60 , 0.01 , 1.14 , 0.208.

Techniques:

Journal: Journal of Clinical Pathology

Article Title: Microbial infections in eight genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis

doi: 10.1136/jcp.2009.072561

Figure Lengend Snippet: Fold-difference values for 88 genes in each of eight subtypes (A–H) in 114 subtyped patients with chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME). Genes without values for the subtypes are those for which there was missing data for one or more subtypes. Bold type indicates genes targeted by existing drugs and those CFS/ME subtypes in which fold-difference values of 1.5 were found

Article Snippet: PIK3R1 , NM_181523 , Hs00236128_m1 , 4.04 , 0.005 , 2.60 , 0.01 , 1.14 , 0.208.

Techniques: