Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 1. (A) Hematoxylin and eosin staining and immunohistochemical staining for PKCα and claudin-1 in normal pancreatic ducts, well- and poorly differentiated pancreatic carcinomas. Bar: 50 μm. (B and C) Western blotting for pPKCα, panPKCα (PKCα), Snail, claudin-1, -4, -7 and occludin in normal pancreatic duct epithelial cells (hTERT-HPDEs) and pancreatic cancer cell lines HPAC, HPAF-II, BXPC-3 and PANC-1. Snail (1): exposed to X-ray film for 1 min, Snail (2): exposed to X-ray film for 20 min. The corresponding expression levels of B and C are shown as bar graphs. n = 3, *P < 0.05 and **P < 0.01 versus hTERT-HPDEs.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Staining, Immunohistochemical staining, Western Blot, Expressing

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 2. (A) Western blotting for Snail, claudin-1, -4, -7 and occludin in PANC-1 cells after treatment with 0–2 μg/ml Gö6976 for 24 h. (B) Real-time PCR for Snail, claudin-1 and occludin in PANC-1 cells after treatment with 0–2 μg/ml Gö6976 for 24 h. (C) Western blotting for claudin-1, -4, -7 and occludin in hTERT- HPDEs after treatment with 1 and 2 μg/ml Gö6976 for 24 h. (D) Real-time PCR for claudin-1, -4, -7 and occludin in hTERT-HPDEs after treatment with 1 μg/ ml Gö6976 for 24 h. (E) TER values in hTERT-HPDEs after treatment with 1 μg/ml Gö6976 for 24 h. iPKCα: PKCα inhibitor Gö6976. n = 3, *P < 0.05 and **P < 0.01 versus control (0 μg/ml).

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 3. Western blotting for pMAPK and panMAPK (MAPK) in PANC-1 cells (A) and hTERT-HPDEs (B) after treatment with 0–2 μg/ml Gö6976 for 24 h. Western blotting for claudin-1, occludin, pMAPK, MAPK and Snail in PANC-1 cells (C) and hTERT-HPDEs (D) pretreated with 20 μM MAPK/ERK inhibitor U0126 before treatment with 1 μg/ml Gö6976 for 24 h. Western blotting for claudin-1 in PANC-1 cells (E) and hTERT-HPDEs (F) pretreated with 20 μM MAPK/ERK inhibitor U0126, 10 μM p38 MAPK inhibitor SB203580 (SB), 10 μM PI3K inhibitor LY294002 (LY), 10 μM panPKC inhibitor GF109203X (GF), 10 μM JNK inhibitor SP600125 (SP) and 0.1 μM NF-κB inhibitor IMD-0354 (IMD) before treatment with 1 μg/ml PKCα inhibitor Gö6976 for 24 h. iPKCα: PKCα inhibitor Gö6976.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 4. (A) Western blotting for Snail, pPKCα, panPKCα (PKCα), pMAPK, panMAPK (MAPK), claudin-1, -4, -7 and occludin in PANC-1 cells after treatment with 100 ng/ml TGF-β1 for 24 and 48 h. (B) Western blotting for claudin-1, -4, -7, occludin, Snail, PKCα, pMAPK and MAPK in hTERT-HPDEs after treatment with 20 ng/ml TGF-β1 for 24 and 48 h. (C) Western blotting for Snail and claudin-1 in PANC-1 cells treated with siRNAs of Snail. (D) Western blotting for Snail and claudin-1 in PANC-1 cells treated with siRNAs of Snail and 100 ng/ml TGF-β1 for 48 h. Control cells in Figure 4C and D are transfected with a scrambled siRNA. KD: knockdown.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Control, Transfection, Knockdown

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 5. (A) Western blotting for Snail, claudin-1, -4, -7, occludin, pMAPK and panMAPK (MAPK) in PANC-1 cells pretreated with 1 μg/ml Gö6976 before treatment with 100 ng/ml TGF-β1 for 24 h. (B) Western blotting for claudin-1, -4, -7, occludin, Snail, panPKCα (PKCα), pMAPK and MAPK in hTERT-HPDEs pretreated with 1 μg/ml Gö6976 before treatment with 20 ng/ml TGF-β1 for 24 h. (C) Western blotting for Snail, claudin-1, occludin, pPKCα, PKCα, pMAPK and MAPK in PANC-1 cells with or without 1 μg/ml Gö6976 under hypoxia for 24 h. (D) Western blotting for claudin-1, -4, -7, occludin, pMAPK, MAPK and Snail in hTERT-HPDEs with or without 1 μg/ml Gö6976 under hypoxia for 24 h. iPKCα: PKCα inhibitor Gö6976.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 6. (A) Western blotting for Snail, pPKCα, panPKCα (PKCα), claudin-1, -4, -7 and occludin in HPAC cells after treatment with 100 μg/ml TGF-β1 for 24 and 48 h. (B) Western blotting for Snail, pPKCα, PKCα, claudin-1, -4, -7 and occludin in HPAC cells pretreated with 1 μg/ml Gö6976 before treatment with 100 ng/ml TGF-β1 for 24 h. (C) TER values in HPAC cells pretreated with 100 ng/ml TGF-β1 for 24 h with or without 1 μg/ml Gö6976. (D) Fence function examined by diffusion of labeled BODIPY-sphingomyelin into HPAC cells pretreated with 100 ng/ml TGF-β1 for 24 h with or without 1 μg/ml Gö6976. In HPAC cells treated with TGF-β1, the probe strongly labels the basolateral surface and appears to penetrate the cells (arrowheads), whereas the probe is effectively retained in the apical domain of the control cells and the cells were treated with TGF-β1 and Gö6976. Bar: 20 μm. iPKCα: PKCα inhibitor Gö6976.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Diffusion-based Assay, Labeling, Control

Journal: Cell reports

Article Title: Integrated genomic and proteomic analyses identify stimulus-dependent molecular changes associated with distinct modes of skeletal muscle atrophy

doi: 10.1016/j.celrep.2021.109971

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-alpha- tubulin (11H10) , Cell Signaling Technologies , 2125S; RRID:AB_2619646.

Techniques: Recombinant, SYBR Green Assay, Membrane, Electron Microscopy, Bicinchoninic Acid Protein Assay, Cell Based Assay, Reverse Transcription, Muscles, Gene Expression, Control, Mass Spectrometry, Software

Journal: Developmental cell

Article Title: SUPPRESSING MITOCHONDRIAL RESPIRATION IS CRITICAL FOR HYPOXIA TOLERANCE IN THE FETAL GROWTH PLATE

doi: 10.1016/j.devcel.2019.04.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti LDHA , Cell Signaling Technology , Cat# 2012, RRID:AB_2137173.

Techniques: Virus, Luciferase, Plasmid Preparation, Recombinant, Saline, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Marker, Western Blot, Stripping, In Situ, Imaging, Live Cell Imaging, Membrane, Cell Based Assay, Software

Journal: Developmental cell

Article Title: SUPPRESSING MITOCHONDRIAL RESPIRATION IS CRITICAL FOR HYPOXIA TOLERANCE IN THE FETAL GROWTH PLATE

doi: 10.1016/j.devcel.2019.04.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti Caspase-3 , Cell Signaling Technology , Cat# 9662, RRID: AB_331439.

Techniques: Virus, Luciferase, Plasmid Preparation, Recombinant, Saline, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Marker, Western Blot, Stripping, In Situ, Imaging, Live Cell Imaging, Membrane, Cell Based Assay, Software

Journal: Cell metabolism

Article Title: TLR8-mediated metabolic control of human Treg function: a mechanistic target for cancer immunotherapy

doi: 10.1016/j.cmet.2018.09.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GAPDH (14C10) Rabbit mAb , Cell Signaling Technology , Cat# 2118; RRID:AB_561053.

Techniques: Derivative Assay, Recombinant, Reverse Transcription, Selection, Cell Based Assay, Software

Journal: PLoS ONE

Article Title: GLI2 Regulates TGF-β1 in Human CD4 + T Cells: Implications in Cancer and HIV Pathogenesis

doi: 10.1371/journal.pone.0040874

Figure Lengend Snippet: The TGF-β and SHH Pathways Exhibit Extensive Cross-Regulation.

Article Snippet: The agarose resin was coated with one of three antibodies: Sigma F1804 anti-FLAG M2 antibody (to bind Tat to bead), CST 71D10 anti-MYC antibody (to bind GLI2 to bead), or an isotype control antibody.

Techniques: Activation Assay, Transgenic Assay

Journal: PLoS ONE

Article Title: GLI2 Regulates TGF-β1 in Human CD4 + T Cells: Implications in Cancer and HIV Pathogenesis

doi: 10.1371/journal.pone.0040874

Figure Lengend Snippet: CD4 + CD25 + FoxP3 + Treg were enriched from PBMC and then incubated for 72 hours with non-targeting (NT) or GLI2 siRNA. The cells were then stimulated (1 αCD3/αCD28 bead: 1 cell) for an additional 24 hours. RT-PCR was used to measure TGF-β1 mRNA levels. Relative TGF-β1 or GLI1 mRNA was normalized to 18S rRNA. (A) GLI2 knockdown in Treg significantly decreased TGF-β1 transcription (n = 7, * p = 0.0189). (B) GLI1 mRNA was also measured as a control GLI2-regulated gene; showing that knockdown of GLI2 in Treg also diminished GLI1 mRNA (n = 5, ** p = 0.0278). (C) The sequence of the GLI2 siRNA was scrambled (SCRAM) as an additional GLI2 siRNA negative control and was not significantly different from the non-targeting control.

Article Snippet: The agarose resin was coated with one of three antibodies: Sigma F1804 anti-FLAG M2 antibody (to bind Tat to bead), CST 71D10 anti-MYC antibody (to bind GLI2 to bead), or an isotype control antibody.

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Negative Control

Journal: PLoS ONE

Article Title: GLI2 Regulates TGF-β1 in Human CD4 + T Cells: Implications in Cancer and HIV Pathogenesis

doi: 10.1371/journal.pone.0040874

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) was done to assess the binding of GLI2 to the human TGF-β1 promoter. Primary human regulatory CD4 + CD25 hi T cells were negatively isolated and stimulated with αCD3/αCD28-coated beads overnight. PCR was done to assess GLI2 binding. Whole DNA was used as a positive control for the PCR. GLI2 bound to the TGF-β1 promoter at ‘Site E’ in the human TGF-β1 promoter. GLI2 did not bind to a non-targeting region in the human IL-10 promoter (Lane 8).

Article Snippet: The agarose resin was coated with one of three antibodies: Sigma F1804 anti-FLAG M2 antibody (to bind Tat to bead), CST 71D10 anti-MYC antibody (to bind GLI2 to bead), or an isotype control antibody.

Techniques: Chromatin Immunoprecipitation, Binding Assay, Isolation, Positive Control

Journal: PLoS ONE

Article Title: GLI2 Regulates TGF-β1 in Human CD4 + T Cells: Implications in Cancer and HIV Pathogenesis

doi: 10.1371/journal.pone.0040874

Figure Lengend Snippet: Co-immunoprecipitation was used to test the binding of HIV-1 Tat to human GLI2. Dual transfections were done in 293T cells (plasmid combinations shown on right) to express the Tat-FLAG and GLI2-6xMYC proteins (pcDNA3 = empty control vector). ( A ) Beads were coated with anti-FLAG antibody and incubated with cell lysates from transfections #1–3 shown on right-hand side. Following washing and elution steps, the bound protein was run on a SDS-PAGE gel followed by Western Blotting to assess GLI2 binding. Anti-myc antibody was used to confirm binding of GLI2 to Tat when Tat was bound to the bead. ( B ) Beads were coated with anti-myc antibody and incubated with cell lysates from transfections #1–3 shown on right-hand side. Following washing and elution steps, the bound protein was run on a SDS-PAGE gel and Western Blotting was done to look for HIV-1 Tat binding. Bands developed at approximately 27 kDa. Anti-flag antibody was used to confirm binding of Tat to GLI2 when GLI2 was bound to the bead.

Article Snippet: The agarose resin was coated with one of three antibodies: Sigma F1804 anti-FLAG M2 antibody (to bind Tat to bead), CST 71D10 anti-MYC antibody (to bind GLI2 to bead), or an isotype control antibody.

Techniques: Immunoprecipitation, Binding Assay, Transfection, Plasmid Preparation, Incubation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling

doi: 10.1101/2024.02.28.582540

Figure Lengend Snippet: Nanopore sequencing results of an AIV viral culture using DNA-nanopores (“cDNA” sequencing through R10 chemistry; direct RNA sequencing through “RNA002” R9 chemistry) and RNA-nanopores (direct RNA sequencing through “RNA004” RNA chemistry). A. Sequencing read length distribution across the cDNA, RNA002, and RNA004 datasets. B. Reference genome coverage of the three sequencing datasets across all AIV segments (PB1: Polymerase basic 1, PB2: Polymerase basic 2, PA: Polymerase acidic, HA: Hemagglutinin, NP: Nucleoprotein, NA: Neuraminidase, M: Matrix, NS: Nonstructural). The horizontal line indicates a coverage of 50x.

Article Snippet: Here we find that the latest direct RNA nanopore sequencing technology (which is based on a unique RNA-nanopore specifically designed for transcriptomic rather than genomic research), provides similar results to cDNA sequencing using Oxford Nanopore Technologies’ established high-accuracy DNA-nanopores (R10 chemistry).

Techniques: Nanopore Sequencing, Sequencing, RNA Sequencing

Journal: bioRxiv

Article Title: Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling

doi: 10.1101/2024.02.28.582540

Figure Lengend Snippet: Evaluation of viral consensus sequence creation from nanopore sequencing datasets (cDNA, RNA002, RNA004) across all data rarefactions ( min , med , max ). The performance of the computational tools BCFtools, iVar, and IRMA, which were the best-performing approaches for the max datasets, is visualized. A. Consensus sequence evaluation across the eight viral AIV segments through normalized BIT scores calculated based on the known AIV reference. B. Consensus sequence evaluation through whole-genome evolutionary distance comparisons with the known AIV reference.

Article Snippet: Here we find that the latest direct RNA nanopore sequencing technology (which is based on a unique RNA-nanopore specifically designed for transcriptomic rather than genomic research), provides similar results to cDNA sequencing using Oxford Nanopore Technologies’ established high-accuracy DNA-nanopores (R10 chemistry).

Techniques: Sequencing, Nanopore Sequencing

Journal: bioRxiv

Article Title: Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling

doi: 10.1101/2024.02.28.582540

Figure Lengend Snippet: Phylogenetic tree of AIV consensus HA segments from four environmental samples (dust samples from turkey farm in France) and known European AIV strains. AIV of the environmental samples was assessed by cDNA nanopore sequencing, and consensus sequence was created through BCFtools.

Article Snippet: Here we find that the latest direct RNA nanopore sequencing technology (which is based on a unique RNA-nanopore specifically designed for transcriptomic rather than genomic research), provides similar results to cDNA sequencing using Oxford Nanopore Technologies’ established high-accuracy DNA-nanopores (R10 chemistry).

Techniques: Environmental Sampling, Nanopore Sequencing, Sequencing

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet:

Article Snippet: AMPKalpha (23A3) Rabbit mAb (Sepharose Bead Conjugate) , Cell Signaling , Cat#6707; RRID: AB_10698749.

Techniques: Produced, FLAG-tag, Control, Recombinant, Membrane, Transfection, Reverse Transcription, Silver Staining, Isolation, Cell Culture, Expressing, Plasmid Preparation, Luciferase, shRNA, Software

Journal: eLife

Article Title: Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition

doi: 10.7554/eLife.44161

Figure Lengend Snippet:

Article Snippet: Antibody , anti-cyclin B1 (rabbit polyclonal) , Cell Signaling Technology , #4138 RRID: AB_2072132 , 1:1000.

Techniques: Luciferase, shRNA, CRISPR, Suspension, Wilms Tumor Assay, Immunohistochemistry, RNA HS Assay, Library Quantification, Reverse Transcription, SYBR Green Assay, Staining, Cell Viability Assay, Cell Based Assay, In Vitro, In Vivo, Software

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: Morphology of cells overexpressing constitutively active IF1-H49K. (A) IF1-H49K is localized in mitochondria. IF1-H49K-HA was visualized by immunostaining using anti-HA antibody in a stable cell line. Second antibody with Cy3 (λ exc. 561 nm, λ em. 580–620 nm). Cells were stained with MitoTracker™ Green (MTG) before fixation. The overlay of the Cy3 and MTG fluorescence channels clearly shows colocalization. (B) Candidate IF1 overexpression clones of human cervical carcinoma cells were subjected to immunoblot analysis with antibodies against IF1, the mitochondrial marker protein VDAC and cellular actin. Endogenous IF1 and IF1-HA/IF1-H49K-HA levels were determined and normalized to actin levels. Right panel: Total IF1/actin levels in the used cells lines. N = 3. (C) Morphological analysis of mitochondria using the ImageJ ® plugin MiNA. Mitochondrial footprint is the total mitochondrial area in one cell. Each symbol corresponds to one analyzed cell. Statistics: N = 3, p ≤ 0.001, ***. p ≤ 0.01, **; n . s .: non significant. Scale bar: 10 μm (A,C).

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Immunostaining, Stable Transfection, Staining, Fluorescence, Over Expression, Clone Assay, Western Blot, Marker

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: Effect of IF1-H49K on mitochondrial function. (A) Mitochondrial membrane potential under the influence of overexpressed IF1-wt and IF1-H49K. HeLa cells were stained with 7 nM TMRE (Scale bars: 10 μm). Right panel: Mean grey values of the TMRE signal per cell (N = 3). (B) Metabolic profiling of control and IF1-H49K cells: both cell lines display a mixed metabolic phenotype with moderate oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) indicating glycolytic activity. The OCR of IF1-H49K cells was corrected for the decreased mitochondrial footprint ( N = 2, SEM are shown). (C) Indirect determination of cytosolic ATP levels by MgGreen staining of free Mg 2+ . The MgGreen fluorescence signal is inversely related to the ATP concentration. N = 2 replicates. Significance levels were determined by One Way ANOVA with post hoc Scheffé test. (D) Determination of mitochondrial ATP levels in mitochondria with active and inhibited ATP synthase via ATP-Red fluorescence. (N = 2 experiments, SD). Every dot is the mean ATP red fluorescence of one cell. (E) Doubling time DT of control cells and cells expressing IF1-H49K. DT is expressed in hours (N = 2 experiments, SD). Scale bars: 10 μm (A, C).

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Membrane, Staining, Control, Activity Assay, Fluorescence, Concentration Assay, Expressing

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: The spatio-temporal organization of ATP synthase is affected by IF1-H49K. (A) Mobility pattern of F 1 F O ATP synthase under control conditions. Recording: 59 Hz, 3000 frames, total recording time 51 s ( – ). The panels show trajectory maps of the F 1 F O ATP synthase in two individual mitochondria. Inset: selected trajectories from a third mitochondrion showing orthogonal trajectories (arrowheads). Trajectories of the F 1 F O ATP synthase were generated by using the Multi-Target-Tracing-Tool (MTT). Every single trajectory in one color. (B) Mobility pattern of F 1 F O ATP synthase in cells expressing IF1-HA, see in . Recording rate: 59 Hz, 3000 frames. (C) Mobility pattern of F 1 F O ATP synthase in cells expressing IF1-H49K-HA. Recording: 59 Hz, 3000 frames (51 s) ( – ). Two types of trajectory maps were distinguishable and classified as state I and state II. Scale bars: 1 μm (A, B, C), 0.5 μm (inset A).

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Control, Generated, Expressing

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: Partial inhibition of ATPase function by constitutively active IF1 and complete inhibition of the F 1 F O ATP synthase by oligomycin mobility in the absence and presence of IF1-H49K-HA. (A) Diffusion coefficients for ATP synthase under control conditions (5.6 mM Glucose, no inhibition), and in the presence of IF1-H49K-HA are shown. The probability density function (PDF) of log D and the cumulative probability function (CPF) are shown. State I and state II refer to the spatio-temporal characteristics of ATP synthase in . Ctrl.: F 1 F O ATP synthase mobility without constitutive expression of IF1-H49K. IF1-H49K state I and state II: F 1 F O ATP synthase mobility in cells expressing IF1-H49K. (B) D 1 for the mobile fraction of inhibited (with IF1-H49K) and non-inhibited ATP synthase is shown. Oligom.: Oligomycin (10 μM, 30 min) as inhibitor for ATP synthase forward and reverse function. (C) Analysis of the step directions in relation to the longitudinal axis ( ± 180° and 0°) and the orthogonal axis (from − 90° and + 90°) of the respective mitochondrion. For the determination of the directionality, the direction of 4 successive steps was integrated and displayed on a graduated disc. Trajectories analyzed: ctrl. #1053; IF1-H49K-state I #1024 and IF1-H49K state II #1300 (A, C). (D) Orientation of trajectories of ATP synthase under control and oligomycin inhibited conditions.

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Inhibition, Diffusion-based Assay, Control, Expressing

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: Mitochondrial ultrastructure in cells with constitutively active IF1. (A) TEM of HeLa WT and IF1-H49K-HA cell monolayer. (B) 100 cristae were analyzed for each condition. Determination of mitochondrial length, mitochondrial width, number of cristae per μm mitochondrial length and cristae with shorter length (truncated). Statistical analysis of the parametric data was performed with one-way ANOVA with post hoc Scheffé test. Statistical analysis of the non-parametric data was performed as a Kruskal-Wallis test. Scale bars: 1 μm (A).

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques:

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: 2D and 3D view of mitochondria in cells expressing IF1. (A–C) TEM after fixation of monolayers of cells. (A) Overview. (B) Magnified view, arrows indicate altered cristae structures such as arches and branches. (D-E) ET of a single mitochondrion with altered ultrastructure in the presence of IF1. (D) Single tomographic slice. (E) 3D reconstruction after segmentation; different view angles are shown. From left to right: Full segmentation. The outer membrane (OM) is depicted in translucent red, the light blue indicates the inner boundary membrane and cristae are in different colors. Mid: view from a different angle without the OM. Right: only cristae are shown. Scale bars: 1 μM (A); 500 nm (B), 200 nm (C); 100 nm (D, E).

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Expressing, Membrane

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: 3D views of mitochondria in cells with constitutively active IF1-H49K. Tomograms of single mitochondria with altered ultrastructure in the presence of IF1-H49K. Three states (I-III) were classified. Left panels: single tomographic slice. Right panels: 3D reconstruction after segmentation, different view angles are shown. From left to right: Full segmentation. The outer membrane is depicted in translucent red, the light blue indicates the inner boundary membrane and cristae are in different colors. Mid: view from a different angle without the OM. Right: only cristae are shown. Scale bars: 100 nm.

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Membrane

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: Semi-quantitative high through put analysis of cristae morphology in control and IF1-H49K expressing cells. (A, B) Live cell Airyscan images of HeLa mitochondria, control (A) and IF1-H49K (B). Cells were stained with NAO (100 nM), which was present during the measurement. Note, that from left to right original images are shown, followed by probability maps of mitochondria, cristae and of matrix space, and the masks used for cristae measurement. Circled areas show zoomed in regions of cristae probability maps, cristae are marked with red arrowheads. Images were recorded with AiryScan2 (Zeiss) in superresolution (SR) mode equipped with an alpha Plan-Apochromat 63×/1.46 Oil DIC M27 objective on the Zeiss LSM 980. Raw images were automatically processed into deconvolved Airyscan images using the Zen software. (C) Quantification of cristae area between control and IF1-H49K expressing cells, stained with NAO. N = 3 experiments, 16 mitochondria per condition. Data are presented as Box and Whisker Blots. P -values are shown in the panels, n . s .: non-significant. Scale bar: 1 μM (all panels).

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Control, Expressing, Staining, Software, Whisker Assay

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: Mitofilin overexpression induces ultrastructural changes but does not alter the fraction of mobile ATP synthase. (A) TEM of HeLa cells stably overexpressing mitofilin-GFP. (B) Single mitochondrion with normal and aberrant cristae, lower panel: normal cristae false-colored in blue and aberrant cristae false-colored in red. (C) Determination of aberrant cristae (cell number: ctrl.: > 10 and mitofilin: 5; mitochondria: ctrl.: > 35 and mitofilin: 21; cristae: ctrl.: > 450 and mitofilin: 166). (D) Trajectory map of F 1 F O ATP synthase (labeled at SU γ-HaloTag with TMR HTL ) in one mitochondrion. Cumulative image of 3000 subsequent frames (recording 100 Hz). (E) Diffusion coefficients of F 1 F O ATP synthase in control cells, cells overexpressing mitofilin and cells expressing IF1-H49K transiently or stably. (F) Percentage of mobile ATP synthase particles under the same condition as in (E). (G) Quantification of mitofilin protein expression levels in ctrl. cells and IF1-H49K-HA expressing cells by quantitative RT-PCR (N = 3 replicates). (H) Immunostaining for IF1 in mitofilin-GFP expressing cells in comparison to other cell lines used in this study. The IF1 levels were not higher when normalized on actin or VDAC. N = 5 replicates. Scale bars: 0.5 μm (A), 300 nm (B, E). Statistics: ANOVA with post hoc Scheffé test, n . s .: non-significant, p ≤ 0.001: ***, error as standard deviation of the mean, SDM. Scale bars: 300 nm (A), 100 nm (B) and 1 μm (E).

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Over Expression, Stable Transfection, Labeling, Diffusion-based Assay, Control, Expressing, Quantitative RT-PCR, Immunostaining, Comparison, Standard Deviation

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: Relative levels of fission and fusion proteins including OPA1 in IF1 overexpressing cells. (A) Semiquantitative RT-PCR blot showing relative mRNA levels of GAPDH, IF1, OPA1, and Drp1 in control cells and cells expressing IF1 (blots: inverse). (C) Immuno-staining of OPA1 in control cells and cells expressing IF1 and IF1-H49K, respectively (antibody α-OPA1: courtesy of A. Reichert lab). OPA1 appears in long and short isoforms due to proteolytic processing of L-OPA1. Line plot profile: relative levels of S- and L-OPA1 protein bands along the cross section indicated with a blue line in the immunoblot. Right panel: Ratio of L-OPA1 and S-OPA1 peak signals indicating the L/S OPA1 relation (peak ratio b/d) for two experiments.

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Immunostaining, Western Blot

Journal: Biochimica et biophysica acta. Bioenergetics

Article Title: Inhibition of the mitochondrial ATPase function by IF1 changes the spatiotemporal organization of ATP synthase

doi: 10.1016/j.bbabio.2020.148322

Figure Lengend Snippet: IF1-H49K expressing cells tend to have more ATP synthase dimers and more IF1 bound to ATP synthase. (A) Equal amounts (60 μg per lane) of digitonin-solubilized mitochondrial extracts from HeLa with the indicated IF1 genotype were separated by BNGE and immunoblotted with the indicated antibodies. (B) Quantitative densitometric analysis of ATP synthase dimer vs. monomer (D/M). (C) Quantitative densitometric analysis of IF1 bound to total ATP synthase. (D) Immunoblots after separation of mitochondrial proteins by SDS-PAGE, antibodies as indicated for subunits e (α-ATP5I), β (α-ATP5B) and VDAC as loading control. (E) Ratio of subunit e and subunit β after normalization to VDAC. N = 3 replicates.

Article Snippet: After separation on a 3–13% gel, the proteins were electro-blotted onto Hybond-P-polyvinylidene fluoride (PVDF) membranes (GE Healthcare) and immune-blotted with a primary antibody against subunit β of ATP synthase (Abcam 14730, mouse monoclonal) and against IF1 (CD6P1Q, Cell Signaling (#13268)).

Techniques: Expressing, Western Blot, SDS Page, Control