Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 1. Comparison of NPRA and NPRC protein expression. (A) Protein expression of NPRA and NPRC were visualized by western blotting in membrane enriched fractions prepared from a variety of mouse tissues: gonadal white adipose tissue (gWAT), inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), heart, liver, kidney, and lung. (B) BAT membrane enriched fractions from wildtype and NPRC−/− mice fed either standard chow or HFD were used to visualize NPRA and NPRC protein levels. Šídák’s multiple comparisons test for two-way ANOVA indicated on graph. * P < 0.05, *** P < 0.001, **** P < 0.0001. (C) BAT protein from chow- and HFD-fed mice was separated into membrane and cytosolic enriched fractions. NPRA and NPRC were assessed by western blotting. (D) HIB-1B cells were transfected with the indicated plasmids and treated with adipocyte differentiation cocktail for 48 h. Cells were then treated with the 2.5 µM MG-132 for 16 h. Flag-NPRA and NPRC were assessed from whole-cell lysates by western blotting. Šídák’s multiple comparisons test for one-way ANOVA indicated on graph. ** P < 0.01, *** P < 0.001.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Comparison, Expressing, Western Blot, Membrane, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 2. NPRC immunoprecipitates with NPRA and NPRB. HEK 293FT cells were transfected with plasmids expressing Flag-tagged natriuretic peptide receptors. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag Affinity agarose. (A) Endogenous NPRC coimmunoprecipitated with hNPRA-Flag, hNPRB-Flag, or VEGFR1-Flag. (B) Endogeneous NPRC coimmunoprecipitated with GC-C-Flag. (C) NPRC band intensity to input of NPRC was calculated through Image J software was presented in the right panel. Šídák’s multiple comparisons test for one-way ANOVA comparing all samples to the negative control VEGFR1 for western blots in A and B are indicated on graph. *P < 0.05, ** P < 0.01, *** P < 0.001. (D) hNPRA-Flag expressing HEK 293FT cells were treated with ANP (ANP1-28, 200 nM) or a truncated form of ANP that preferentially binds NPRC (ANP4-23, 200 nM) for 30 min. Neither ANP1-28 or ANP4-23 affected the ability of endogenous NPRC to coimmunoprecipitate with hNPRA-Flag. (E) Endogenous NPRC coimmunoprecipitated with Flag-rNPRA and a truncated version of Flag- rNPRA lacking the c-terminal amino acids 528-1057 [Flag-rNPRA(1-527)].

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Transfection, Expressing, Immunoprecipitation, Software, Negative Control, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 3. Endogenous natriuretic peptide receptor expression in cell models used. Expression of NPRA, NPRB, and NPRC were detected by western blot in HeLa, HEK293FT, and HAP-1 cells. β-actin was used as the loading control.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Expressing, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 6. Coexpression of NPRC reduces ANP-evoked cGMP production in plasma membranes. Plasma membranes were made from HEK-293T cells transfected with either NPRA-flag plus pcDNA3-Clover control or NPRA-flag plus NPRC-flag. Plasma membranes were made without or with (SI Appendix, Fig. S3) phosphatase inhibitors. Co-expression of hNPRC along with hNPRA reduced the cGMP generated in the presence of 100 nM ANP (P = 0.0163, test for difference in slopes between NPRA and NPRA+NPRC; Šídák’s multiple comparisons test comparing NPRA vs. NPRA+NPRC are indicated on the graphs, ** P < 0.01.). Untransfected cells did not appear to generate cGMP. Western blot of membranes used in the ANP-stimulated cGMP assay.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Clinical Proteomics, Transfection, Control, Expressing, Generated, Western Blot

Journal: Biomolecules

Article Title: Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia

doi: 10.3390/biom14050523

Figure Lengend Snippet: Light microscopy images and the expression of herpesvirus entry mediator (HVEM) surface protein on breast cancer (MCF-7), hepatocellular carcinomas (HepG2), chronic myelogenous leukemia (K562), and human embryonic kidney (Hek293) cells. The left column represents the microscope and a magnification of 100 pt using NIS-Elements F 4.00.00 software (Nikon Instruments, USA) under a light microscope (Nikon Eclipse, Fujisawa, Japan). Data collected from four separate experiments represents cell proliferation, morphology, and attachment nature. The right column shows histogram graphs representing the measurements of HVEM expression after staining with anti-HVEM-PE. The dotted-line histograms show the unstained control cells, whereas the line histograms represent the HVEM-stained cells. The numbers at the top of each histogram indicate the MFI of the unstained ( left ) and HVEM-PE ( right ). The number in the middle represents the percentage of HVEM-positive cells. The results were collected from two wells in three separate experiments. Magnification: 100 pt.

Article Snippet: The efficacy of blocking HVEM expressed on K562 leukemic cells by NBP1-76690PEP (Novus Biologicals, USA) on the proliferation of naïve CFSE CD4 + T cells showed an increase in CD4 + T cell proliferation in vitro.

Techniques: Light Microscopy, Expressing, Microscopy, Software, Staining, Control

Journal: Biomolecules

Article Title: Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia

doi: 10.3390/biom14050523

Figure Lengend Snippet: The relative gene expression level of HVEM , normalized to the housekeeping gene GAPDH , is represented as a fold change.

Article Snippet: The efficacy of blocking HVEM expressed on K562 leukemic cells by NBP1-76690PEP (Novus Biologicals, USA) on the proliferation of naïve CFSE CD4 + T cells showed an increase in CD4 + T cell proliferation in vitro.

Techniques: Gene Expression

Journal: Biomolecules

Article Title: Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia

doi: 10.3390/biom14050523

Figure Lengend Snippet: Light microscopic images of K562 ( left ) and Hek293 ( right ): ( A ) alone, ( B ) HVEM untreated cells with CFSE CD4 + T cells at one tumor or normal to ten T cells, or ( C ) HVEM blocker treated cells with CFSE CD4 + T cells after K562 and Hek293 for 72 h of incubation. Data are representative of three separate experiments. Magnification: 100 μm.

Article Snippet: The efficacy of blocking HVEM expressed on K562 leukemic cells by NBP1-76690PEP (Novus Biologicals, USA) on the proliferation of naïve CFSE CD4 + T cells showed an increase in CD4 + T cell proliferation in vitro.

Techniques: Incubation

Journal: Biomolecules

Article Title: Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia

doi: 10.3390/biom14050523

Figure Lengend Snippet: Proliferation of CFSE-labeled CD4 + T cells in response to ( A ) K562 tumor cells or ( B ) Hek293 healthy control cells without HVEM blocker treatment ( left column), or after K562 and Hek293 treatment with HVEM blocker ( middle column); and non-proliferated CFSE CD4 + T cells ( right column) after 72 h of incubation at 37 °C. Data are representative of two separate experiments.

Article Snippet: The efficacy of blocking HVEM expressed on K562 leukemic cells by NBP1-76690PEP (Novus Biologicals, USA) on the proliferation of naïve CFSE CD4 + T cells showed an increase in CD4 + T cell proliferation in vitro.

Techniques: Labeling, Control, Incubation

Journal: Biomolecules

Article Title: Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia

doi: 10.3390/biom14050523

Figure Lengend Snippet: Mean ± SD of fluorescence intensity of CFSE-labeled CD4 + T cells before proliferation on day zero and after 72 h of incubation with either K562 tumor cells or with Hek293 normal cells that were left untreated or treated with 20 ng HVEM blocker. Cells were cultured at a ratio of one tumor or normal cell to ten CFSE CD4 + T cells. ** indicates strong significant differences between groups with p = 0.0033, whereas “ns” indicates no significant differences with p = 0.5918. The results were collected from three separate experiments.

Article Snippet: The efficacy of blocking HVEM expressed on K562 leukemic cells by NBP1-76690PEP (Novus Biologicals, USA) on the proliferation of naïve CFSE CD4 + T cells showed an increase in CD4 + T cell proliferation in vitro.

Techniques: Fluorescence, Labeling, Incubation, Cell Culture

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: ( A ) Histology of untreated cultures (UT) show (a) low-grade RCC with small round tumor cells and a clear cytoplasm surrounded by a distinct cell membrane and round uniform nuclei with inconspicuous or absent nucleoli. (b) R1TNF- and (c) wtTNF-treated cultures show elevated level of death in mTECs compared with (d) R2TNF-treated cultures. ( B ) Morphological features of necrosis are evident in R1TNF-treated cultures such as large/small cytoplasmic vacuoles (x), condensation of the chromatin into small, irregular, circumscribed patches (white arrow), increasing translucent cytoplasm, swollen organelles (orange arrow) and disruption of the plasma membrane (black arrows). ( C ) Expression of RIPK1 and RIPK3 in tissue biopsies comprising RCC and NK; NK show a rare signal for RIPK1 and RIPK3 in MNCs within glomeruli (arrows) and with interstitium (small arrows). In contrast, RCC grade 3 shows marked signal in mTECs (arrows), MNCs (small arrows) and in some VECs (arrowheads). ( D and E ) Representative immunoblot of samples from nine patients with similar results and bar graph of relative RIPK1 and RIPK3 protein levels in grades 3/4 RCC (G3/G4) and non-tumor kidney (NK). Bars=mean+S.E.M.; * P <0.05 versus NK; paired Student's t -test. Glom-glomeruli; MNCs-mononuclear cells; TECs-tubular epithelial cells

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Membrane, Disruption, Clinical Proteomics, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: Representative confocal images and light micrographs of the effect of R1TNF on protein and mRNA expression for RIPK1 and RIPK3 in organ cultures of RCC grade 1 and adjacent non-tumor kidney (NK). ( a and b ) Untreated (UT) cultures from RCC show a rare signal for RIPK1 and RIPK3 protein and mRNA. In contrast, R1TNF-treated cultures show a marked signal of both proteins mainly confined to mTECs (arrows), with RIPK3 also present in nuclei (white shaded arrow). Similarly, UT cultures of NK show a rare signal for both proteins; increased expression of both protein and mRNA is detected in R1TNF-treated cultures mainly confined to normal TECs (white arrows), peritubular capillaries (white arrowheads) and in infiltrating mononuclear cells (black arrowhead) but not in glomeruli (Glom). ( c ) Representative mean fluorescence intensity (MFI) for RIPK1 and RIPK3 expression in RCC and NK organ cultures. ** P <0.01 versus UT, * P <0.05 versus UT, ± P <0.05 versus R1TNF; Bars=mean±S.E.M.; n =3 independent experiments from six separate organ culture experiments with similar results. Nuclei stained with Hoechst 33342. Confocal images: × 40 and × 63 original magnifications; photomicrographs: × 400 magnification

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Expressing, Fluorescence, Organ Culture, Staining

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: PLA of organ culture of RCC grade 1. ( a ) In comparison with untreated controls (UT), R1TNF induced a strong interaction of RIPK1-RIPK3 and RIPK3-pMLKL Ser358 appearing as strong red fluorescence spots mainly within the cytoplasm of mTECs. Each individual interacting protein pair observed as a red spot by confocal microscopy is expressed as the number of signals/cell (PLA spots/cell). ( b and c ) Quantification of PLA spots in TECs in the two study groups; RCC (RCCoC) and normal kidney (NKoC) show a statistically significant difference, more pronounced in RCCoC. ** P <0.01 versus UT, * P <0.05 versus UT, ± P <0.05 versus R1TNF (NKoC). ( d and e ) Representative confocal images of pMLKL Ser358 or pDrp1 Ser637 and TUNEL in organ cultures of RCC grade 1. Compared with UT cultures, R1TNF induced an increase in the level of TUNEL+ mTECs ( green ) associated with pMLKL Ser358 and pDrp1 Ser616 ( red ) expression (arrows). ( f and g ) Quantification of TUNEL+ mTECs/pMLKL Ser358+ and TUNEL+ mTECs/pDrp1 Ser616+ shows statistical significant differences between cultures. *** P <0.0001 versus UT, ** P <0.001 versus UT, * P <0.01 versus UT, ± P <0.05 versus R1TNF (NKoC). ( h ) Combined immunofluorescence of R1TNF-treated cultures shows co-localization of pMLKL Ser358 and pDrp1 Ser616 in mTECs (arrow). ( i ) Immunogold electron microscopy demonstrate close proximity of gold particles for pMLKL Ser358 (5 nm) and pDrp1 Ser616 (15 nm) in mitochondria (m) ( inset zoomed × 2.5 ). Bars=mean±S.E.M.; images are representative of n =3 independent experiments from six separate organ culture experiments with similar results

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Organ Culture, Comparison, Fluorescence, Confocal Microscopy, TUNEL Assay, Expressing, Immunofluorescence, Electron Microscopy

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: Expression of necrosomal signaling components regulated by wtTNF, R1TNF and R2TNF in malignant tubular epithelial cells in human RCC grade 1 organ cultures

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Expressing, Immunostaining

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: ( a ) Effect of necrosulfonamide (NSA) and mdivi-1 (m) in grade 1 RCC organ cultures treated with R1TNF. R1TNF alone without NSA (no NSA) induced increased levels of TUNEL+ mTECs, which were significantly reduced with 50 μ M NSA, and to a lesser extent, with 10 μ M or 20 μ M NSA (arrows). Cultures pretreated with 5 μ M NSA showed comparable levels of TUNEL+ mTECs as untreated cultures (UT). ( b ) The percentage of TUNEL+ mTECs and ( c ) pMLKL Ser358 expression presented as mean fluorescent intensity (MFI) in similar cultures. *** P <0.0001 versus UT, ** P <0.001 versus R1TNF; * P <0.05 versus R1TNF; ¥ P <0.05 versus R1TNF (+20 or 10 μ M); ± P <0.001 versus R1TNF (+20 or 10 μ M); ns, not significant. ( d ) In comparison with UT, which show a rare TUNEL+ mTECs, R1TNF alone (without m) induced increased levels of TUNEL+ mTECs, significantly reduced by m (10 μ M) with no effect by zVAD.fmk (m+z) but a marked reduction by nec-1 (m+n) comparable with cultures pretreated with a combination of zVAD.fmk and nec-1 (m+n+z). ( e ) The percentage of TUNEL+ mTECs and ( f and g ) the mean fluorescence intensity (MFI) for pDrp1 Ser616 and pDrp1 Ser637 in similar cultures. *** P <0.0001 versus UT ( e ), ** P <0.01 versus UT ( f ), * P <0.05 versus R1TNF, ± P <0.05 versus R1TNF+m. ( g ) * P <0.05 versus UT, ± P <0.5 versus R1TNF, ¥ P <0.05 versus R1TNF+m; ns, not significant. Bars=mean±S.E.M.; n =3 independent experiments from six separate organ culture experiments with similar results. ( h ) Schematic diagram of the consequences of R1TNF-mediated necroptosis in mTEC in RCC; ligation of TNFR1 results in the recruitment of RIPK1, facilitating its interaction with RIPK3, which in turn recruits and phosphorylates MLKL at Ser358 and Drp1 at Ser616 thus causing their co-localization with the mitochondria. A separate process causes a reduction in pDrp1 at ser637. Nec-1 inhibits RIPK1, and NSA inhibits MLKL and mdivi-1 inhibits Drp1 inhibiting cell death

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: TUNEL Assay, Expressing, Comparison, Fluorescence, Organ Culture, Ligation