Journal: Journal of proteome research

Article Title: Smelling the Dark Proteome: Functional Characterization of PITH Domain-Containing Protein 1 (C1orf128) in Olfactory Metabolism.

doi: 10.1021/acs.jproteome.0c00452

Figure Lengend Snippet: Figure 3. Targeted phosphoproteomic analysis of olfactory neuroepithelial cells upon PITHD1 stimulation. (A) rPITHD1 detection in the cell media of ON cells after 60 min of treatment. (B) rPITHD1 induces changes in the phosphorylation levels of specific transcription factors and kinases in ON cells (60 min treatment). Three independent experiments were performed. Data are presented as mean ± SEM *p < 0.05, **p < 0.01 vs unstimulated ON cells. Representative images of phosphokinase arrays are shown.

Article Snippet: Cytokine antibody arrays were obtained from Abcam (ab133998) and human phosphokinase antibody arrays from R&D systems (ARY003B).

Techniques: Phospho-proteomics

Journal: Nature Communications

Article Title: The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets

doi: 10.1038/s41467-021-23499-w

Figure Lengend Snippet: a Washed murine WT platelets carefully isolated with inhibitors of platelet activation were stimulated with C5a. The supernatant was analyzed by a membrane-based antibody array. Specific mediators, such as CXCL4 (red circles), were upregulated after stimulation with C5a (bottom) compared with the vehicle control (top). b Results of four array repeats were quantified. For most factors, C5a stimulation (gray) induced a slight upregulation compared to vehicle control (black). The strongest increase was observed for CXCL4. Data are shown as the mean ± SEM ( n = 4 independent experiments) and are displayed as intensity values. * p < 0.05. Bonferroni’s post hoc analysis was performed; results are displayed in Supplementary Fig. . c Single-cell staining revealed that murine platelets are activated upon C5a stimulation and release CXCL4. Upon stimulation with ADP and C5a, a ring-like staining pattern was observed, indicating platelet activation (arrows). However, C5a-stimulated platelets exhibit reduced CXCL4 content after stimulation with C5a, which cannot be observed to the same extent in the ADP-stimulated group. ×630 magnification, scale bars represent 5 µm. Images are representative of four independent experiments. d Quantification of P-selectin intensity in each platelet reveals upregulation, indicating platelet activation upon stimulation with ADP and C5a. However, no significant difference was detected between ADP and C5a. Data are shown as the mean ± SEM ( n = 5 images analyzed) and are displayed as the percentage of control. The P-selectin intensity in vehicle control-stimulated WT platelets represents 100%. * p < 0.05 e Quantification of CXCL4 intensity in relation to P-selectin intensity in each platelet reveals relatively stronger secretion of CXCL4 upon C5a stimulation compared to ADP. Data are shown as the mean ± SEM ( n = 5 images analyzed) and are displayed as the percentage of control. The CXCL4 intensity in relation to P-selectin intensity in vehicle control-stimulated WT platelets represents 100%. * p < 0.05. f Single platelets from mice were stimulated with C5a or vehicle control. Granules were quantified by calculating the area of predominantly green staining within the platelets (P-selectin-predominant), predominantly red areas (CXCL4-predominant) as well as overlayed yellow areas (P-selectin-CXCL4 double positive). ×630 magnification, scale bars represent 1 µm. Images are representative of >100 analyzed single platelets. g As described in Supplementary Fig. , granule area fractions were quantified. We observed a distinct distribution pattern of granules, with a similar amount of CXCL4-predominant as well as P-selectin-CXCL4 double-positive granules and a significantly lower number of P-selectin-predominant granules, which contain P-selectin over CXCL4. Data are shown as the mean ± SEM ( n = 112 single platelets analyzed) and are displayed as the area fraction. p (CXCL4 versus double positive) < 0.001, p (CD62P versus double positive) = 0.0022. h Upon stimulation with C5a, analysis revealed a reduction in CXCL4-predominant granules, while the area fraction of double-positive granules remains unchanged, while the relative area fraction of P-selectin predominant granules increases, thus demonstrating C5a-induced secretion of a subset of α-granules, which contain CXCL4 over P-selectin. Data are shown as the mean ± SEM ( n = 166 single platelets analyzed) and are displayed as the area fraction. p (CD62P versus CXCL4) < 0.001. i Conventional ELISA confirmed a significant dose-dependent increase in CXCL4 secretion from human platelets after C5a stimulation. Maximum CXCL4 release is reached at a C5a concentration of 2000 ng/ml. Data are shown as the mean ± SEM ( n = 8 independent experiments containing separate donors) and are displayed as the percentage of control. The CXCL4 protein level of vehicle-stimulated platelet supernatant represents 100%. * p < 0.05. j Murine megakaryocytes from WT versus C5ar1 −/− mice were assessed for distribution of CXCL4-predominant granules (red). Displayed are representative images, nuclei are shown in green. ×630 magnification, scale bars represent 10 µm. k As described in Supplementary Fig. , granule area fractions were quantified in megakaryocytes from WT versus C5ar1 −/− mice. No significant difference could be detected for CXCL4-predominant granules. Data are shown as the mean ± SEM ( n = 10–12 single megakaryocytes analyzed) and are displayed as the area fraction. l RT-PCR analysis detected similar mRNA levels of CXCL4 in megakaryocytes from WT versus C5ar1 −/− mice. Data are presented as the mean ± SEM ( n = 5 independent experiments) and are shown as relative expression in relation to GAPDH. m Citrated whole blood from WT and C5ar1 −/− mice was stimulated using different concentrations of C5a (20, 200, 2000 ng/ml) and vehicle control and assessed for platelet activation markers by flow cytometry. For the gating strategy, please refer to the “Methods” section. Activated platelets are detected as CD62PJONA ++ , whereas JONA detects activated GPIIbIIIa. C5a induced platelet activation in WT platelets but not in C5ar1 −/− platelets. Data are shown as the mean ± SEM ( n = 4 independent experiments) and are displayed as the percentage of control. The percentage gated CD62PJONA ++ platelets in the vehicle-stimulated group represents 100%. * p < 0.05. n In order to uncover the signaling downstream of C5aR1 leading to CXCL4 secretion, lysates of WT platelets were generated after vehicle control or C5a stimulation and samples were probed at equal protein concentrations for phospho-proteins as well as non-phosphorylated controls. Platelet C5aR1 ligation induced reproducible PKC substrate phosphorylation (PKC phosphor Ser). As a control, we used the PKC activator PMA. PKC activation was quantified by dividing the phosphorylation signal by the total protein signal as well as unphosphorylated PKC α (Supplementary Fig. ). Displayed are representative images of at least four independent experiments. o We also checked whether PKC activation also entails CXCL4 release by stimulating washed platelets with C5a and PMA. Both C5a and PMA induced significant CXCL4 release. Data are shown as the mean ± SEM ( n = 5–16 independent experiments) and are displayed as the percentage of control of CXCL4 concentration in platelet supernatant measured by ELISA. The mean fluorescence intensity (MFI) of platelets in the vehicle-stimulated group represents 100%. * p < 0.01. p Furthermore, C5a induced an upregulation of phosphorylated Akt (Ser473), PI3K (Ser 47), GSK-3β (Ser 9), p44/42 MAPK (Thr202/Tyr204), PLCβ3 (Ser537). Displayed are representative images of at least four independent experiments. For quantification of phosphorylation, please refer to Supplementary Fig. . q Interestingly, C5a induced no regulation of phosphorylated PLCγ2 (Tyr1217) and PKA (α/β/γ catalytic subunit phospho T197). Activated Rap1 (Rap1-GTP) was analyzed using a Rap1 pulldown assay. No significantly altered amounts of Rap1-GTP could be detected. Displayed are representative images of at least four independent experiments. One-way ANOVA with Bonferroni’s post hoc test in b , d , e , g – i , m . Two-sided Student’s t test in k , l , o .

Article Snippet: Membranes were then blocked with 5% skim milk or 5% BSA (for the detection of phosphorylated antigens) in Tris-buffered saline 0.05% Tween-20 and incubated with the following primary antibodies: anti-mouse C3aR (Santa Cruz Biotechnologies; clone D12; 2 μg/ml); thrombospondin 1 (rabbit polyclonal Anti-Thrombospondin-1 Antibody, Proteintech, Chicago, IL, USA, #1 18304-1-AP, 1:2000); phospho PLCγ2 (Tyr1217, Cell Signaling Technology, #3871S, 1:1000), total PLCγ2 (Cell Signaling Technology, Danvers, USA, #3872S, 1:1000); phospho PKA (PKA α/β/γ catalytic subunit phospho T197, Abcam, #ab75991, 1:3000), total PKA (α/β catalytic subunits, Abcam, #ab216572, 1:500); phospho Akt (Ser473, Cell Signaling Technology, #9271S, 1:1000), total Akt (Rabbit monoclonal Akt pan C67E7, Cell Signaling Technology, # 4691S, 1:1000); phospho PI3K (Phospho PI3 Kinase p85 Tyr458/p55 Tyr199, Cell Signaling Technology, #4228S, 1:1000), total PI3K (rabbit monoclonal PI3 Kinase p85 19H8, Cell Signaling Technology, #4257S, 1:1000); phospho GSK-3β (Ser9, Cell Signaling Technology, #9336S, 1:1000), total GSK-3β (rabbit monoclonal anti GSK-3β 27C10, Cell Signaling Technology, #9315S, 1:1000); phospho p44/42 MAPK (Erk1/2 Thr202/Tyr204, Cell Signaling Technology, #9101S, 1:1000), total p44/42 MAPK (Erk1/2, Cell Signaling Technology, #9102S, 1:1000); phospho PLCβ3 (Ser537, Cell Signaling Technology, #29021S, 1:1000), total PLCβ3 (rabbit monoclonal PLCβ3 D9D6S, Cell Signaling Technology, #14247, 1:1000); phospho PKC (Ser) substrate (Cell Signaling Technology, #2261S, 1:1000), total PKCα (Cell Signaling Technology, #2056S, 1:1000).

Techniques: Isolation, Activation Assay, Membrane, Ab Array, Control, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry, Generated, Ligation, Phospho-proteomics, Fluorescence

Journal: Molecular cancer research : MCR

Article Title: EXOC4 Promotes Diffuse-Type Gastric Cancer Metastasis via Activating FAK Signal.

doi: 10.1158/1541-7786.MCR-21-0441

Figure Lengend Snippet: Figure 4. EXOC4 regulates FAK phosphorylation at Tyr397. A and B, MKN45 cells were transfected with control or independent siRNAs against EXOC4 for 48 hours and the WCLs were subjected to phosphokinase antibody array analysis (A), and the quantification were shown on (B). C and D, Immunoblotting (IB) analysis of whole-cell lysates (WCL) derived from MKN45 cells transfected with the indicated siRNA (C) or plasmids (D) for 48 hours. E and F, Co-IP experiments in MKN45 cells were performed using anti-FAK (E) or anti-EXOC4 (F) antibodies. IgG was used as a negative control. G, Co-IP analysis. IB analysis of WCLs and IPs derived from 293T cells transfected with HA-FAK together with the indicated Flag-tagged EXOC4. H, Co-IP analysis. IB analysis of WCLs and IPs derived from MKN45 cells transfected with Flag-EXOC4 together with the indicated constructs of HA-FAK. I, IB analysis of the FAK Y397 phosphorylation protein expression in different gastric cancer cells.

Article Snippet: On this premise, a human phosphokinase antibody array (catalog no. ARY003B, R&D Systems) was employed, and alterations in 43 phosphokinase sites that were likely crucial in the regulation of various cell processes, including cell migration and invasion, were evaluated.

Techniques: Phospho-proteomics, Transfection, Control, Ab Array, Western Blot, Derivative Assay, Co-Immunoprecipitation Assay, Negative Control, Construct, Expressing

Journal: Nature Communications

Article Title: The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets

doi: 10.1038/s41467-021-23499-w

Figure Lengend Snippet: a Washed murine WT platelets carefully isolated with inhibitors of platelet activation were stimulated with C5a. The supernatant was analyzed by a membrane-based antibody array. Specific mediators, such as CXCL4 (red circles), were upregulated after stimulation with C5a (bottom) compared with the vehicle control (top). b Results of four array repeats were quantified. For most factors, C5a stimulation (gray) induced a slight upregulation compared to vehicle control (black). The strongest increase was observed for CXCL4. Data are shown as the mean ± SEM ( n = 4 independent experiments) and are displayed as intensity values. * p < 0.05. Bonferroni’s post hoc analysis was performed; results are displayed in Supplementary Fig. . c Single-cell staining revealed that murine platelets are activated upon C5a stimulation and release CXCL4. Upon stimulation with ADP and C5a, a ring-like staining pattern was observed, indicating platelet activation (arrows). However, C5a-stimulated platelets exhibit reduced CXCL4 content after stimulation with C5a, which cannot be observed to the same extent in the ADP-stimulated group. ×630 magnification, scale bars represent 5 µm. Images are representative of four independent experiments. d Quantification of P-selectin intensity in each platelet reveals upregulation, indicating platelet activation upon stimulation with ADP and C5a. However, no significant difference was detected between ADP and C5a. Data are shown as the mean ± SEM ( n = 5 images analyzed) and are displayed as the percentage of control. The P-selectin intensity in vehicle control-stimulated WT platelets represents 100%. * p < 0.05 e Quantification of CXCL4 intensity in relation to P-selectin intensity in each platelet reveals relatively stronger secretion of CXCL4 upon C5a stimulation compared to ADP. Data are shown as the mean ± SEM ( n = 5 images analyzed) and are displayed as the percentage of control. The CXCL4 intensity in relation to P-selectin intensity in vehicle control-stimulated WT platelets represents 100%. * p < 0.05. f Single platelets from mice were stimulated with C5a or vehicle control. Granules were quantified by calculating the area of predominantly green staining within the platelets (P-selectin-predominant), predominantly red areas (CXCL4-predominant) as well as overlayed yellow areas (P-selectin-CXCL4 double positive). ×630 magnification, scale bars represent 1 µm. Images are representative of >100 analyzed single platelets. g As described in Supplementary Fig. , granule area fractions were quantified. We observed a distinct distribution pattern of granules, with a similar amount of CXCL4-predominant as well as P-selectin-CXCL4 double-positive granules and a significantly lower number of P-selectin-predominant granules, which contain P-selectin over CXCL4. Data are shown as the mean ± SEM ( n = 112 single platelets analyzed) and are displayed as the area fraction. p (CXCL4 versus double positive) < 0.001, p (CD62P versus double positive) = 0.0022. h Upon stimulation with C5a, analysis revealed a reduction in CXCL4-predominant granules, while the area fraction of double-positive granules remains unchanged, while the relative area fraction of P-selectin predominant granules increases, thus demonstrating C5a-induced secretion of a subset of α-granules, which contain CXCL4 over P-selectin. Data are shown as the mean ± SEM ( n = 166 single platelets analyzed) and are displayed as the area fraction. p (CD62P versus CXCL4) < 0.001. i Conventional ELISA confirmed a significant dose-dependent increase in CXCL4 secretion from human platelets after C5a stimulation. Maximum CXCL4 release is reached at a C5a concentration of 2000 ng/ml. Data are shown as the mean ± SEM ( n = 8 independent experiments containing separate donors) and are displayed as the percentage of control. The CXCL4 protein level of vehicle-stimulated platelet supernatant represents 100%. * p < 0.05. j Murine megakaryocytes from WT versus C5ar1 −/− mice were assessed for distribution of CXCL4-predominant granules (red). Displayed are representative images, nuclei are shown in green. ×630 magnification, scale bars represent 10 µm. k As described in Supplementary Fig. , granule area fractions were quantified in megakaryocytes from WT versus C5ar1 −/− mice. No significant difference could be detected for CXCL4-predominant granules. Data are shown as the mean ± SEM ( n = 10–12 single megakaryocytes analyzed) and are displayed as the area fraction. l RT-PCR analysis detected similar mRNA levels of CXCL4 in megakaryocytes from WT versus C5ar1 −/− mice. Data are presented as the mean ± SEM ( n = 5 independent experiments) and are shown as relative expression in relation to GAPDH. m Citrated whole blood from WT and C5ar1 −/− mice was stimulated using different concentrations of C5a (20, 200, 2000 ng/ml) and vehicle control and assessed for platelet activation markers by flow cytometry. For the gating strategy, please refer to the “Methods” section. Activated platelets are detected as CD62PJONA ++ , whereas JONA detects activated GPIIbIIIa. C5a induced platelet activation in WT platelets but not in C5ar1 −/− platelets. Data are shown as the mean ± SEM ( n = 4 independent experiments) and are displayed as the percentage of control. The percentage gated CD62PJONA ++ platelets in the vehicle-stimulated group represents 100%. * p < 0.05. n In order to uncover the signaling downstream of C5aR1 leading to CXCL4 secretion, lysates of WT platelets were generated after vehicle control or C5a stimulation and samples were probed at equal protein concentrations for phospho-proteins as well as non-phosphorylated controls. Platelet C5aR1 ligation induced reproducible PKC substrate phosphorylation (PKC phosphor Ser). As a control, we used the PKC activator PMA. PKC activation was quantified by dividing the phosphorylation signal by the total protein signal as well as unphosphorylated PKC α (Supplementary Fig. ). Displayed are representative images of at least four independent experiments. o We also checked whether PKC activation also entails CXCL4 release by stimulating washed platelets with C5a and PMA. Both C5a and PMA induced significant CXCL4 release. Data are shown as the mean ± SEM ( n = 5–16 independent experiments) and are displayed as the percentage of control of CXCL4 concentration in platelet supernatant measured by ELISA. The mean fluorescence intensity (MFI) of platelets in the vehicle-stimulated group represents 100%. * p < 0.01. p Furthermore, C5a induced an upregulation of phosphorylated Akt (Ser473), PI3K (Ser 47), GSK-3β (Ser 9), p44/42 MAPK (Thr202/Tyr204), PLCβ3 (Ser537). Displayed are representative images of at least four independent experiments. For quantification of phosphorylation, please refer to Supplementary Fig. . q Interestingly, C5a induced no regulation of phosphorylated PLCγ2 (Tyr1217) and PKA (α/β/γ catalytic subunit phospho T197). Activated Rap1 (Rap1-GTP) was analyzed using a Rap1 pulldown assay. No significantly altered amounts of Rap1-GTP could be detected. Displayed are representative images of at least four independent experiments. One-way ANOVA with Bonferroni’s post hoc test in b , d , e , g – i , m . Two-sided Student’s t test in k , l , o .

Article Snippet: Membranes were then blocked with 5% skim milk or 5% BSA (for the detection of phosphorylated antigens) in Tris-buffered saline 0.05% Tween-20 and incubated with the following primary antibodies: anti-mouse C3aR (Santa Cruz Biotechnologies; clone D12; 2 µg/ml); thrombospondin 1 (rabbit polyclonal Anti-Thrombospondin-1 Antibody, Proteintech, Chicago, IL, USA, #1 18304-1-AP, 1:2000); phospho PLCγ2 (Tyr1217, Cell Signaling Technology, #3871S, 1:1000), total PLCγ2 (Cell Signaling Technology, Danvers, USA, #3872S, 1:1000); phospho PKA (PKA α/β/γ catalytic subunit phospho T197, Abcam, #ab75991, 1:3000), total PKA (α/β catalytic subunits, Abcam, #ab216572, 1:500); phospho Akt (Ser473, Cell Signaling Technology, #9271S, 1:1000), total Akt (Rabbit monoclonal Akt pan C67E7, Cell Signaling Technology, # 4691S, 1:1000); phospho PI3K (Phospho PI3 Kinase p85 Tyr458/p55 Tyr199, Cell Signaling Technology, #4228S, 1:1000), total PI3K (rabbit monoclonal PI3 Kinase p85 19H8, Cell Signaling Technology, #4257S, 1:1000); phospho GSK-3β (Ser9, Cell Signaling Technology, #9336S, 1:1000), total GSK-3β (rabbit monoclonal anti GSK-3β 27C10, Cell Signaling Technology, #9315S, 1:1000); phospho p44/42 MAPK (Erk1/2 Thr202/Tyr204, Cell Signaling Technology, #9101S, 1:1000), total p44/42 MAPK (Erk1/2, Cell Signaling Technology, #9102S, 1:1000); phospho PLCβ3 (Ser537, Cell Signaling Technology, #29021S, 1:1000), total PLCβ3 (rabbit monoclonal PLCβ3 D9D6S, Cell Signaling Technology, #14247, 1:1000); phospho PKC (Ser) substrate (Cell Signaling Technology, #2261S, 1:1000), total PKCα (Cell Signaling Technology, #2056S, 1:1000).

Techniques: Isolation, Activation Assay, Ab Array, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry, Generated, Ligation, Fluorescence

Journal: Nature Communications

Article Title: The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets

doi: 10.1038/s41467-021-23499-w

Figure Lengend Snippet: a Washed murine WT platelets carefully isolated with inhibitors of platelet activation were stimulated with C5a. The supernatant was analyzed by a membrane-based antibody array. Specific mediators, such as CXCL4 (red circles), were upregulated after stimulation with C5a (bottom) compared with the vehicle control (top). b Results of four array repeats were quantified. For most factors, C5a stimulation (gray) induced a slight upregulation compared to vehicle control (black). The strongest increase was observed for CXCL4. Data are shown as the mean ± SEM ( n = 4 independent experiments) and are displayed as intensity values. * p < 0.05. Bonferroni’s post hoc analysis was performed; results are displayed in Supplementary Fig. . c Single-cell staining revealed that murine platelets are activated upon C5a stimulation and release CXCL4. Upon stimulation with ADP and C5a, a ring-like staining pattern was observed, indicating platelet activation (arrows). However, C5a-stimulated platelets exhibit reduced CXCL4 content after stimulation with C5a, which cannot be observed to the same extent in the ADP-stimulated group. ×630 magnification, scale bars represent 5 µm. Images are representative of four independent experiments. d Quantification of P-selectin intensity in each platelet reveals upregulation, indicating platelet activation upon stimulation with ADP and C5a. However, no significant difference was detected between ADP and C5a. Data are shown as the mean ± SEM ( n = 5 images analyzed) and are displayed as the percentage of control. The P-selectin intensity in vehicle control-stimulated WT platelets represents 100%. * p < 0.05 e Quantification of CXCL4 intensity in relation to P-selectin intensity in each platelet reveals relatively stronger secretion of CXCL4 upon C5a stimulation compared to ADP. Data are shown as the mean ± SEM ( n = 5 images analyzed) and are displayed as the percentage of control. The CXCL4 intensity in relation to P-selectin intensity in vehicle control-stimulated WT platelets represents 100%. * p < 0.05. f Single platelets from mice were stimulated with C5a or vehicle control. Granules were quantified by calculating the area of predominantly green staining within the platelets (P-selectin-predominant), predominantly red areas (CXCL4-predominant) as well as overlayed yellow areas (P-selectin-CXCL4 double positive). ×630 magnification, scale bars represent 1 µm. Images are representative of >100 analyzed single platelets. g As described in Supplementary Fig. , granule area fractions were quantified. We observed a distinct distribution pattern of granules, with a similar amount of CXCL4-predominant as well as P-selectin-CXCL4 double-positive granules and a significantly lower number of P-selectin-predominant granules, which contain P-selectin over CXCL4. Data are shown as the mean ± SEM ( n = 112 single platelets analyzed) and are displayed as the area fraction. p (CXCL4 versus double positive) < 0.001, p (CD62P versus double positive) = 0.0022. h Upon stimulation with C5a, analysis revealed a reduction in CXCL4-predominant granules, while the area fraction of double-positive granules remains unchanged, while the relative area fraction of P-selectin predominant granules increases, thus demonstrating C5a-induced secretion of a subset of α-granules, which contain CXCL4 over P-selectin. Data are shown as the mean ± SEM ( n = 166 single platelets analyzed) and are displayed as the area fraction. p (CD62P versus CXCL4) < 0.001. i Conventional ELISA confirmed a significant dose-dependent increase in CXCL4 secretion from human platelets after C5a stimulation. Maximum CXCL4 release is reached at a C5a concentration of 2000 ng/ml. Data are shown as the mean ± SEM ( n = 8 independent experiments containing separate donors) and are displayed as the percentage of control. The CXCL4 protein level of vehicle-stimulated platelet supernatant represents 100%. * p < 0.05. j Murine megakaryocytes from WT versus C5ar1 −/− mice were assessed for distribution of CXCL4-predominant granules (red). Displayed are representative images, nuclei are shown in green. ×630 magnification, scale bars represent 10 µm. k As described in Supplementary Fig. , granule area fractions were quantified in megakaryocytes from WT versus C5ar1 −/− mice. No significant difference could be detected for CXCL4-predominant granules. Data are shown as the mean ± SEM ( n = 10–12 single megakaryocytes analyzed) and are displayed as the area fraction. l RT-PCR analysis detected similar mRNA levels of CXCL4 in megakaryocytes from WT versus C5ar1 −/− mice. Data are presented as the mean ± SEM ( n = 5 independent experiments) and are shown as relative expression in relation to GAPDH. m Citrated whole blood from WT and C5ar1 −/− mice was stimulated using different concentrations of C5a (20, 200, 2000 ng/ml) and vehicle control and assessed for platelet activation markers by flow cytometry. For the gating strategy, please refer to the “Methods” section. Activated platelets are detected as CD62PJONA ++ , whereas JONA detects activated GPIIbIIIa. C5a induced platelet activation in WT platelets but not in C5ar1 −/− platelets. Data are shown as the mean ± SEM ( n = 4 independent experiments) and are displayed as the percentage of control. The percentage gated CD62PJONA ++ platelets in the vehicle-stimulated group represents 100%. * p < 0.05. n In order to uncover the signaling downstream of C5aR1 leading to CXCL4 secretion, lysates of WT platelets were generated after vehicle control or C5a stimulation and samples were probed at equal protein concentrations for phospho-proteins as well as non-phosphorylated controls. Platelet C5aR1 ligation induced reproducible PKC substrate phosphorylation (PKC phosphor Ser). As a control, we used the PKC activator PMA. PKC activation was quantified by dividing the phosphorylation signal by the total protein signal as well as unphosphorylated PKC α (Supplementary Fig. ). Displayed are representative images of at least four independent experiments. o We also checked whether PKC activation also entails CXCL4 release by stimulating washed platelets with C5a and PMA. Both C5a and PMA induced significant CXCL4 release. Data are shown as the mean ± SEM ( n = 5–16 independent experiments) and are displayed as the percentage of control of CXCL4 concentration in platelet supernatant measured by ELISA. The mean fluorescence intensity (MFI) of platelets in the vehicle-stimulated group represents 100%. * p < 0.01. p Furthermore, C5a induced an upregulation of phosphorylated Akt (Ser473), PI3K (Ser 47), GSK-3β (Ser 9), p44/42 MAPK (Thr202/Tyr204), PLCβ3 (Ser537). Displayed are representative images of at least four independent experiments. For quantification of phosphorylation, please refer to Supplementary Fig. . q Interestingly, C5a induced no regulation of phosphorylated PLCγ2 (Tyr1217) and PKA (α/β/γ catalytic subunit phospho T197). Activated Rap1 (Rap1-GTP) was analyzed using a Rap1 pulldown assay. No significantly altered amounts of Rap1-GTP could be detected. Displayed are representative images of at least four independent experiments. One-way ANOVA with Bonferroni’s post hoc test in b , d , e , g – i , m . Two-sided Student’s t test in k , l , o .

Article Snippet: Membranes were then blocked with 5% skim milk or 5% BSA (for the detection of phosphorylated antigens) in Tris-buffered saline 0.05% Tween-20 and incubated with the following primary antibodies: anti-mouse C3aR (Santa Cruz Biotechnologies; clone D12; 2 µg/ml); thrombospondin 1 (rabbit polyclonal Anti-Thrombospondin-1 Antibody, Proteintech, Chicago, IL, USA, #1 18304-1-AP, 1:2000); phospho PLCγ2 (Tyr1217, Cell Signaling Technology, #3871S, 1:1000), total PLCγ2 (Cell Signaling Technology, Danvers, USA, #3872S, 1:1000); phospho PKA (PKA α/β/γ catalytic subunit phospho T197, Abcam, #ab75991, 1:3000), total PKA (α/β catalytic subunits, Abcam, #ab216572, 1:500); phospho Akt (Ser473, Cell Signaling Technology, #9271S, 1:1000), total Akt (Rabbit monoclonal Akt pan C67E7, Cell Signaling Technology, # 4691S, 1:1000); phospho PI3K (Phospho PI3 Kinase p85 Tyr458/p55 Tyr199, Cell Signaling Technology, #4228S, 1:1000), total PI3K (rabbit monoclonal PI3 Kinase p85 19H8, Cell Signaling Technology, #4257S, 1:1000); phospho GSK-3β (Ser9, Cell Signaling Technology, #9336S, 1:1000), total GSK-3β (rabbit monoclonal anti GSK-3β 27C10, Cell Signaling Technology, #9315S, 1:1000); phospho p44/42 MAPK (Erk1/2 Thr202/Tyr204, Cell Signaling Technology, #9101S, 1:1000), total p44/42 MAPK (Erk1/2, Cell Signaling Technology, #9102S, 1:1000); phospho PLCβ3 (Ser537, Cell Signaling Technology, #29021S, 1:1000), total PLCβ3 (rabbit monoclonal PLCβ3 D9D6S, Cell Signaling Technology, #14247, 1:1000); phospho PKC (Ser) substrate (Cell Signaling Technology, #2261S, 1:1000), total PKCα (Cell Signaling Technology, #2056S, 1:1000).

Techniques: Isolation, Activation Assay, Ab Array, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry, Generated, Ligation, Fluorescence

Journal: Cancer research

Article Title: Host Wnt5a Potentiates Microenvironmental Regulation of Ovarian Cancer Metastasis

doi: 10.1158/0008-5472.CAN-19-1601

Figure Lengend Snippet: Identification of Fgr as a downstream mediator of Wnt5a prometastatic cellular behavior. A, OVCAR5 and OVCAR8 cells were treated with SFM or rWnt5a protein (0.4 μg/mL, 24 hours) prior to lysis. Lysates were analyzed using the Proteome Profiler Human Phospho-Kinase Array according to the manufacturer’s specifications. Log2-fold change of phosphorylated proteins in the phosphokinase array is shown. B, Quantification of Fgr (Y412) phosphorylation by measuring pixel density for duplicate spots on the phospho-array blot using ImageJ. Data are presented as mean ± SEM. C, Validation of Fgr expression and phosphorylation. OVCAR5 and OVCAR8 cells were serum-starved for 2 hours then incubated with the Fgr inhibitor TL02–59 (0.01 μmol/L) for 2.5 hours. After washing, cells in SFM were incubated with rWnt5a protein (0.4 μg/mL) for the time points indicated. Ctrl, cells without rWnt5a treatment. Lysates were electrophoresed on SDS-polyacrylamide gels and immunoblotted with the antibodies noted. One representative blot from three independent biological replicates is shown. D and E, RFP-tagged OVCAR5 and OVCAR8 cells were treated with SFM, rWnt5a (0.4 mg/mL), rWnt5a (0.4 μg/mL), and the specific Fgr kinase inhibitor TL02–59 (1 μmol/L) or TL02–59 (1 μmol/L) for 24 hours, as indicated, prior to incubation with mesomimetic culture for 20 minutes or 1 hour (D), respectively, or with murine peritoneal explants in an ex vivo adhesion assay for 30 or 90 minutes (E), respectively. Quantification of adherent cells is shown for each condition. F, OVCAR5 and OVCAR8 cells were added to transwell migration chambers containing SFM, rWnt5a (0.4 μg/mL), rWnt5a (0.4 μg/mL), and TL02–59 (1 μmol/L), or TL02–59 (1 μmol/L) and incubated for 12 and 18 hours, respectively. Quantification of migrated cells in each condition.

Article Snippet: Proteome profiler human phosphokinase array Human phosphokinase antibody array purchased from R&D Systems was used to detect the expression of 43 kinase phosphorylation sites on proteins isolated from OVCAR5 and OVCAR8 cells treated with SFM or 0.4 μg/mL rWnt5a for 24 hours.

Techniques: Lysis, Phospho-proteomics, Biomarker Discovery, Expressing, Incubation, Ex Vivo, Cell Adhesion Assay, Migration

Journal: Cancer research

Article Title: Host Wnt5a Potentiates Microenvironmental Regulation of Ovarian Cancer Metastasis

doi: 10.1158/0008-5472.CAN-19-1601

Figure Lengend Snippet: Tumor-bearing WNT5A knockout mice have altered peritoneal cytokine profiles. A, Peritoneal lavage obtained from mice with conditional WNT5A knockout or controls was analyzed using the murine Proteome Profiler Cytokine Array according to the manufacturer’s specifications. Spots showing differential expression are boxed.B, Each cytokine has duplicate spots on the blot and each spot intensity was quantified by measuring pixel density using ImageJ following the manufacturer’s protocol. All results are presented as mean ± SEM.

Article Snippet: Proteome profiler human phosphokinase array Human phosphokinase antibody array purchased from R&D Systems was used to detect the expression of 43 kinase phosphorylation sites on proteins isolated from OVCAR5 and OVCAR8 cells treated with SFM or 0.4 μg/mL rWnt5a for 24 hours.

Techniques: Knock-Out, Quantitative Proteomics