Journal: EMBO Molecular Medicine

Article Title: Gene signature driving invasive mucinous adenocarcinoma of the lung

doi: 10.15252/emmm.201606711

Figure Lengend Snippet: A549 cells were infected with Nkx2‐1 ‐expressing lentivirus as previously reported (Maeda et al , ). ChIP‐seq indicates that NKX2‐1 bound to the locus of PD‐L1 and PD‐L2 . PD‐L1 and PD‐L2 mRNAs were significantly induced by NKX2‐1. Results are expressed as mean ± SEM of biological triplicates for each group. P < 0.05 versus control was considered significant (Student's t ‐test). Gene expression was normalized by comparison with the constitutive expression of GAPDH . Control: control lentivirus; Nkx2‐1 : Nkx2‐1 ‐expressing lentivirus. A549 cells stably expressing Nkx2‐1 were developed as described in (A) and (B). Protein expression was confirmed by IB. PD‐L1 was detected by antibodies from Cell Signaling (CST), Spring Bioscience (Spring) and Sino Biological (Sino) as described in . ACTA1 was used as a loading control. Shown is a representative image from three independent experiments. NKX2‐1 induced PD‐L1 in A549 cells. H2122, Calu‐3, and H292 mucus‐producing lung cancer cells were infected with control lentivirus or Nkx2‐1 ‐expressing lentivirus. Protein expression was confirmed by IB. PD‐L1 antibody from Cell Signaling (CST) was used to detect PD‐L1 protein. ACTA1 was used as a loading control. Shown is a representative image from two independent experiments. NKX2‐1 induced PD‐L1 in the mucus‐producing lung cancer cells. NKX2‐1 (black) was expressed in the nucleus of non‐mucinous lung tumor cells (non‐IMA) but not in mucinous lung tumor cells (IMA) in human. Scale bar: 50 μm. PD‐L1 was expressed in NKX2‐1‐positive lung tumor cells compared to NKX2‐1‐negative lung tumor cells in human (two‐tailed Fisher's exact test: P ‐value = 2.078E‐02). PD‐L1‐positive cases include > 5% of tumor cells expressing PD‐L1 in cell membrane. PD‐L1 (black) was expressed in cell membranes of non‐mucinous lung tumor cells (non‐IMA) but not in mucinous lung tumor cells (IMA) in human. Scale bar: 50 μm. PD‐L1 was expressed in non‐mucinous lung tumor cells (non‐IMA) but not in mucinous lung tumor cells (IMA) in human. PD‐L1‐positive cases were determined as described in (F). Source data are available online for this figure.

Article Snippet: A549 lung carcinoma cells (Lot# F‐10600), H2122 lung adenocarcinoma cells (Lot# 59399669), Calu‐3 lung adenocarcinoma cells (Lot# 57814093), and H292 lung mucoepidermoid carcinoma cells (Lot# 3895200) were obtained directly from ATCC (Manassas, VA).

Techniques: Infection, Expressing, ChIP-sequencing, Control, Gene Expression, Comparison, Stable Transfection, Two Tailed Test, Membrane

Journal: Molecular Metabolism

Article Title: Enhanced dynorphin expression and secretion in pancreatic beta-cells under hyperglycemic conditions

doi: 10.1016/j.molmet.2024.102088

Figure Lengend Snippet: Inhibition of CamKII decreases Pdyn levels in mouse and human islets . Pdyn mRNA expression measured by RT-PCR in islets treated with (A) LG, HG, CREBi with HG, STO-609 (10 μM) with HG and KN93 (10 μM) with HG; (B) LG, HG, KN92 (1 and 10 μM) and KN93 (1 and 10 μM) with HG; (C) LG, 5 mM glucose, KN92 (10 μM) and KN93 (10 μM) and Verapamil (50 μM) with 5 mM glucose. (D–E) Camk2b and Pdyn expression at the mRNA level by RT-PCR in MIN6 cells transfected with Camk2b -siRNA for 72 h. (F) Pdyn expression at the mRNA level by RT-PCR in islets treated with LG, HG with or without palmitate (Palm; 0.4 mM) and KN93 with HG with or without palmitate for 24 h. Immunoblotting and quantification of pCreb, pCamKII, Pdyn and Actin in (G) mouse and (H) human islets treated with LG, HG and HG + KN93 for 24 h. LG: grey bars and HG: blue bars. Data expressed as means ± s.e.m., ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The lyophilized small interfering RNA (siRNA) targeting CamKIIβ (Santa Cruz; sc-38952) was resuspended in 330 μl of the RNAse-free water, which makes a 10 μM solution, according to the manufacturer’s protocol.

Techniques: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot

Journal: PLoS ONE

Article Title: Small Heterodimer Partner-Targeting Therapy Inhibits Systemic Inflammatory Responses through Mitochondrial Uncoupling Protein 2

doi: 10.1371/journal.pone.0063435

Figure Lengend Snippet: ( A ) WT mice ( n = 3) received fenofibrate (100 mg/kg; administrated orally) or vehicle only for the indicated period. The expression of phosphorylated AMPKα in liver and spleen was assessed by immunoblotting (IB). Whole cell lysates (WCL) were used for IB with anti-Actin. ( B ) BMDMs were treated with fenofibrate (50 µM) for various times, followed by IB with phosphorylated forms of AMPKα, ACC, and SHP. WCL were used for IB with anti-β-actin as the loading control. ( C ) BMDMs were treated with fenofibrate (50 µM for 6 h) in the presence or absence of the AMPK inhibitor compound C (Comp C; 5, 10, or 25 µM). Total RNA was extracted from WCL and used for RT-PCR analyses of Shp and Actb mRNA; below, densitometry. ( D ) BMDMs were transduced for 48 h with adenovirus encoding GFP only (Ad-GFP), constitutively active AMPK (Ad-AMPK), or dominant negative AMPK (Ad-DN-AMPK; MOI = 10) and were then treated with fenofibrate (50 µM) for 4 h. Total RNA was extracted from WCL and analyzed by semi-quantitative RT-PCR for Shp and Actb mRNA. Representative gel images ( top ); densitometric analyses ( bottom ). ( E and F ) BMDMs were transduced with shNS or shLKB1 (E) or shCAMKKβ (F) prior to treatment with fenofibrate (50 µM) for the indicated time periods, followed by IB with antibodies against the phosphorylated forms of AMPKα and SHP. WCL were used for IB with anti-β-actin as the loading control. Total LKB1 (D) and CAMKKβ (E) protein expression was measured for transduction efficiency of lentiviral vectors. The data are representative of at least three independent experiments with similar results. Quantitative data are shown as the mean ± SD of three experiments ( C and D bottom ). Statistical differences (*, p <0.05; **, p <0.01), as compared to the control cultures, are indicated (paired t -test with Bonferroni adjustment). FF, fenofibrate. n.s. , non-specific.

Article Snippet: For silencing murine CaMKKβ, LKB1, or UCP2 in primary cells, pLKO.1-based lentiviral CaMKKβ shRNA constructs (sc-38952-SH) and LKB1 shRNA constructs (sc-35817-SH) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Dominant Negative Mutation, Quantitative RT-PCR, Transduction

Journal: IBRO Neuroscience Reports

Article Title: CaMKIIβ knockdown decreases store-operated calcium entry in hippocampal dendritic spines

doi: 10.1016/j.ibneur.2022.01.001

Figure Lengend Snippet: Analysis of the specificity and effectiveness of CaMKIIβ knockdown in HEK293T cells. Western blot analysis of the expression of Venus-CaMKIIα (A) and GFP-CaMKIIβ (B) proteins in HEK293T cell lysates after co-transfection with Venus-CaMKIIα or GFP-CaMKIIβ plasmids together with either plasmid encoding control shRNA (shControl), or shRNA specific for CaMKIIβ (shCaMKIIβ). Quantitative analysis of the expression of Venus-CaMKIIα (C) and GFP-CaMKIIβ (D) on Western blot results depicted on panels A and B. The expression levels of the proteins are normalized to tubulin. Results are presented as mean ± SEM with individual data points. Statistical analysis was performed using Student's t-test, p value is indicated on the figure. Each band corresponds to a cell lysate taken from single well of 24 well plate.

Article Snippet: CaMKIIβ shRNA (shCaMKIIβ) plasmid was obtained from Santa Cruz Biotechnology (#sc-38951-SH).

Techniques: Knockdown, Western Blot, Expressing, Cotransfection, Plasmid Preparation, Control, shRNA

Journal: IBRO Neuroscience Reports

Article Title: CaMKIIβ knockdown decreases store-operated calcium entry in hippocampal dendritic spines

doi: 10.1016/j.ibneur.2022.01.001

Figure Lengend Snippet: CaMKIIβ knockdown causes mushroom and stubby spines loss and increases percentage of thin spines in hippocampal cultures. (A) representative confocal images of dendritic spines in control neurons (Control, co-transfected with TD-Tomato and shControl plasmids) and in neurons with CaMKIIβ knockdown (co-transfected with TD-Tomato and shCaMKIIβ plasmids). Histograms with average percentages (B) and spine densities (C) of mushroom, stubby and thin spines in two experimental groups, control neurons and in neurons with CaMKIIβ knockdown (shCaMKIIβ), quantified with Neuronstudio. Results are presented as mean ± SEM, experiments were repeated at least 4 times. n (neurons) ≥ 27. Statistical analysis was performed using Student's t-test, * p < 0.05, *** p < 0.0001. Scale bar = 10 µm.

Article Snippet: CaMKIIβ shRNA (shCaMKIIβ) plasmid was obtained from Santa Cruz Biotechnology (#sc-38951-SH).

Techniques: Knockdown, Control, Transfection

Journal: IBRO Neuroscience Reports

Article Title: CaMKIIβ knockdown decreases store-operated calcium entry in hippocampal dendritic spines

doi: 10.1016/j.ibneur.2022.01.001

Figure Lengend Snippet: CaMKIIβ knockdown decreases store-operated calcium entry in postsynaptic spines in primary hippocampal cultures. (A) Time course of GCaMP5.3 relative fluorescence signal changes (F/F0) in individual spines of hippocampal neurons. Presence of Ca 2+ channels blockers (Tg, TTX, Nifedipine, D -AP5, CNQX) is indicated by grey bars. The time of extracellular Ca 2+ readdition is indicated by a black bar above the traces. Neurons were co-transfected with either shControl + GCamp5.3(Control) or shCaMKIIβ+ GCamp5.3 (shCaMKIIβ) plasmids. For each experimental group, individual spine (gray) and average (red) fluorescence traces are shown. (B), Average nSOCE spine peak amplitude is shown for each group of cells shown in A panel. Mean F/F0 peak amplitude signals for each group are presented as mean ± SEM (n ≥ 107 spines from 3 independent experiment). Statistical analysis was performed using Student's t-test, *** p < 0.0001.

Article Snippet: CaMKIIβ shRNA (shCaMKIIβ) plasmid was obtained from Santa Cruz Biotechnology (#sc-38951-SH).

Techniques: Knockdown, Fluorescence, Transfection, Control

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: Primer sets for PCR and qPCR.

Article Snippet: To silence CaMKIIβ expression, CaMKIIβ small-interfering RNA (CaMKIIβ siRNA) (Santa Cruz, Dallas, TX, USA, sc-38951) was transfected using INTERFERin ® siRNA transfection reagent (Polyplus, 409-10), according to the manufacturer’s protocol.

Techniques: Sequencing

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: The CaMKII blocker KN-93 decreases the migration and chloride currents of U251 cells. ( A ) Representative photographic images showing the migration of U251 cells treated with KN-93 as compared with the migration of the cells treated with dimethyl sulfoxide (DMSO). Scale bar, 200 μm. ( B ) The summary bar graph shows the reduced migration of U251 cells after treatment with KN-93 compared to that of cells treated with DMSO. ( C ) Averaged traces of whole-cell currents of U251 cells treated with KN-93. ( D ) The summary bar graph shows the inhibitory effect of KN-93 on the amplitude of the chloride current at ± 100 mV. Number on each bar indicates n for each condition. All values are mean ± s.e.m. P -values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To silence CaMKIIβ expression, CaMKIIβ small-interfering RNA (CaMKIIβ siRNA) (Santa Cruz, Dallas, TX, USA, sc-38951) was transfected using INTERFERin ® siRNA transfection reagent (Polyplus, 409-10), according to the manufacturer’s protocol.

Techniques: Migration

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: CaMKIIβ specifically increases the surface expression and activity of ANO1 in U251 cells. ( A ) The mRNA expression of CaMKII isoforms in U251 cells were detected with specific primers for CaMKIIα, β, γ and δ. ( B ) Normalized expression of CaMKII isoforms as compared to that of GAPDH in U251 cells. ( C ) Effect of CaMKII isoform overexpression on ANO1-mediated whole cell chloride currents in U251 cells. ( D ) The summary bar graph showing ANO1-mediated current density at ± 100 mV. ( E ) Cell surface biotinylation results from membrane protein fractions from U251 cells transfected with mCh-CaMKIIα and mCh-CaMKIIβ. ( F ) The summary bar graph showing data obtained from at least three independent experiments as in ( E ). Number on each bar indicates n for each condition. All values are mean ± s.e.m. P -values were obtained with Student’s t-test. ** p < 0.01 and *** p < 0.001. n.s means not significant.

Article Snippet: To silence CaMKIIβ expression, CaMKIIβ small-interfering RNA (CaMKIIβ siRNA) (Santa Cruz, Dallas, TX, USA, sc-38951) was transfected using INTERFERin ® siRNA transfection reagent (Polyplus, 409-10), according to the manufacturer’s protocol.

Techniques: Expressing, Activity Assay, Over Expression, Membrane, Transfection

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: CaMKIIβ knockdown reduces the surface expression of ANO1 in U251 cells. ( A,B ) Validation of the silencing efficiency of the siRNA against CaMKIIβ using qPCR and Western blotting. ( C ) U251 cells transfected with Sc shRNA or CaMKIIβ siRNA were imaged using antibodies against ANO1 and WGA. Nuclei were stained using DAPI staining solution. Scale bar, 20 μm. ( D ) Cell surface biotinylation results from U251 cells transfected with Sc shRNA or CaMKIIβ siRNA. ( E ) The summary bar graph shows the summary of ( D ), data obtained from three independent experiments. ( F ) Averaged traces of whole-cell currents of U251 cells transfected with Sc shRNA, CaMKIIβ siRNA, or T16Ainh-A01 (A01). ( G ) The summary bar graph shows the inhibitory effect of CaMKIIβ siRNA on ANO1–mediated current amplitude at ±100 mV. The bar graph shows normalized A01-sensitive current densities at + 100 mV. Number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with Student’s t-test. * p < 0.05 and ** p < 0.01.

Article Snippet: To silence CaMKIIβ expression, CaMKIIβ small-interfering RNA (CaMKIIβ siRNA) (Santa Cruz, Dallas, TX, USA, sc-38951) was transfected using INTERFERin ® siRNA transfection reagent (Polyplus, 409-10), according to the manufacturer’s protocol.

Techniques: Knockdown, Expressing, Biomarker Discovery, Western Blot, Transfection, shRNA, Staining

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: Gene silencing of ANO1 or CaMKIIβ leads to the attenuation of migration and invasion in U251 cells. ( A,B ) Validation of the silencing efficiency of the shRNA against ANO1 using qPCR and Western blotting. ( C ) Cell invasion assay of U251 cells infected with Lenti-ANO1 shRNA and transfected with CaMKIIβ siRNA. ( D ) The averaged bar graph shows the summary of ( C ), data obtained from three independent experiments. ( E ) Representative photographic images of the migration of U251 cells infected with Lenti-ANO1 shRNA and transfected with CaMKIIβ siRNA and the cells with Lenti-Sc shRNA and Sc siRNA control. Scale bar, 100 μm. ( F ) The averaged bar graph shows the summary of ( E ), data obtained from three independent experiments. All values are mean ± s.e.m. p -values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To silence CaMKIIβ expression, CaMKIIβ small-interfering RNA (CaMKIIβ siRNA) (Santa Cruz, Dallas, TX, USA, sc-38951) was transfected using INTERFERin ® siRNA transfection reagent (Polyplus, 409-10), according to the manufacturer’s protocol.

Techniques: Migration, Biomarker Discovery, shRNA, Western Blot, Invasion Assay, Infection, Transfection, Control

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: Inhibition of CaMKIIβ and/or ANO1 decreased migration and invasion of U87MG cells. ( A,B ) Validation of the silencing efficiency of the Lenti-ANO1shRNA and the CaMKIIβ siRNA with qPCR and Western blot in U87MG cells. ( C ) Cell invasion assay of U87MG cells infected with Lenti-ANO1shRNA and transfected with CaMKIIβ siRNA. ( D ) The averaged bar graph shows the summary of (C), data obtained from three independent experiments. ( E ) Representative photographic images of the migration of U87MG cells infected with Lenti-ANO1shRNA and transfected with CaMKIIβ siRNA as compared to the cells with Lenti-Sc shRNA and Sc siRNA control. Scale bar, 100 μm. ( F ) The averaged bar graph shows the summary of ( E ), data obtained from three independent experiments. All values are mean ± s.e.m. p -values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To silence CaMKIIβ expression, CaMKIIβ small-interfering RNA (CaMKIIβ siRNA) (Santa Cruz, Dallas, TX, USA, sc-38951) was transfected using INTERFERin ® siRNA transfection reagent (Polyplus, 409-10), according to the manufacturer’s protocol.

Techniques: Inhibition, Migration, Biomarker Discovery, Western Blot, Invasion Assay, Infection, Transfection, shRNA, Control