Journal: Stem Cell Research & Therapy

Article Title: Inositol pyrophosphates mediate the effects of aging on bone marrow mesenchymal stem cells by inhibiting Akt signaling

doi: 10.1186/scrt431

Figure Lengend Snippet: Aging increased the apoptosis of MSCs induced by H/SD injury. (A) Representative immunofluorescence images of apoptosis (TUNEL, green fluorescent) and DAPI (blue fluorescence) in young and aged MSCs under hypoxic or normoxic conditions (Scale bars: 50 μm). (B) Apoptosis was also analyzed by flow cytometry after staining with Annexin V and propidium iodide (PI). (C) Quantification of the apoptotic MSCs is presented as the percentage of apoptotic cells. (D) Quantification of the FACS analysis. Viable cells: Annexin V - /PI - ; early apoptosis: Annexin V + /PI - ; late apoptosis: V + /PI + ; necrotic: V - /PI + . (E) : Caspase-3 activity of MSCs in all groups. Data expressed as mean ± SEM. n = 5, * P <0.05, # P <0.05 versus young + H/SD. DAPI, 4,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; H/SD, hypoxia and serum deprivation; MSC, mesenchymal stem cells; SEM, standard error of the mean; TUNEL, terminal- deoxynucleotidyl transferase mediated nick end labeling.

Article Snippet: In brief, after being incubated with monoclonal phycoerythrin (PE)-conjugated antibodies against CD markers (BD, San Jose, CA, USA) for one hour, BM-MSCs isolated from young and aged mice were processed through a FACS Caliper system (BD) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, TUNEL Assay, Fluorescence, Flow Cytometry, Staining, Activity Assay, FACS, End Labeling

Journal: Stem Cell Research & Therapy

Article Title: Inositol pyrophosphates mediate the effects of aging on bone marrow mesenchymal stem cells by inhibiting Akt signaling

doi: 10.1186/scrt431

Figure Lengend Snippet: TNP decreased apoptosis of aged MSCs. (A) Representative confocal microscopic images of TUNEL stains for the hypoxia-induced apoptosis in aged MSCs with or without the IP6Ks inhibitor TNP. (B) Apoptosis was also analyzed by flow cytometry after staining with Annexin V and PI. (C) Quantification of the TUNEL assay is presented as the percentage of apoptotic cells. (D) Quantification of the FACS analysis. Viable cells: Annexin V - /PI - ; early apoptosis: Annexin V + /PI - ; late apoptosis: V + /PI + ; necrotic: V - /PI + . (E) Caspase-3 activity of MSCs in all groups. Data are expressed as mean ± SEM. n = 5, * P <0.05, # P <0.05 versus H/SD. FACS, fluorescence-activated cell sorting; H/SD, hypoxia and serum deprivation; IP6Ks, inositol hexakis phosphate kinases; MSCs, mesenchymal stem cells; SEM, standard error of the mean; TNP, N6-(p-nitrobenzyl) purine; TUNEL, terminal- deoxynucleotidyl transferase mediated nick end labeling.

Article Snippet: In brief, after being incubated with monoclonal phycoerythrin (PE)-conjugated antibodies against CD markers (BD, San Jose, CA, USA) for one hour, BM-MSCs isolated from young and aged mice were processed through a FACS Caliper system (BD) according to the manufacturer’s protocol.

Techniques: TUNEL Assay, Flow Cytometry, Staining, Activity Assay, Fluorescence, FACS, End Labeling

Journal: Nature Communications

Article Title: A one-two punch targeting reactive oxygen species and fibril for rescuing Alzheimer’s disease

doi: 10.1038/s41467-024-44737-x

Figure Lengend Snippet: (i) The co-assembly of nanodrug (Ang-AIE NCs, termed Ang-NCs). (ii) the NIR-II imaging monitored the BBB-traversing process of Ang-NCs. (iii) the inflammation-associated reactive oxygen species (ROS) environment stimulated the controllable release of Ang-NCs. iv) the activation of the self-enhanced dual-targeting program: compound 3 specifically inhibits the formation of amyloid-beta (Aβ) fibrils, degrades toxic fibrils, and inhibits reaggregation. Compound 6 relieves harmful inflammation-associated ROS and promotes the inhibition of Aβ fibrils generation.

Article Snippet: After blocking with 5% defatted milk for 1 h at room temperature, the membranes were respectively incubated with Anti-GPX4 antibody (Abcam, ab125066, 1: 1000 dilution), Anti-iL-1β antibody (Proteintech, 26048-1-AP, 1: 1000 dilution), Anti-NLRP3 antibody (Hangzhou Huaan Biotechnology, ER1706-72, 1: 1000 dilution), and β-actin antibody (ABclonal AC026, 1: 50000 dilution) overnight at 4 °C.

Techniques: Imaging, Activation Assay, Inhibition

Journal: Nature Communications

Article Title: A one-two punch targeting reactive oxygen species and fibril for rescuing Alzheimer’s disease

doi: 10.1038/s41467-024-44737-x

Figure Lengend Snippet: a Chemical formula of 3 and 6 . b Illustration of the HOMO/LUMO and the corresponding energy gap of 3 in the ground-state (S 0 ). c Increase ratio ( α AIE ) of relative photoluminescence (PL) intensity of 3 at 1110 nm and 6 at 916 nm. d Representative size distribution and morphology image of NCs. The data was presented as mean ± SEM. e , f were respectively the absorption and emission spectra (excitation: 808 nm) of two AIEgens and NCs. g Elements (Ce and O) mapping of NCs. h NIR imaging at 1350 nm for various concentrations of NCs aqueous solution (0.1, 0.3, and 0.6 mg/mL). i In vivo NIR imaging at 1350 nm of cerebral vessels in mice without removing the scalp or skull after i.v . injection of Ang-NCs. Source data are provided as a Source Data file.

Article Snippet: After blocking with 5% defatted milk for 1 h at room temperature, the membranes were respectively incubated with Anti-GPX4 antibody (Abcam, ab125066, 1: 1000 dilution), Anti-iL-1β antibody (Proteintech, 26048-1-AP, 1: 1000 dilution), Anti-NLRP3 antibody (Hangzhou Huaan Biotechnology, ER1706-72, 1: 1000 dilution), and β-actin antibody (ABclonal AC026, 1: 50000 dilution) overnight at 4 °C.

Techniques: Imaging, In Vivo, Injection

Journal: Nature Communications

Article Title: A one-two punch targeting reactive oxygen species and fibril for rescuing Alzheimer’s disease

doi: 10.1038/s41467-024-44737-x

Figure Lengend Snippet: a Cell viability of various cell lines treated with NCs and Ang-NCs ( n = 3 biologically independent samples). b The confocal images and quantification in the 96-well plates for the uptake of NCs and Ang-NCs in PC12 cells ( n = 3). c Confocal images of in vitro BBB penetration of two NCs by PC12 and HT-22 cells via Transwell model. d Confocal images of Aβ fibril degradation in PC12 cells with different treatments and ThT staining (green). e Cellular ROS in PC12 cells with various treatments for 48 h and stained with DCFH-DA (green). f , g Confocal images of the superoxide generation and mitochondrial membrane potential in HT-22 cells with various treatments for 48 h and staining by MitoSOX ( f ) and JC-1 probes ( g ). The confocal images were representative data of three independent experiments in c – g . h The relative expression of mRNA (TNF-α, IL-1β, and IL-6) in HT-22 cells tested by RT-PCR ( n = 3). The P values ( **** ) were the comparison of three parameters between the Aβ fibril group and the other two groups, respectively. i IL-1β level in HT-22 cells tested by ELISA kit ( n = 3). j The expression of IL-1β and NLRP3 in HT-22 cells, and GPX4 in PC12 cells by Western blot analyses. k Cell viability of PC12 cells treated with various concentrations of Ang-NCs ( n = 3). l Cell viability of PC12 cells treated with Aβ fibril, Aβ fibril with 6 (100 μg/mL), and Aβ fibril with 3 (100 μg/mL) for 48 h ( n = 3). m Cell viability ( n = 3) of PC12 and HT-22 cells incubated with Aβ oligomer (10 μM), Aβ fibril, and degraded Aβ fragment (10 μM). n Representative crystal violet staining images of HT-22 cells after various treatments for 48 h. o The corresponding biological transmission electron microscopy (Bio-TEM) images of mitochondria in HT-22 cells with various treatments. Unless otherwise stated, all concentrations of Aβ fibril and Ang-NCs were respectively 10 μM and 100 μg/mL. All data were presented as mean ± SD. NS, no significance, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. All assays ( n = 3) were biologically independent samples. All statistical analyses were compared by analyses of one-way ANOVA except the two-tailed Student’s t -test in a and b . Source data are provided a s a Source Data file.

Article Snippet: After blocking with 5% defatted milk for 1 h at room temperature, the membranes were respectively incubated with Anti-GPX4 antibody (Abcam, ab125066, 1: 1000 dilution), Anti-iL-1β antibody (Proteintech, 26048-1-AP, 1: 1000 dilution), Anti-NLRP3 antibody (Hangzhou Huaan Biotechnology, ER1706-72, 1: 1000 dilution), and β-actin antibody (ABclonal AC026, 1: 50000 dilution) overnight at 4 °C.

Techniques: In Vitro, Staining, Membrane, Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: A one-two punch targeting reactive oxygen species and fibril for rescuing Alzheimer’s disease

doi: 10.1038/s41467-024-44737-x

Figure Lengend Snippet: a The outline of the experiment design and sequences for animal behavior evaluation. b In vivo pharmacokinetic analyses ( n = 3 biologically independent mice). c NIR imaging at 1350 nm of the brain in APP/PS1 mice at various time points (0 and 12 h) post i.v . injection of NCs and Ang-NCs (dosage: 10 mg/kg). d , e NIR images and corresponding fluorescence quantification for main organs of AD mice treated with NCs and Ang-NCs ( n = 4 biologically independent mice), heart (H), liver (Li), spleen (S), lung (L), and kidney (K). f Fluorescence images of ROS in ex vivo brains from AD mice post the injection of PBS and Ang-NCs. The DCFH-DA was dropped onto the surface of ex vivo brains before imaging. g The levels of TNF-α, IL-1β, and Aβ 1–42 in the various mice ( n = 5 biologically independent samples). h Fluorescence quantification of Aβ plaques of brain slices (3 biologically independent mice and respectively sampling two tested sites in the hippocampus in one slice of each mouse, the same quantification for cortex site). i Immunofluorescence images of Aβ plaques, Iba-1, and GFAP in hippocampus and cortex regions of ex vivo brain slices, red (Aβ plaque, Iba-1, and GFAP) and cell nucleus (blue), all scale bars: 100 μm. j , k The corresponding fluorescence quantification of Iba-1 and GFAP from the hippocampus and cortex of the brain slices (3 biologically independent mice and respectively sampling two tested sites in the hippocampus in one slice of each mouse, the same quantification for cortex site). l , m The representative images and corresponding scoring comparisons of various groups in the nesting test ( n = 8 biologically independent mice). n Swimming tracks of various groups. o – q were respectively the escape latency ( o ), the staying time in the target quadrant ( p ), and the crossing number through the platform ( q ) in the Morris water maze (MWM) test ( n = 8 biologically independent mice). r Recognition index in the NOR test ( n = 8 biologically independent mice). All dosages were 10 mg/kg. All data were presented as mean ± SEM. NS, no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. All statistical analyses were compared by analyses of one-way ANOVA except the two-tailed Student’s t -test in e . Source data are provided as a Source Data file.

Article Snippet: After blocking with 5% defatted milk for 1 h at room temperature, the membranes were respectively incubated with Anti-GPX4 antibody (Abcam, ab125066, 1: 1000 dilution), Anti-iL-1β antibody (Proteintech, 26048-1-AP, 1: 1000 dilution), Anti-NLRP3 antibody (Hangzhou Huaan Biotechnology, ER1706-72, 1: 1000 dilution), and β-actin antibody (ABclonal AC026, 1: 50000 dilution) overnight at 4 °C.

Techniques: In Vivo, Imaging, Injection, Fluorescence, Ex Vivo, Sampling, Immunofluorescence, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Profiling hypoxia signaling reveals a lncRNA signature contributing to immunosuppression in high-grade glioma

doi: 10.3389/fimmu.2024.1471388

Figure Lengend Snippet: TP73-AS1’s role in HGG cell malignancy and interaction with TAM. (A) Fluorescence in situ hybridization of TP73-AS1, immunohistochemistry of HIF1α, TGF-β, IBA-1, and quantitative correlation analyses of HIF1α expression, TGF-β expression, IBA-1 + TAM infiltration, and TP73-AS1 expression in glioma patients. Scale bar: 20 μm. (B) Multicolor immunofluorescence staining examined the relationship between TP73-AS1, HIF1α, and IBA-1 in glioma patients. Scale bar: 20 μm. (C) TP73-AS1 expression in NHA, LN229, and U87 cells was measured by qRT-PCR. (D) Cellular hypoxia model validation by western blot analysis of HIF1α expression in LN229 and U87 at various time points. (E) qRT-PCR analysis of TP73-AS1 expression in LN229 and U87 over different time points under CoCl 2 -induced hypoxia condition. (F) Efficiency of si-TP73-AS1-1, -2, and -3 in LN229 and U87 glioma cells validated by qRT-PCR. (G) Western blot analysis of HIF1α expression in LN229 and U87 treated with CoCl 2 or TP73-AS1 targeted siRNAs. (H, I) Cell proliferation after si-TP73-AS1-1 and -2 transfection, determined by colony formation and CCK-8 assays. (J, K) Migration analysis of THP-1 and flow cytometry of THP-1 polarization markers (M1: MHCII, M2: CD206), co-cultured with LN229 treated with DMSO, CoCl 2 , TP73-AS1 targeted siRNAs, or control siRNA. **p < 0.01; ***p < 0.001; ****p < 0.0001; DMSO, dimethyl sulfoxide.

Article Snippet: Sections were incubated with primary antibodies against IBA-1 (1:500; 10904-1-AP, Proteintech), HIF-1A (1:500; 20960-1-AP, Proteintech), and TGF-β (1:500; 21898-1-AP, Proteintech).

Techniques: Fluorescence, In Situ Hybridization, Immunohistochemistry, Expressing, Multicolor Immunofluorescence Staining, Quantitative RT-PCR, Biomarker Discovery, Western Blot, Transfection, CCK-8 Assay, Migration, Flow Cytometry, Cell Culture, Control

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) Cartoon of AC9-POPDC complex. ( B , C ) Proximity ligation assay was performed with HEK293 cells expressing pCDNA3 (background control), POPDC1-Myc alone, and YFP-AC9 in the presence of either POPDC1-Myc or POPDC2-Myc. The interaction between AC9 and Gβγ served as a positive control . ( B ) Images of PLA signal (red) and DAPI (blue). ( C ) Mean cellular fluorescence intensity was quantified by high content microscopy and shown as Box and Whisker plots. Kruskal-Wallis One Way ANOVA analysis was performed (n=7 experiments, P=0.003 between groups) with multiple comparisons by Bonferroni t-test (*P<0.05) ( D ) Quantification of COS7 cells expressing BiFC constructs for AC9, POPDC 1-3. The expressed proteins tagged with VN (top line) and VC (bottom line) are shown. Kruskal-Wallis One Way ANOVA analysis was performed (n=4 experiments, ***P<0.001 between groups) with multiple comparisons to control by Dunn’s method (**P<0.01, *P<0.05) ( E ) Representative live-cell images of indicated BiFC combinations in HEK293 cells (n>20 cells; scale bar is 10 um). Quantification of POPDC1-VN:AC9-VC is shown in and .

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Proximity Ligation Assay, Expressing, Positive Control, Fluorescence, Microscopy, Whisker Assay, Construct

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) Cell lysates from HEK293 cells expressing Flag-AC9 in the presence or absence of Myc-tagged POPDC1 or −2 were subjected to co-immunoprecipitation (Co-IP) with anti-MYC and assayed for AC activity with 300 nM Gαs-GTPγS. Kruskal-Wallis One Way ANOVA analysis on Ranks was performed (n=6 experiments, P=0.003 between all groups) with multiple comparisons to AC9 control by Dunn’s method (**P<0.01). ( B ) A portion of the lysates and Co-IP from ( A ) were subjected to western blot (WB) analysis with anti-MYC (POPDC) and anti-FLAG (AC9). Membranes isolated from Sf9 cells expressing Flag-AC9 served as a positive WB control. Note, POPDC protein runs as multiple bands (48-65 kDa) with altered sizes/patterns in different tissues due to changes in glycosylation patterns . Molecular weight markers are denoted as M. Quantitation of Flag-AC9 WB by One Way ANOVA, n=3-4 experiments, with comparisons by Tukey test, **P<0.01). ( C ) HEK293 cells expressing Flag-AC9 +/- POPDC1-Myc or POPDC2-Myc were subjected to Co-IP with anti-FLAG and subjected to WB analysis with anti-MYC and anti-FLAG. Quantitation of anti-MYC WB by One Way ANOVA, n=3 experiments, with comparisons by Tukey test, **P<0.01). ( D ) POPDC1 does not interact with control TM proteins. HEK293 cells expressing POPDC1-Myc +/- GFP-tagged AC9, EGFR, or LAMP1 were subjected to Co-IP with anti-MYC. Western blotting of lysates and Co-IPs for GFP (top) and Myc (bottom) are shown (n=3 experiments). ( E ) Schematic of POPDC1 truncations. ( F ) BiFC of AC9 and indicated POPDC1 truncations in COS-7 cells. Kruskal-Wallis One Way ANOVA analysis was performed (n=4 experiments, ***P<0.001 between groups) with multiple comparisons by Tukey test (**P=0.008). ( G ) Co-IP with anti-MYC in COS-7 cells expressing Flag-AC9 and indicated Myc-tagged POPDC1 truncations. Western blotting with anti-AC9 and anti-MYC (POPDC1) of Co-IP and lysates is shown. Kruskal-Wallis One Way ANOVA analysis was performed (n=3-5 experiments, P=0.003), with multiple comparisons by Dunn’s method (*P<0.05).

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Activity Assay, Western Blot, Isolation, Molecular Weight, Quantitation Assay

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) Co-localization of CFP-TREK-1 with the BiFC signal from AC9-VN:AC9-VC homodimer (top) and POPDC1-VN:AC9-VC complex (bottom) in HEK293 cells. Scale bar is 10 um. ( B ) The lifetime distribution of Cerulean-tagged proteins +/- the indicated YFP-tagged proteins expressed in HEK293 cells are displayed as box and whisker plots with all outliers shown. Mann-Whitney Rank Sum Test was performed (***P<0.001; n=cell number indicated on each bar). ( C ) Co-IP of Myc-tagged POPDC1 (anti-MYC) pulls down both Flag-AC9 and GFP-tagged TREK-1 (n=3 experiments). ( D ) Co-IP of TREK-1 (anti-GFP) pulls down Flag-AC9 and endogenous POPDC1. AKAP79 is not pulled down in complex (n=3 experiments). Quantification of Flag-AC9 WB (IP/total expression in lysate) is shown to the right of ( C ) and ( D ).

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Whisker Assay, MANN-WHITNEY, Co-Immunoprecipitation Assay, Expressing

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) BiFC signal between the indicated VN- and VC-tagged proteins expressed in HEK293 cells (POPDC1, P1). Kruskal-Wallis One-Way ANOVA on Ranks (n=5 experiments, P<0.001 between groups) with multiple comparison to VN and VC controls by Student-Newman-Keuls tests (*P<0.05). ( B ) Percent decrease of BiFC signal in ( A ) with ISO treatment (10 μM, 10 min at 37°C). Box and whisker plots are shown. Paired t-test was performed for each condition (vehicle versus ISO) using raw data prior to calculation of percent of vehicle control (n=6; *P<0.05; **P<0.01). ( C ) ISO dose-response curves for BiFC interactions between POPDC1:AC9, TREK-1:AC9, and TREK-1 with a catalytically inactive AC9 (TREK-1:AC9d). Data were analyzed by Two-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4; *P<0.05). ( D ) TREK-1:AC9 BiFC signal is absent in COS-7 cells lacking detectable POPDC1 (TREK-1:TREK-1 BiFC is shown as a positive control). One-way ANOVA with multiple comparisons by Holm Sidak method (n=3 experiments; ***P<0.001); ( E ) Overexpression of POPDC1 truncation of Popeye domain (POPDC1-Δ172), but not WT POPDC1, abolishes ISO reduction of TREK-1:AC9 BiFC signal. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4 experiments; ***P<0.001 compared to control). ( F ) Model of cAMP effects on AC9-POPDC1-TREK-1 complex formation.

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Whisker Assay, Positive Control, Over Expression

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) IP-AC assay with IgG versus anti-TREK-1 in heart homogenates from WT and Adcy9 -/- mice; AC activity stimulated with 300 nM Gαs-GTPγS. IP-AC with anti-AKAP150 is shown as a positive control. Two-way ANOVA (overall P<0.001, n=3 mice per genotype) with multiple comparisons by Holm-Sidak test; # P<0.001 for indicated comparison or to respective IgG controls. IgG versus anti-TREK-1 in Adcy9 -/- was analyzed by Student’s t-test (**P=0.009, n=3). ( B ) IP-AC assay with IgG versus anti-TREK in WT and Popdc1 -/- mouse heart homogenates; AC activity stimulated as in (A). Statistics as in A, n=4 mice per genotype, #P<0.001. Paired t-test within Popdc1 -/ - , P=0.123. ( C ) Total AC activity in heart homogenates from WT or Popdc1 -/- ( P1 -/- ) mice stimulated with 300 nM Gαs-GTPγS or 100 μM calcium and 300 nM calmodulin. Analyzed by Student’s t-test (n=4, n.s.) ( D ) Cell lysates from HEK293 cells expressing AC1 or AC8 in the presence or absence of Myc-tagged POPDC1 were subjected to co-IP with anti-MYC and assayed for AC activity with 100 μM calcium and 300 nM calmodulin. Student’s t-test (n=3 experiments, **P=0.0011 and *P=0.012 for AC1 and AC8, respectively). (E) IP-AC assay with IgG versus anti-TREK-1 in WT and Popdc1 -/- mouse heart homogenates; AC activity stimulated 100 μM calcium and 300 nM calmodulin. Student’s t-test (*P=0.03; **P=0.005, n=4 mice per genotype).

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Activity Assay, Positive Control, Expressing, Co-Immunoprecipitation Assay

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Sequences of the primers used for reverse transcription polymerase chain reaction.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Reverse Transcription, Sequencing

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: The expression of CCL3 and its receptors in cornea after alkali injury. A : Semi-quantitative RT–PCR was performed to assess mRNA expression of CCL3 and its receptors, CCR1 and CCR5 , and the ratios of target gene expression to β-actin were determined. All values represent the mean±SEM of three to five independent measurements. B : Whole eyes were obtained at 0, 2, 4, and 7 days after alkali injury and processed for immunohistochemical analysis using anti-CCL3 (upper panels) or anti-CCR1 antibodies (lower panels). Representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Targeted Gene Expression, Immunohistochemical staining

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Alkali-induced corneal neovascularization and macrophage infiltration. A : The macroscopic appearances of WT, CCL3-KO, CCR1-KO, and CCR5-KO mouse eyes two weeks after alkali injury are illustrated. Representative results from at least 10 animals in each group are shown here. B : Corneal tissues were obtained from WT, CCR1-KO, and CCL3-KO mice two weeks after the injury. Tissues were stained with hematoxylin and eosin (left panels) or immunostained with anti-CD31 antibodies (right panels), and representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm. C : CNV numbers per mm 2 in hot spots (upper panel) and % CNV areas in hot spots (lower panel) were determined on corneas obtained from WT or KO mice two weeks after the injury. Each value represents the mean and SEM (n=5 animals). An asterisk represents a p<0.05 and that the value was obtained comparing WT and CCL3-KO mice. D : The numbers of infiltrated F4/80 positive macrophages were determined two and four days after the injury. Each value represents the mean and SEM (n=5). The double asterisk indicates a p<0.01 and that the value was obtained comparing WT and CCL3-KO mice.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Staining

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Angiogenic factor expression. A : RT–PCR analysis of pro-angiogenic and anti-angiogenic gene expressions in the injured corneas of WT and CCL3-KO mice. RT–PCR analysis was performed on total RNAs extracted from eyes 0, 2, 4, and 7 days after alkali injury, and then the ratios of VEGF to β-actin, bFGF to β-actin, and TSP-1 to β-actin of WT (black bars) and CCL3-KO mice (open bars) were determined. All values represent the mean and SEM (n=3-5 animals). The asterisk denotes a p<0.05; the hash mark denotes a p<0.01 and that the value was obtained comparing WT and KO mice. The effects of CCL3 on VEGF expression by murine peritoneal macrophages is shown in B and C . B : RT–PCR was performed on macrophages incubated with the indicated concentrations of CCL3 for 12 h, and the ratio of VEGF to β-actin was calculated. Each value represents the mean and SEM (n=3). C : Murine macrophages were stimulated with either 0, 10, or 100 ng/ml of CCL3 for 24 h. VEGF concentrations in the supernatants were determined with ELISA as described in Methods. The representative results from three independent experiments are shown. The asterisk denotes a p<0.05 and the double asterisk denotes a p<0.01 when compared to untreated.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Intracorneal CCR5 positive cell infiltration. A : A double-color immunofluorescence analysis of CCR5-expressing cells is illustrated. Corneas were obtained from WT mice 0 and 4 days after the injury. The samples were immunostained with a combination of anti-CCR5 and anti-CCR2 antibodies as described in Methods and observed with fluorescence microscopy (original magnification, 400X). Signals were digitally merged in the right panels. Arrows indicate the double, positively stained cells. Representative results from three independent experiments are shown. B : Corneal tissues from WT mice (left panel) or CCL3-KO mice (right panel) obtained four days after the injury were stained with anti-CCR5 Ab. Scale bar, 100 μm. C : The numbers of intracorneal CCR5 positive cells four days after the injury were determined as described in Methods, and the mean and SEM are shown here (n=5).

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Immunofluorescence, Expressing, Fluorescence, Microscopy, Staining

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: The effects of topical CCL3 application on corneal neovascularization. A : Macroscopic appearances of WT, CCL3-KO mice, and CCL3-KO mice topically applied with CCL3 two weeks after alkali injury are shown. Representative results from five animals from each group are shown here. B : Corneal tissues were obtained two weeks after the injury from WT, CCL3-KO, and CCL3-KO mice topically applied with CCL3 and were immunostained with anti-CD31 antibodies. Representative results from five individual mice from each group are shown. Original magnification, 400X. Scale bar, 50 μm. C : The CNV numbers per mm 2 in hot spots (left panel) and % CNV areas in hot spots (right panel) were determined. Each value represents the mean and SEM (n=5 animals). D : The number of infiltrated F4/80 positive macrophages was determined on WT, CCL3-KO, and CCL3-KO, which were all treated with CCL3, two days after the injury. Each value mean represents both the mean and SEM (n=5). The asterisk denotes a p<0.05, and the double asterisk means a p<0.01 when compared with CCL3-KO (this applies to both C and D ).

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques:

Journal: Autophagy

Article Title: Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis

doi: 10.1080/15548627.2015.1096485

Figure Lengend Snippet: Defective autophagy in VSMCs elicits cellular hypertrophy, and increases migration capacity and total collagen amount. ( A ) VSMCs isolated from Atg7 +/+ Tagln-Cre + (+/+) and Atg7 F/F Tagln-Cre + (−/−) aorta were labeled with calcein AM and visualized by confocal fluorescence microscopy. Scale bar: 10 µm. Cell size was measured using z-stack images (*, P < 0.05; n = 2 experiments; Student t test). ( B ) Thoracic aorta of Atg7 +/+ and atg7 −/− mice were stained with H&E to measure the width of the media (white arrows) (**, P < 0.01; n = 6 regions/aorta; Univariate). Scale bar: 25 µm. Note that the number of VSMC layers between Atg7 +/+ and atg7 −/− aorta was not different. ( C ) Migratory capacity of Atg7 +/+ and atg7 −/− VSMCs was analyzed using an Oris Migration Assay (***, P < 0.001; n = 2 experiments in triplicate; Student t test). Western blot analysis of TGFB and CXCL12 in Atg7 +/+ and atg7 −/− VSMCs. ( D ) Atg7 +/+ and atg7 −/− VSMCs were left untreated or treated with 10 ng/ml TGFB for 48 h and stained with Sirius red to examine total collagen amount (***, P < 0.001 vs. Atg7 +/+ ; ### , P < 0.001 vs. control; n = 4 experiments in triplicate; two-way ANOVA with genotype and treatment as category factors).

Article Snippet: Membranes were probed with the following primary antibodies: goat anti-GSTA (ab53940), rabbit anti-NFE2L2 (ab137550), mouse anti-CDKN2A/p16 (ab54210) and rabbit anti-CDKN1A/p21 (ab7960) from Abcam; rabbit anti-CXCL12 (Bioss, bs-4938); rabbit anti-GAPDH (14C10), rabbit anti-TGFB (3711) and rabbit anti-phospho RB (8516) from Cell Signaling Technology; mouse anti-MAP1LC3B (Nanotools, clone 5F10, 0231-100); rabbit anti-NQO1 (Novus Biologicals, NBP1-40663); rabbit anti-PARP1 (sc-7150), rabbit anti-CDKN2A/p16 (sc-1207) and rabbit anti-total RB (sc-50) from Santa Cruz Biotechnology; mouse anti-ACTB (clone AC-15, A5441), rabbit anti-ATG7 (A2856), rabbit anti-ATG5 (A0856), rabbit anti-SQSTM1/p62 (P0067) and rabbit anti-acetyl-TP53 (SAB4503014) from Sigma-Aldrich.

Techniques: Migration, Isolation, Labeling, Fluorescence, Microscopy, Staining, Western Blot

Journal: Autophagy

Article Title: Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis

doi: 10.1080/15548627.2015.1096485

Figure Lengend Snippet: Defective VSMC autophagy promotes upregulation of MMP9, TGFB and CXCL12, 5 d after ligation-induced injury. ( A ) The left common carotid artery (LCCA) of Atg7 +/+ Tagln-Cre + (+/+) and Atg7 F/F Tagln-Cre + (−/−) mice (n = 3) was ligated for 5 d. Gelatin zymographic analysis of the LCCA to detect MMP9 and MMP2 activity followed by densitometric analysis (***, P < 0.001; NS, not significant; Student t test). ( B ) Western blot analysis of the LCCA for TGFB, CXCL12 and GAPDH. Relative expression of TGFB/GAPDH and CXCL12/GAPDH was determined by densitometric analysis (*, P < 0.05; ***, P < 0.001; Student t test).

Article Snippet: Membranes were probed with the following primary antibodies: goat anti-GSTA (ab53940), rabbit anti-NFE2L2 (ab137550), mouse anti-CDKN2A/p16 (ab54210) and rabbit anti-CDKN1A/p21 (ab7960) from Abcam; rabbit anti-CXCL12 (Bioss, bs-4938); rabbit anti-GAPDH (14C10), rabbit anti-TGFB (3711) and rabbit anti-phospho RB (8516) from Cell Signaling Technology; mouse anti-MAP1LC3B (Nanotools, clone 5F10, 0231-100); rabbit anti-NQO1 (Novus Biologicals, NBP1-40663); rabbit anti-PARP1 (sc-7150), rabbit anti-CDKN2A/p16 (sc-1207) and rabbit anti-total RB (sc-50) from Santa Cruz Biotechnology; mouse anti-ACTB (clone AC-15, A5441), rabbit anti-ATG7 (A2856), rabbit anti-ATG5 (A0856), rabbit anti-SQSTM1/p62 (P0067) and rabbit anti-acetyl-TP53 (SAB4503014) from Sigma-Aldrich.

Techniques: Ligation, Activity Assay, Western Blot, Expressing

Journal: Autophagy

Article Title: Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis

doi: 10.1080/15548627.2015.1096485

Figure Lengend Snippet: Overview of the mechanisms by which defective VSMC autophagy accelerates senescence and promotes postinjury neointima formation and diet-induced atherogenesis. SQSTM1 accumulation in autophagy defective VSMCs triggers NFE2L2 activation and transcription of multiple antioxidative enzymes including GSTA and NQO1. Upregulation of GSTA and NQO1 promotes VSMC survival against oxidative stress under defective autophagy conditions. SQSTM1 accumulation in autophagy defective VSMCs triggers the development of stress-induced premature senescence. Autophagy defective VSMCs are characterized by CDKN2A-RB-mediated G 1 proliferation arrest, increased migration and changes in VSMC phenotype. Enhanced migration is associated with increased secretion of MMP9, TGFB and CXCL12. The phenotype of autophagy defective VSMCs is defined by nuclear and cellular hypertrophy, and by increased collagen content. Defective autophagy in VSMCs accelerates postinjury neointima formation and diet-induced atherogenesis.

Article Snippet: Membranes were probed with the following primary antibodies: goat anti-GSTA (ab53940), rabbit anti-NFE2L2 (ab137550), mouse anti-CDKN2A/p16 (ab54210) and rabbit anti-CDKN1A/p21 (ab7960) from Abcam; rabbit anti-CXCL12 (Bioss, bs-4938); rabbit anti-GAPDH (14C10), rabbit anti-TGFB (3711) and rabbit anti-phospho RB (8516) from Cell Signaling Technology; mouse anti-MAP1LC3B (Nanotools, clone 5F10, 0231-100); rabbit anti-NQO1 (Novus Biologicals, NBP1-40663); rabbit anti-PARP1 (sc-7150), rabbit anti-CDKN2A/p16 (sc-1207) and rabbit anti-total RB (sc-50) from Santa Cruz Biotechnology; mouse anti-ACTB (clone AC-15, A5441), rabbit anti-ATG7 (A2856), rabbit anti-ATG5 (A0856), rabbit anti-SQSTM1/p62 (P0067) and rabbit anti-acetyl-TP53 (SAB4503014) from Sigma-Aldrich.

Techniques: Activation Assay, Migration