Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Western blotting (left panel) for total and phosphorylated SFK in the lungs from control IgG- and 38–2 mAb-treated mice at 3 dpi with 200 IFU of IAV/PR8. Actb, β-actin. The percent density of total SFK (middle panel) and SFK phosphorylated at tyrosine 416 (right panel) against the densities of the corresponding proteins in IgG-treated, infected lungs after being normalized by the densities of β-actin. **, p<0.01. ( B ) Survival rate (%, upper panels) and body weight loss (%, lower panels) of WT mice intraperitoneally administrated with control IgG (left panels) and 38–2 mAb (right panel) together with 20 mg/mouse of DS 1 day before intranasal infection with 200 IFU of IAV/PR8. Error bars, SD. ( C ) Western blotting (upper panel) for total SFK, phosphorylated SFK (Tyr416), the cleaved caspase 3, and the viral protein M2 in the lungs from control IgG/DMSO-, 38–2 mAb/DMSO-, control IgG/DS-, and 38–2 mAb/DS-treated mice at 3 dpi with 200 IFU of IAV/PR8. Actb, β-actin. Lungs from uninfected, saline/DMSO-treated mice were used as a control. The percent density (lower panels) of total SFK, phosphorylated SFK (Tyr416), the cleaved caspase 3, and the viral protein M2 against the densities of the corresponding proteins in uninfected, saline/DMSO-treated lungs after being normalized by the densities of β-actin. *, p<0.05; **, p<0.01.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Control, Infection, Saline

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Double immunofluorescent staining for phosphorylated SFK (Tyr416) together with MGL1/2, CD3, and MPO in the lungs from control IgG- and 38–2 mAb-treated mice uninfected and at 3 dpi with 200 IFU of IAV/PR8. Un, uninfected. Bar, 200 μ m. ( B ) Fold mRNA expression (2 -ΔCt ) analyzed by real-time RT-PCR for IL-6, TNF-α, IFN-α, INF-γ, MCP-1, iNOS, IFIT1, MxA, ARG1, MGL1, and IL-10 in the lungs from control IgG/DMSO, control IgG/DS-, 38–2 mAb/DMSO-, and 38–2 mAb/DS-treated mice uninfected and at 3 dpi with 200 IFU of IAV/PR8 (n = 3 in each group). Un, uninfected.*, p<0.05;**, p<0.01.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Staining, Control, Expressing, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Uncropped, full picture of Western blotting for PrP C with 38–2 mAb in the lungs (Lg) and peritoneal macrophages (PM) from WT and Prnp 0/0 mice. Actb, β-actin. ( B ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages from WT and Prnp 0/0 mice 3 hrs after treatment with control IgG and 38–2 mAb together with DS or control DMSO. ( C ) ELISA for INF-γ, TNF-α, IL-4, and IL-10 in the culture medium from WT peritoneal macrophages at 0.5, 4, 16, and 24 hours (hrs) after treatment with control IgG and 38–2 mAb.*, p<0.05;** p<0.01. ( D ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages (PM) from WT and Prnp 0/0 mice 1 and 3 days after peritoneal injection with control IgG and 38–2 mAb together with control DMSO and DS.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Injection

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages from WT and Prnp 0/0 mice 3 hrs after treatment with control IgG and 3S9 and 2H9 mAbs together with control DMSO and DS. ( B ) Survival rate (%, upper panels) and body weight loss (%, lower panels) of WT mice intraperitoneally administered with control IgG and 3S9 and 2H9 mAbs 1 day before intranasal infection with 100 (left panels) or 200 IFU (right panels) of IAV/PR8. Error bars, SD. *, p<0.05; **, p<0.01; IgG vs 3S9. #, <0.05; IgG vs 2H9.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Control, Infection

Journal: Immunology and Cell Biology

Article Title: An alternative downstream translation start site in the non‐TIR adaptor Scimp enables selective amplification of CpG DNA responses in mouse macrophages

doi: 10.1111/imcb.12540

Figure Lengend Snippet: Scimp is required for sustained LPS responses and Scimp TV1 is basally, rather than LPS‐inducibly, phosphorylated. (a, b) BMMs from wild‐type (WT) and Scimp TV1 mice were treated with LPS (1 ng mL −1 ) for the indicated time points, before being washed of LPS and replated in fresh media for a further 24 h. Supernatants were collected and assessed for IL‐12p40 production by ELISA. Data (mean ± s.e.m., n = 3 or 4 mice per genotype) are combined from three or four independent experiments. (c) BMMs from WT and Scimp TV1 (TV1) mice were differentiated with either CSF‐1 (10 ng mL −1 ; lanes at left) or GM‐CSF (10 ng mL −1 ; lanes at right) for 6 days. CSF‐1‐BMMs were stimulated with LPS (10 ng mL −1 ) and IFNγ (5 ng mL −1 ) or GM‐CSF (10 ng mL −1 ) for 24 h. Total protein was collected and assessed for Scimp expression via western blot. Data are representative of two independent experiments collected from two mice per genotype. (d) BMMs from WT and Scimp TV1 mice were differentiated using CSF‐1, then primed overnight with GM‐CSF (10 ng mL −1 ). BMMs were stimulated with LPS (10 ng mL −1 ) for the indicated time points before being lysed. Collected protein was immunoprecipitated using a rabbit anti‐Scimp antibody or an isotype control (rabbit anti‐Myc tag). Immunoprecipitated samples were assessed for levels of phosphorylated Scimp (p‐Tyr) and coimmunoprecipitated TLR4 and Lyn via western blot. Data are representative of two independent experiments. (e) TLR4 and (f) phospho‐Scimp levels from d were quantified using Image Lab and displayed relative to the corresponding immunoprecipitated Scimp band (the IC background signal in control lanes was subtracted from the appropriate sample band intensity before quantifying). Data are representative of two independent experiments collected from two mice per genotype. (g) SH2 pulldown assays were used to assess LPS‐inducible phosphorylation of Y58 (Grb2‐SH2) and Y120 (SLP‐SH2) on Scimp‐V5 in RAW264.7 cells expressing Scimp‐V5 or Scimp TV1‐V5. Data are representative of three independent experiments. Statistical significance in a and b was determined using two‐way ANOVA, followed by Bonferroni’s multiple comparison test using GraphPad Prism 9 (*** P < 0.001). GM‐CSF‐BMM, granulocyte macrophage colony‐stimulating factor‐derived bone marrow‐derived macrophages; IC, isotype control; IFN, interferon; LPS, lipopolysaccharide.

Article Snippet: Lysates were passed through a 28‐gauge needle and centrifuged at 10 000 g for 15 min. 30 µL of sample was added to columns that contained 200 µg (20 µL of 10 µg per µL stock solution) of SH2 probe [SH2 domain of either human CSK, GRB2 or SLP65 conjugated to GST and bound to Pierce protein G plus agarose beads (Life Technologies) as described in Luo et al. ].

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Immunoprecipitation, Derivative Assay

Journal: Immunology and Cell Biology

Article Title: An alternative downstream translation start site in the non‐TIR adaptor Scimp enables selective amplification of CpG DNA responses in mouse macrophages

doi: 10.1111/imcb.12540

Figure Lengend Snippet: Assessment of Scimp cellular localization and phosphorylation in response to CpG DNA. (a–c) Wild‐type (WT) BMMs retrovirally transduced with either an empty vector, Scimp‐V5 or Scimp TV1‐V5 expression construct. Cells were treated with 0.3 µM fluorescently labeled (Alexa‐647‐conjugated) CpG DNA (CpG) for 30 min. Cellular localization of V5‐tagged Scimp proteins and CpG DNA were assessed via immunofluorescence microscopy (anti‐V5/Scimp: green; CpG‐647: red; DAPI: blue; scale bars: 10 µm). White arrows indicate filopodia‐localized Scimp‐V5. Similar results were observed in three independent experiments in which a total of three WT C57Bl/6 mice were retrovirally transduced (1 mouse per experiment). (b) SH2 pulldown assays were used to assess CpG DNA‐inducible phosphorylation of Y58 (Grb2‐SH2) and Y96 (CSK‐SH2) on Scimp‐V5 in CSF‐1‐BMMs expressing Scimp‐V5 or Scimp TV1‐V5. Data are representative of three independent experiments and three mice per genotype. (c) CSK‐bound Scimp and Scimp TV1 bands from b were quantified and normalized to the 0‐min control of the WT. Data (mean ± s.e.m., n = 3 mice per genotype) are combined from three independent experiments. BMM, bone marrow‐derived macrophages.

Article Snippet: Lysates were passed through a 28‐gauge needle and centrifuged at 10 000 g for 15 min. 30 µL of sample was added to columns that contained 200 µg (20 µL of 10 µg per µL stock solution) of SH2 probe [SH2 domain of either human CSK, GRB2 or SLP65 conjugated to GST and bound to Pierce protein G plus agarose beads (Life Technologies) as described in Luo et al. ].

Techniques: Transduction, Plasmid Preparation, Expressing, Construct, Labeling, Immunofluorescence, Microscopy, Derivative Assay

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the healthy population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the HT and CVD population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the HT population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the CVD population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Antioxidants

Article Title: Glyoxalase 2 Drives D-Lactate Oncometabolite Signaling to Promote Prostate Cancer Aggressiveness via FAK/Src Activation

doi: 10.3390/antiox15020171

Figure Lengend Snippet: Effect of Glyoxalase 2 (Glo2) silencing on FAK/Src activation in PC3 cells. Glo2 silencing affected both ( a ) FAK (Tyr397) and ( b ) Src (Tyr416) phosphorylation, assessed using ELISA kits in total cell lysates. Histograms represent mean ± SD of three different independent cultures. *** p < 0.001, **** p < 0.0001.

Article Snippet: Phosphorylated FAK levels were measured using the Human FAK [Phospho] [pY397] ELISA Kit (cat. KHO0441, Thermo Fisher Scientific, Milan, Italy), while phosphorylated Src levels were determined using PathScan ® Phospho-Src [Tyr416] Sandwich ELISA Kit (cat. 7953, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Antioxidants

Article Title: Glyoxalase 2 Drives D-Lactate Oncometabolite Signaling to Promote Prostate Cancer Aggressiveness via FAK/Src Activation

doi: 10.3390/antiox15020171

Figure Lengend Snippet: Effects of Glyoxalase 2 (Glo2) silencing and exogenous D-lactate supplementation (2.5 mM) on FAK/Src activation and migration/invasion in PC3 cells. ( a ) FAK (Tyr397) and Src (Tyr416) phosphorylation, assessed using ELISA kits in total cell lysates, ( b ) cell migration and invasion capabilities, evaluated by specific assays. Histograms represent mean ± SD of three different independent cultures. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Phosphorylated FAK levels were measured using the Human FAK [Phospho] [pY397] ELISA Kit (cat. KHO0441, Thermo Fisher Scientific, Milan, Italy), while phosphorylated Src levels were determined using PathScan ® Phospho-Src [Tyr416] Sandwich ELISA Kit (cat. 7953, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activation Assay, Migration, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Antioxidants

Article Title: Glyoxalase 2 Drives D-Lactate Oncometabolite Signaling to Promote Prostate Cancer Aggressiveness via FAK/Src Activation

doi: 10.3390/antiox15020171

Figure Lengend Snippet: Effects of FAK inhibition on Src activation, migration, and invasion in Glyoxaalse 2 (Glo2)-silenced PC3 cells treated with exogenous D-lactate (2.5 mM). ( a ) Src (Tyr416) phosphorylation, assessed using ELISA kit in total cell lysate, ( b ) cell migration and invasion capabilities, evaluated by specific assays. Histograms represent mean ± SD of three different independent cultures. siGlo2: Glo2 transient silencing; PF-573228: FAK inhibitor (5 µM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Phosphorylated FAK levels were measured using the Human FAK [Phospho] [pY397] ELISA Kit (cat. KHO0441, Thermo Fisher Scientific, Milan, Italy), while phosphorylated Src levels were determined using PathScan ® Phospho-Src [Tyr416] Sandwich ELISA Kit (cat. 7953, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Inhibition, Activation Assay, Migration, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cancer Research

Article Title: Protein Kinase G Type Iα Activity in Human Ovarian Cancer Cells Significantly Contributes to Enhanced Src Activation and DNA Synthesis/Cell Proliferation

doi: 10.1158/1541-7786.mcr-09-0178

Figure Lengend Snippet: FIGURE 4. Inhibition of endogenous basal PKG-Iα activity using DT-2 in OV2008 ovarian cancer cells reduces the basal level of autophosphorylation of endogenous SFKs and completely inhibits EGF-induced stimulation of SFK autophosphorylation and DNA synthesis. A, the PKG-Iα kinase inhibitor DT-2 dramatically lowered the basal SFK autophosphorylation level, assessed by both Western blot analysis determining autophosphorylation (the equivalent of Tyr416; top image) and ELISA measuring Tyr416 autophosphorylation versus total Src (graph). B, EGF (10 ng/mL) elevated VASP Ser239

Article Snippet: The samples were analyzed by Western blot using anti–phospho-Tyr416-Src antibody and anti– total Src antibody (Cell Signaling Technology).

Techniques: Inhibition, Activity Assay, DNA Synthesis, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cancer Research

Article Title: Protein Kinase G Type Iα Activity in Human Ovarian Cancer Cells Significantly Contributes to Enhanced Src Activation and DNA Synthesis/Cell Proliferation

doi: 10.1158/1541-7786.mcr-09-0178

Figure Lengend Snippet: FIGURE 6. EGF stimulates both Src/SFK autophosphorylation and PKG-Iα kinase activity (VASP Ser239 phosphorylation) in OV2008 ovarian cancer cells and these responses are inhibited by SU6656, a SFK-selective tyrosine kinase inhibitor. A, Western blot showing that EGF (10 and 50 ng/mL) increases Src/SFK autophosphorylation (equivalent of Tyr416) in OV2008 cells in a time-dependent manner, with the phosphorylation reaching a peak at 15 to 30 min. B, Western blot showing that EGF (10 and 50 ng/mL) increases both Src/SFK activity (autophosphorylation) and PKG-Iα kinase activity (assessed by downstream phosphorylation of the PKG-Iα substrate, VASP at Ser239) in OV2008 cells in a concentration-dependent manner. C, SU6656 (1 and 5 μmol/L) inhibited the EGF-induced stimulation of the EGFR phosphorylation at Tyr845 (site phosphorylated by Src), the Ser239

Article Snippet: The samples were analyzed by Western blot using anti–phospho-Tyr416-Src antibody and anti– total Src antibody (Cell Signaling Technology).

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Concentration Assay

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet: GH induces WIP1 through GHR signaling (A) Western blots of hNCC line #1 pretreated with 20 mg/mL pegvisomant (Peg) for 1 h then treated with GH (500 ng/mL) for an additional 24 h. C, untreated control. (B) Colon tissue derived from 3- and 24-month-old WT and GHR −/− male mice. Each lane represents sample analysis derived from an individual animal. (C) hNCC line #1 transduced with lentivirus expressing GH (Lenti-GH) or empty vector (Lenti-V) and analyzed 10 days later. (D) iPSC-derived human intestinal organoid lines from 3 different patients were transduced with Lenti-V or Lenti-GH and analyzed 5 weeks later. Representative blots from one human intestinal organoid line are shown. (E) hNCC line #1 transduced with lentivirus expressing control shRNA (shControl) or GH shRNA (shGH). (F) Three different human intestinal organoid lines were transduced with GFP-expressing Lenti-V or Lenti-GH and cultured for 5 weeks, then sorted for GFP-negative cells. GFP-negative cells co-existing in organoids with either Lenti-GH or Lenti-V expressing cells were analyzed. All lines exhibited similar results. Representative blots from one human intestinal organoid line are shown. Representative blots from at least 3 independent experiments are depicted. ImageJ quantifications of western blots are depicted in Figure S2 .

Article Snippet: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

Techniques: Western Blot, Control, Derivative Assay, Transduction, Expressing, Plasmid Preparation, shRNA, Cell Culture

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet: GH induces WIP1 by activating Src/AMPK (A and B) Western blots of (A) hNCC line #1 treated with 1 μM AMPK inhibitor (Compound C) for 1 h followed by GH (500 ng/mL) for an additional 24 h and (B) human intestinal organoids treated with 2 μM AMPK inhibitor and GH (500 ng/mL) for 48 h. (C–F) Western blots of hNCC line #1 was treated with (C) GH (500 ng/mL) for up to 60 min; (D) 25 μM Src inhibitor for 24 h followed by GH (500 ng/mL) for 30 min–60 min; (E) 25 μM Src inhibitor for 1 h followed by GH (500 ng/mL) for 24 h; and (F) 25 μM Src inhibitor for 24 h followed by GH (500 ng/mL) for 3 h. Untreated cells/organoids served as control (C). (G–I) Western blots of hNCC line #1 transduced with lentivirus expressing Control shRNA (shControl) or Src shRNA (shSrc) (G) analyzed 72 h after transduction; (H) treated with GH (500 ng/mL) for 3 h; and (I) treated with GH (500 ng/mL) for 24 h. ImageJ quantifications of western blots are depicted in Figure S5 .

Article Snippet: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

Techniques: Western Blot, Control, Transduction, Expressing, shRNA

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet:

Article Snippet: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

Techniques: Virus, Control, shRNA, Recombinant, Cell Recovery, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, DC Protein Assay, Extraction, Immunoprecipitation, cDNA Synthesis, Single Cell Gel Electrophoresis, Derivative Assay, Generated, Software, Imaging, Real-time Polymerase Chain Reaction, Microscopy