Journal: Journal of Ovarian Research

Article Title: MSCs–derived EVs protect against chemotherapy-induced ovarian toxicity: role of PI3K/AKT/mTOR axis

doi: 10.1186/s13048-024-01545-7

Figure Lengend Snippet: Effect of MSCs-EVs, rapamycin, and quercetin on ( A ) phosphorylation of mTOR, PI3K, and AKT as detected by western blot analysis, ( B - D ) Intensity of immunoreactivity of selected proteins as quantified by densitometry. Results are expressed as mean ± SEM. **significant vs. Control group at p<0.01, **** at p<0.0001, # significant vs. OF group at p<0.05, ## at p<0.01, ### at p<0.001, #### at p<0.0001, & significant vs. OF + Rapamycin group at p<0.05

Article Snippet: Next, the blots were incubated with the appropriate primary antibodies for an entire night at 4 °C, PTEN (E-AB-63495, Elabscience, USA), FOXO3 (NBP2-16521, Novus Biologicals USA), mTOR (sc-517464, Santa Cruz Biotechnology, USA), Phospho-mTOR (sc-293133, Santa Cruz Biotechnology, USA), PI3K (E-AB-64202, Elabscience, USA), Phospho-PI3K (E-AB-20966, Elabscience, USA), AKT (E-AB-15441, Elabscience, USA), Phospho-AKT (E-AB-20804, Elabscience, USA), β-actin (E-AB-20031, Elabscience, USA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Cell

Article Title: A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits

doi: 10.1016/j.cell.2018.05.020

Figure Lengend Snippet: FCHSD2 Is Not Directly Recruited to CCPs by PI3KC2α or Its Kinase Activity, Related to Figure 2 (A) Top: Transferrin uptake assay by flow cytometry comparing wild-type and FCHSD2 KO cells silenced for PI3KC2α. Uptake of Alexa488 labeled transferrin normalized by the amount of surface transferrin receptor for each condition and against uptake for the wild-type cells in each experiment. Each value represents median fluorescence from at least 5000 cells (n = 12, mean ± SD). ∗∗∗ p > 0.001, ns = non-significant. One-way ANOVA with Tukey’s post hoc analysis. Bottom: Immunoblots showing PI3KC2α knockdown in wild-type and FCHSD2 KO cells. (B) Kymographs of HeLa FCHSD2-Venus stables silenced for PI3KC2α and control cells. Cells were transfected with mCherry-ClathrinLC 24 hs before imaging. Kymographs generated from 120 s movies at 1Hz. Note the elongated CCP lifetimes in PI3KC2α knockdown cells as described in Posor et al. (2013) . (C) Autoinhibition of FCHSD2. Representative images of a center slice from cells expressing different FCHSD2 truncation constructs and co-stained with phalloidin (Actin). The bar graph (upper right) shows the quantification of cellular protrusions/μm for each construct. The non-inhibited BAR domain produces many protrusions. Numbers inside bars represent number of cells measured. The line graph (bottom right) shows the fluorescence profile of sum intensity projections for cells expressing each construct. Due to the natural thinning of cells from their centers to the edge, a gradually decaying line indicates that the fluorescent protein is primarily cytosolic while a flat line with an abrupt fall on the cell edge indicates that the fluorescent protein is primarily bound to the membrane. While the presence of SH3-1 significantly reduces the generation of cellular protrusions generated by the FCHSD2 F-BAR, a significant fraction of the protein remains bound to the membrane (green line). Only the combined presence of SH3-1 and SH3-2 is capable to avoid promiscuous binding of the BAR domain to the membrane. Data is shown as mean ± SD in bar graph and as ± SEM in fluorescence profiles. ∗∗∗ p > 0.001, ns = non-significant. One-way ANOVA with Tukey’s post hoc analysis. (D) Immunoblots for intersectin knockdown in FCHSD2-Venus HeLa cells.

Article Snippet: PI3KC2α siRNA pool , Origene , Cat# SR303516.

Techniques: Activity Assay, Flow Cytometry, Labeling, Fluorescence, Western Blot, Knockdown, Control, Transfection, Imaging, Generated, Expressing, Construct, Staining, Membrane, Binding Assay

Journal: Cell

Article Title: A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits

doi: 10.1016/j.cell.2018.05.020

Figure Lengend Snippet:

Article Snippet: PI3KC2α siRNA pool , Origene , Cat# SR303516.

Techniques: Virus, Recombinant, Labeling, Cloning, Stable Transfection, Expressing, esiRNA, Plasmid Preparation, Software

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: DRI-Pep #20 is a potent PI3Kγ/PKA disruptor peptide. A , chemical structure of DRI-Pep #20. The amino acid sequence of DRI-Pep #20 comprises the nonnatural D-peptide RHQGK, the D-retroinverso (DRI)-isoform of the cell penetrating peptide Penetratin 1 (P1) and a glycine (G) linker. B , schematic representation of the fluorescence spectroscopy assays for the characterization of the interaction between DRI-Pep #20 (or PI3Kγ MP) and the recombinant fluorescein 5-maleimide–labeled PKA-RIIα (PKA-F5M). C , steady-state emission spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep #20 (0–20 μM). K D : dissociation constant. Inset, nonlinear fitting of the fluorescence intensity maxima obtained at various concentrations of DRI-Pep #20 for the monitoring of bio-labeled PKA. K A : association constant. D , for kinetic analysis, fluorescence spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep #20 or PI3Kγ MP (inset) were analyzed and fitted to a single exponential function to obtain the observed rate constant ( k obs ). The binding of DRI-Pep #20 or PI3Kγ MP to biolabeled PKA was investigated under pseudo -first-order conditions, and the kinetic constants, k on and k off , were determined. E , schematic representation of the displacement assay between DRI-Pep #20 (or PI3Kγ MP) and the PI3Kγ/PKA-F5M complex. F , percentage displacement of the PI3Kγ/PKA-RIIα complex by DRI-Pep #20 or PI3Kγ MP, calculated from steady-state emission spectra of the PI3Kγ/PKA-F5M complex in the presence of increasing concentrations of the peptides (0–5 μM). The displacement efficiency was expressed as percentage of the binding between PI3Kγ and PKA-F5M relative to that in the absence of peptides. G , cAMP concentrations in peritoneal macrophages from WT (in green ) and PI3Kγ −/− mice (in gray ) treated with DRI-Pep #20 (1–25 μM) for 30 min. The amount of cAMP was expressed as percentage of cAMP accumulation observed in untreated PI3Kγ −/− cells. n ≥ 6 technical replicates from N > 3 independent experiments. ∗∗∗ p < 0.001 WT versus PI3Kγ −/− and # p < 0.05, ## p < 0.01, and ### p < 0.001 UT versus DRI-Pep #20 by one-way ANOVA, followed by Bonferroni’s post hoc test. Data are means ± SD. AU, arbitrary units; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα; PI3Kγ, phosphoinositide 3-kinase gamma; PKA-F5M, fluorescein 5-maleimide–labeled PKA-RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Sequencing, Fluorescence, Spectroscopy, Recombinant, Labeling, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Binding kinetics of the interaction between DRI-Pep #20 or PI3Kγ MP and PKA-RIIα

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Structural prediction of the binding between DRI-Pep #20 and PKA-RIIα. A , DRI-Pep #20 structure prediction by PEP-FOLD3.5. P1-G and RHQGK domains are shown as cartoons in gray and red , respectively. R-1, H-2, Q-3, and K-5 residues are indicated and shown as sticks . B , circular dichroism spectra of DRI-Pep #20 showing a peak at 190–240 nm. The percentage of α-helical and β-sheet secondary structures calculated by the K2D3 software are indicated. C , molecular docking simulation of the interaction between DRI-Pep #20 and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of DRI-Pep #20 in complex with residues 2 to 44 of PKA-RIIα (cartoon in green ) is shown. The key residues involved in the binding are indicated and shown as sticks , with DRI-Pep #20 residues in bold . Hydrogen bonds between DRI-Pep #20 and PKA-RIIα are indicated by yellow dashed lines . In ( A and C ), the structural models were developed using PyMOL. DRI, D-retroinverso; HADDOCK, high ambiguity driven biomolecular DOCKing; PI3Kγ, phosphoinositide 3-kinase gamma; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Structural Proteomics, Binding Assay, Circular Dichroism, Software

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Structural prediction of the native binding between the N-terminal domain of PI3Kγ and PKA-RIIα. A , molecular docking simulation of the interaction between PI3Kγ and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of residues 109 to 159 of PI3Kγ in complex with residues 2 to 44 of the PKA-RIIα dimer ( green cartoon) is shown. The amino acids critical for the binding between the two proteins are shown and indicated as sticks , with the residues of PI3Kγ in bold . The putative PKA-binding motif of PI3Kγ (126–150) is shown in orange and blue . The sequence in orange indicates the region of PI3Kγ that was identified as being at the core of the interaction (KATHR). Hydrogen bonds between PI3Kγ and PKA-RIIα are indicated by yellow dashed lines . B , structural prediction of the KATHR sequence by PEP-FOLD3.5. KATHR and P1-G domains are shown as cartoons in orange and gray , respectively. K-18, H-21 and R-22 residues of the KATHR sequence (corresponding to K-126, H-129 and R-130 of native PI3Kγ) are indicated and shown as sticks . C , molecular docking simulation of the interaction between KATHR and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of KATHR in complex with residues 2 to 44 of PKA-RIIα (cartoon in green ) is shown. Yellow dashed lines indicate hydrogen bonds between KATHR and 2 to 44 PKA-RIIα. The amino acids critical for the binding are indicated and shown as sticks , with KATHR residues in bold . Throughout, the structural models were developed using PyMOL. HADDOCK, high ambiguity driven biomolecular DOCKing; PI3Kγ, phosphoinositide 3-kinase gamma; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Structural Proteomics, Binding Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: DRI-Pep #20 increases cAMP levels locally in vivo in the airway tract of mice. A , schematic representation of the treatment schedule. Mice received DRI-Pep #20 through intratracheal (i.t.) instillation. B – D , cAMP concentrations in tracheas ( B ), lungs ( C ) and hearts ( D ) from BALB/c mice 24 h after i.t. instillation of different doses of DRI-Pep #20 (0–750 mg/kg). Values in brackets indicate the dose of DRI-Pep #20 expressed as mg/kg. The number of mice (n) ranged from three to six per group. EC 50 , median effective concentration. E – G , cAMP concentrations in tracheas ( E ), lungs ( F ) and hearts ( G ) from WT and PI3Kγ −/− mice 24 h after i.t. instillation of 10 μg/Kg DRI-Pep #20 (in green ) or PBS (in gray ). The number of mice (n) ranged from three to four per group. In ( A and B ), ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 by one-way ANOVA, followed by Bonferroni’s post hoc test. In ( E and F ) ∗ p < 0.05 and ∗∗ p < 0.01 PBS versus DRI-Pep #20 by two-way ANOVA test, followed by Bonferroni’s post hoc analysis. Throughout, data are means ± SD. DRI, D-retroinverso; PI3Kγ, phosphoinositide 3-kinase γ.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: In Vivo, Concentration Assay

Journal: Molecular Cancer Research

Article Title: AKT1 E17K Inhibits Cancer Cell Migration by Abrogating β-Catenin Signaling

doi: 10.1158/1541-7786.mcr-20-0623

Figure Lengend Snippet: Figure 4. AKT2, but not AKT1, increases the transcriptional activity of b-catenin on the ZEB1 promoter, and decreases E-cadherin expression. A, p53ko/E17K cells were infected with a CRISPR/Cas9 lentivirus targeting AKT1 and p53ko cells were infected with a CRISPR/Cas9 lentivirus targeting AKT2. Cells lysates were then subjected to ChIP using a b-catenin antibody. Top: Western blot of whole cell lysates from the indicated p53ko/E17K cells and p53ko cells before and after AKT1 knockdown (AKT1KD) or AKT2 knockdown (AKT2KD) as specified. Bottom: Relative fold of ChIP pull-down of the ZEB1 promoter (b-catenin fold enrichment) was quantified by real-time qPCR, normalized by both input DNA and IgG values. Error bars, mean SEM (n ¼ 3). P values were determined by one-way ANOVA followed by Tukey post hoc test. , P < 0.0001. B, Western blots of lysates from MCF-10A p53ko/E17K or MCF7 PIK3CA WT cells expressing HA-tagged Myr-AKT2 and FLAG-tagged Myr-AKT1 alone and in combination. C, Western blot of lysates from p53ko cells: Left: infected with PTEN CRISPR/Cas9 lentivirus; Right: infected with PIK3CA E545K lentivirus. Cell lysate of parental MCF-10A cells was used as an E-cadherin–positive control.

Article Snippet: PIK3CA E545K–mutant cDNA (Cat#RC400348), purchased from OriGene, was inserted into the AsiS I and Mlu I sites of plasmid Lenti-C-Myc-DDK-IRES-Neo (Cat#PS100081, OriGene), to generate PIK3CA-E545K lentiviral expression vector.

Techniques: Activity Assay, Expressing, Infection, CRISPR, Western Blot, Knockdown, Positive Control

Journal: International journal of molecular sciences

Article Title: Increased Levels of the Parkinson's Disease-Associated Gene ITPKB Correlate with Higher Expression Levels of α-Synuclein, Independent of Mutation Status.

doi: 10.3390/ijms24031984

Figure Lengend Snippet: Figure 2. The ITPKB level is increased in SK-N-SH cell lines overexpressing wild-type or mutated A53T or A30P α-synuclein. SK-N-SH cells stably expressing wild-type (wt) or mutated A53T or A30P α-Synuclein were generated in house [24]. (A) Bar graphs represent ITPKB mRNA quantification in SK-N-SH cells or SK-N-SH cells overexpressing wild-type α-synuclein (wt) or A53T/A30P isoforms (top); ITPKB mRNA was also quantified in SH-SY5Y cells, and its level was compared with cells overexpressing LRRK2 or LRRK2 G2019S (bottom). N = 3–4 independent experiments were performed (B) ITPKB representative Western blot of 4–5 independent experiments. (C) Bar graphs represent ITPKB protein quantification in SK-N-SH cells or SK-N-SH cells overexpressing wild-type α-synuclein (wt) or A53T/A30P isoforms. N = 4–5 independent experiments were performed. Means ± SEM are shown. Statistical significance was calculated using a 1-way ANOVA with Bonferroni’s correction for multiple comparisons (ddPCR) or the Student’s t-test with Welch’s correction for unequal distribution (western blot). NS not significant, * p ≤0.05, ** p ≤0.01. **** p ≤0.0001.

Article Snippet: Membranes were stained with anti ITPKB (1:2000, 12816-1-AP, Proteintech, DBA, Milan, Italy), anti α-synuclein (1:2000, MAB5383, Abnova, Taipei City, Taiwan) or anti β-actin (1:8000, A-5316, Sigma-Aldrich, Milan, Italy).

Techniques: Stable Transfection, Expressing, Generated, Western Blot

Journal: International journal of molecular sciences

Article Title: Increased Levels of the Parkinson's Disease-Associated Gene ITPKB Correlate with Higher Expression Levels of α-Synuclein, Independent of Mutation Status.

doi: 10.3390/ijms24031984

Figure Lengend Snippet: Figure 3. The ITPKB level is increased in the nucleus and in the cytoplasm of SK-N-SH cell lines overexpressing wild-type or mutated A53T or A30P α-synuclein. (A) Representative images of SK-N-SH cells stained for ITPKB (green), α-synuclein (red) and DAPI (blue). Wild-type SK-N-SH (top, left) or SK-N-SK cells transfected with a plasmid carrying wild-type (top, right), A53T (bottom, left) or A3 (bottom, right) mutants are shown. Scale bar = 50 µm. (B) Bar graphs represent ITPKB (left) or α-synuclein (right) quantification in the nucleus. (C) Bar graphs show ITPKB (left) or α-synuclein (right) quantification in the cytoplasm (D) Bar graphs represent the ratio between nuclear (N) and cytoplasmatic (C) quantification for ITPKB (left) or α-synuclein (right). N = 10–12 different fields were acquired. Mean ± s.e.m. are shown. Statistical significance was calculated using a 1-way ANOVA with Bonferroni’s correction for multiple comparisons. * p ≤0.05, *** p ≤0.001. **** p ≤0.0001.

Article Snippet: Membranes were stained with anti ITPKB (1:2000, 12816-1-AP, Proteintech, DBA, Milan, Italy), anti α-synuclein (1:2000, MAB5383, Abnova, Taipei City, Taiwan) or anti β-actin (1:8000, A-5316, Sigma-Aldrich, Milan, Italy).

Techniques: Staining, Transfection, Plasmid Preparation

Journal: International journal of molecular sciences

Article Title: Increased Levels of the Parkinson's Disease-Associated Gene ITPKB Correlate with Higher Expression Levels of α-Synuclein, Independent of Mutation Status.

doi: 10.3390/ijms24031984

Figure Lengend Snippet: Figure 6. Experimental workflow. IP3K2 expression was reduced in flies overexpressing human α-synuclein. Geotaxis and lifespan PD-related phenotypes were measured (A). ITPKB was quantified in SK-N-SH cells overexpressing wild-type or mutated A53T or A30P α-synuclein by western blot (B, top). ITPKB protein level was measured also in the cytoplasm and in the nucleus of cells, separately (B, bottom). ITPKB and SNCA mRNA levels were quantified in cortex from PD patients and controls (C).

Article Snippet: Membranes were stained with anti ITPKB (1:2000, 12816-1-AP, Proteintech, DBA, Milan, Italy), anti α-synuclein (1:2000, MAB5383, Abnova, Taipei City, Taiwan) or anti β-actin (1:8000, A-5316, Sigma-Aldrich, Milan, Italy).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: A four-gene signature predicts survival in clear-cell renal-cell carcinoma

doi: 10.18632/oncotarget.12631

Figure Lengend Snippet: A, B. Low expression of PTEN and PIK3C2A were correlated with high risk, poor prognosis and shorter OS time. C, D. High expression of ITPA and BCL3 indicated high risk, poor prognosis and shorter OS time. Kaplan-Meier survival curves were also constructed to reveal the relationship between predicted risk of ccRCC patients and the OS time. The results showed that patients with high risk had a significantly shorter OS time than those with low risk (A-D). Green and red lines indicated low- and high-risk groups, respectively. P <0.05 was considered to be statistically significant. Cens: Censored; Event: Death; Prog. Idx.: Prognosis Index.

Article Snippet: Subsequently, the sections were first incubated with protein blocker for 1 h at 37°C, and then incubated with anti-PTEN antibody (Ready-to-use, ZSGB-BIO, ZA-0251, China), anti-PIK3C2A antibody (1:50, Proteintech, 22028-1-AP, China), anti-ITPA antibody (1:50, Proteintech, 16134-1-AP, China) and anti-BCL3 antibody (1:50, Proteintech, 23959-1-AP, China) at 4°C overnight.

Techniques: Expressing, Construct

Journal: Oncotarget

Article Title: A four-gene signature predicts survival in clear-cell renal-cell carcinoma

doi: 10.18632/oncotarget.12631

Figure Lengend Snippet: IHC analysis on tissue microarrays and Kaplan-Meier survival curves were constructed to verify the relationship between PTEN A. , PIK3C2A B. , ITPA C. and BCL3 D. expression with regard to OS and DFS. (A, B) Negative expression of PTEN and PIK3C2A were correlated with shorter OS and DFS time and worse prognosis. (C, D) Positive expression of ITPA and BCL3 were correlated with shorter OS and DFS time and worse prognosis. Green and blue lines indicated positive and negative expression groups, respectively. P <0.05 was considered to be statistically significant.

Article Snippet: Subsequently, the sections were first incubated with protein blocker for 1 h at 37°C, and then incubated with anti-PTEN antibody (Ready-to-use, ZSGB-BIO, ZA-0251, China), anti-PIK3C2A antibody (1:50, Proteintech, 22028-1-AP, China), anti-ITPA antibody (1:50, Proteintech, 16134-1-AP, China) and anti-BCL3 antibody (1:50, Proteintech, 23959-1-AP, China) at 4°C overnight.

Techniques: Construct, Expressing

Journal: Oncotarget

Article Title: A four-gene signature predicts survival in clear-cell renal-cell carcinoma

doi: 10.18632/oncotarget.12631

Figure Lengend Snippet: A. SurvExpress was used to analyze the association of the four-gene signature with the predicted risk, survival time and prognosis. B. The gene expression level of PTEN, PIK3C2A, ITPA and BCL3 were detected in high risk and low risk group. C. Kaplan-Meier survival curves showed that patients with predicted high risk ( n = 219) had significantly shorter OS time than those with low risk ( n = 249) ( P <0.05). D. ROC analysis was performed to compare the sensitivity and specificity of the survival prediction between our models. P <0.05 was considered to be statistically significant. Cens: Censored; Event: Death; Prog. Idx.: Prognosis Index; Sur.(M): Survival status (Month).

Article Snippet: Subsequently, the sections were first incubated with protein blocker for 1 h at 37°C, and then incubated with anti-PTEN antibody (Ready-to-use, ZSGB-BIO, ZA-0251, China), anti-PIK3C2A antibody (1:50, Proteintech, 22028-1-AP, China), anti-ITPA antibody (1:50, Proteintech, 16134-1-AP, China) and anti-BCL3 antibody (1:50, Proteintech, 23959-1-AP, China) at 4°C overnight.

Techniques: Gene Expression

Journal: Oncotarget

Article Title: A four-gene signature predicts survival in clear-cell renal-cell carcinoma

doi: 10.18632/oncotarget.12631

Figure Lengend Snippet: Correlation between clinicopathological features and the four proteins expression in ccRCC ( n =174)

Article Snippet: Subsequently, the sections were first incubated with protein blocker for 1 h at 37°C, and then incubated with anti-PTEN antibody (Ready-to-use, ZSGB-BIO, ZA-0251, China), anti-PIK3C2A antibody (1:50, Proteintech, 22028-1-AP, China), anti-ITPA antibody (1:50, Proteintech, 16134-1-AP, China) and anti-BCL3 antibody (1:50, Proteintech, 23959-1-AP, China) at 4°C overnight.

Techniques: Expressing

Journal: Oncotarget

Article Title: A four-gene signature predicts survival in clear-cell renal-cell carcinoma

doi: 10.18632/oncotarget.12631

Figure Lengend Snippet: Univariate and multivariate Cox proportional hazard regression analyses of the association of clinicopathological characteristics and the four genes' expression levels with OS

Article Snippet: Subsequently, the sections were first incubated with protein blocker for 1 h at 37°C, and then incubated with anti-PTEN antibody (Ready-to-use, ZSGB-BIO, ZA-0251, China), anti-PIK3C2A antibody (1:50, Proteintech, 22028-1-AP, China), anti-ITPA antibody (1:50, Proteintech, 16134-1-AP, China) and anti-BCL3 antibody (1:50, Proteintech, 23959-1-AP, China) at 4°C overnight.

Techniques: Expressing

Journal: Oncotarget

Article Title: A four-gene signature predicts survival in clear-cell renal-cell carcinoma

doi: 10.18632/oncotarget.12631

Figure Lengend Snippet: Univariate and multivariate Cox proportional hazard regression analyses of the association of clinicopathological characteristics and the four genes' expression levels with DFS

Article Snippet: Subsequently, the sections were first incubated with protein blocker for 1 h at 37°C, and then incubated with anti-PTEN antibody (Ready-to-use, ZSGB-BIO, ZA-0251, China), anti-PIK3C2A antibody (1:50, Proteintech, 22028-1-AP, China), anti-ITPA antibody (1:50, Proteintech, 16134-1-AP, China) and anti-BCL3 antibody (1:50, Proteintech, 23959-1-AP, China) at 4°C overnight.

Techniques: Expressing

Journal: OncoTargets and Therapy

Article Title:

Antitumor Effect of miR-1294/Pyruvate Kinase M2 Signaling Cascade in Osteosarcoma Cells

doi: 10.2147/ott.s232718

Figure Lengend Snippet: Figure 4 PKM2 was a target of miR-1294 in osteosarcoma cells. (A) PKM2 expression in human mesenchymal stem cells (hMSCs) and five osteosarcoma cell lines (Saos-2, MG63, U2OS, HOS, and 143B) was assessed by real-time PCR. (B) The miR-1294 binding site predictions for PKM2 3ʹUTR by microRNA.org. (C) A dual luciferase reporter assay was performed to confirm the binding in 293T cells. (D and E) PKM2 expression in U2OS and HOS cells transfected with miR-1294 mimic by real-time PCR and Western blot assays. (F and G) PKM2 expression in 143B cells transfected with miR-1294 inhibitor by real-time PCR and Western blot assays. Data are shown as mean ± SD. ∗P < 0.05, ∗∗∗P < 0.001.

Article Snippet: After dewaxing with xylene and rehydration with gradient ethanol, the sections were subjected to antigen retrieval and incubation with one of the primary antibodies against c-Myc (1:200; Proteintech), cyclin D1 (1:200; Boster), MMP2; (1:200; Proteintech), MMP-9 (1:200; Proteintech), and PKM2 (1:200; Proteintech) overnight at 4°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Luciferase, Reporter Assay, Transfection, Western Blot

Journal: OncoTargets and Therapy

Article Title:

Antitumor Effect of miR-1294/Pyruvate Kinase M2 Signaling Cascade in Osteosarcoma Cells

doi: 10.2147/ott.s232718

Figure Lengend Snippet: Figure 5 Overexpression of PKM2 reversed the actions of miR-1294 upregulation on osteosarcoma cells. (A) Cell proliferation was evaluated by CCK-8 assay. (B) Cell apoptosis was examined by flow cytometry using Annexin Vand PI staining. (C) Cell migration was assessed by a wound-healing assay. (D) Cell invasiveness was determined by transwell assay. (E) Expression levels of c-Myc, cyclin D1, cleaved-caspase 3, MMP-2, and MMP-9 were texted by Western blotting. Data are shown as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: After dewaxing with xylene and rehydration with gradient ethanol, the sections were subjected to antigen retrieval and incubation with one of the primary antibodies against c-Myc (1:200; Proteintech), cyclin D1 (1:200; Boster), MMP2; (1:200; Proteintech), MMP-9 (1:200; Proteintech), and PKM2 (1:200; Proteintech) overnight at 4°C.

Techniques: Over Expression, CCK-8 Assay, Cytometry, Staining, Migration, Wound Healing Assay, Transwell Assay, Expressing, Western Blot

Journal: OncoTargets and Therapy

Article Title:

Antitumor Effect of miR-1294/Pyruvate Kinase M2 Signaling Cascade in Osteosarcoma Cells

doi: 10.2147/ott.s232718

Figure Lengend Snippet: Figure 6 Overexpression of miR-1294 inhibited tumorigenicity of osteosarcoma cells. (A, B) Tumor volume in nude mice after subcutaneous injection of ME63 and U2O2 cells with or without overexpression of miR-1294. (C) The expression of miR-1294 in tumor tissues was determined by real-time PCR. (D, E) The expression of PKM2, c-Myc, cyclin D1, MMP-2, and MMP-9 in tumor tissues was assessed using immunohistochemistry and Western blot analysis. Data are shown as mean ± SD from 6 mice/ group. ∗P < 0.05, ∗∗∗P < 0.001.

Article Snippet: After dewaxing with xylene and rehydration with gradient ethanol, the sections were subjected to antigen retrieval and incubation with one of the primary antibodies against c-Myc (1:200; Proteintech), cyclin D1 (1:200; Boster), MMP2; (1:200; Proteintech), MMP-9 (1:200; Proteintech), and PKM2 (1:200; Proteintech) overnight at 4°C.

Techniques: Over Expression, Injection, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Western Blot