Journal: PLoS ONE

Article Title: Pre-Analytical Parameters Affecting Vascular Endothelial Growth Factor Measurement in Plasma: Identifying Confounders

doi: 10.1371/journal.pone.0145375

Figure Lengend Snippet: Investigated parameters.

Article Snippet: Platelet-factor-4 (PF-4) was measured using an ELISA kit from R&D Systems (Cat. No. DPF40).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Centrifugation, Sampling, Luminex, Activation Assay, Saline, Recombinant, Biomarker Discovery

Journal: PLoS ONE

Article Title: Pre-Analytical Parameters Affecting Vascular Endothelial Growth Factor Measurement in Plasma: Identifying Confounders

doi: 10.1371/journal.pone.0145375

Figure Lengend Snippet: 2.1 (a-c) Paired plots (light orange) and box plots (blue) of VEGF-A levels measured at blood taking centers, measuring centers and at the central laboratory (CL). Connecting lines illustrate the measured VEGF-A levels from identical aliquots of samples measured at the acquisition center, the measuring center and the central laboratory; 2.2 Impact of the anticoagulant (EDTA, PECT, CTAD) on measurement of VEGF-A, PF-4 and IGF-1 from plasma. VEGF-A levels measured from PECT (a) and CTAD (b) plasma are much lower than from EDTA plasma. Similar to VEGF-A, PF-4 values are higher in EDTA plasma than in PECT (c) or CTAD (d) plasma, indicating increased thrombolysis in EDTA samples compared to PECT or CTAD. IGF-1 levels are not affected by the choice of anticoagulant (e, f) indicating that the choice of coagulant is not a general confounder for all biomarkers. (g) VEGF-A levels obtained after spiking VEGF-A into PBS containing EDTA, PECT or CTAD yielded comparable results thus confirming that CTAD or PECT do not interfere with the VEGF ELISA assay. It was noted, however, that VEGF stored at -80°C for over one year yields lower recovery rates in all samples, irrespective of the anticagulant used; 2.3 Impact of the kind of centrifuge used for separating plasma from the cell pellet (equivalent settings were applied). VEGF-A (a) and PF-4 (b) levels were higher in some samples from the fixed angle rotor compared to the swing-out rotor. IGF-1 levels (c) were not affected by the type of centrifuge; 2.4 Comparison of measurements obtained with two methods: ELISA vs. multiplex bead array (Luminex). (a) box-plot analysis showing that absolute values differ between the two measurement methods; (b, c) scatter plot and Bland-Altmann plot, however, demonstrate a good correlation of relative measurement results from the two methods; (d, e) VEGF-A protein standards from both the ELISA kit and the Luminex kit were measured in both assays. In either assay, the VEGF-A standard provided with the Luminex kit resulted in higher signals for identical protein concentrations compared to the VEGF-A standard provided with the ELISA kit. This translates in lower measurements of patient sample VEGF-A levels when these are normalized to the Luminex VEGF-A standard as opposed to the ELISA standard (irrespective of the assay used).

Article Snippet: Platelet-factor-4 (PF-4) was measured using an ELISA kit from R&D Systems (Cat. No. DPF40).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Comparison, Multiplex Assay, Luminex

Journal: International Journal of Molecular Sciences

Article Title: TGFBI Protein Is Increased in the Urine of Patients with High-Grade Urothelial Carcinomas, and Promotes Cell Proliferation and Migration

doi: 10.3390/ijms20184483

Figure Lengend Snippet: Distribution of creatinine-normalized TGFBI and PF4 concentrations (in pg/mg creatinine) corresponding to individual sample characteristics for TGFBI ( n = 489) and PF4 ( n = 486). IQR, interquartile range; N , sample number; $ erythrocytes/µL urine; 1 two tumors represent nested variants with bland cytomorphology but potentially poor outcomes; 2 one tumor represented a nested variant; 3 all low-grade; 4 71 out of 102 pT1 tumors (non-muscle invasive) were low-grade (69.6%); 5 2 out of 39 muscle invasive tumors were low-grade (5.1%); 6 all high-grade, as per definition.

Article Snippet: For TGFBI quantification in the supernatant of cells or urine specimens, we applied the human TGFBI DuoSet ELISA Kit, whereas for quantification of PF4 we employed the human PF4 DuoSet ELISA KIT (both BioTechne, Wiesbaden, Germany).

Techniques: Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: TGFBI Protein Is Increased in the Urine of Patients with High-Grade Urothelial Carcinomas, and Promotes Cell Proliferation and Migration

doi: 10.3390/ijms20184483

Figure Lengend Snippet: Creatinine-normalized PF4 concentration in urine of UC patients, and hospital and population controls ( A ), in de novo UC patients and those with UC history ( B ), in hospital controls compared to high and low-grade UC ( C ) and compared to non-muscle invasive (≤pT1) and muscle invasive UC patients (>pT1) ( D ). Group differences were calculated by Wilcoxon rank-sum tests (non-parametric).

Article Snippet: For TGFBI quantification in the supernatant of cells or urine specimens, we applied the human TGFBI DuoSet ELISA Kit, whereas for quantification of PF4 we employed the human PF4 DuoSet ELISA KIT (both BioTechne, Wiesbaden, Germany).

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: TGFBI Protein Is Increased in the Urine of Patients with High-Grade Urothelial Carcinomas, and Promotes Cell Proliferation and Migration

doi: 10.3390/ijms20184483

Figure Lengend Snippet: Receiving operator characteristic (ROC) curves of urinary TGFBI and PF4 alone and in combination. UC patients were compared with samples of the general population ( A ) and urologic controls from the hospital ( B ). For both curves, TGFBI alone and in combination with PF4, are nearly identical.

Article Snippet: For TGFBI quantification in the supernatant of cells or urine specimens, we applied the human TGFBI DuoSet ELISA Kit, whereas for quantification of PF4 we employed the human PF4 DuoSet ELISA KIT (both BioTechne, Wiesbaden, Germany).

Techniques:

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: For human samples, we used a human CXCL4/PF4 Quantikine ELISA Kit (R&D), a human thrombospondin-1 Quantikine ELISA Kit (R&D), a human PDGF BB ELISA Kit (ab100624, Abcam), and a human Endostatin ELISA Kit (RayBiotech, Peachtree Corners, GA, USA).

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 MHEC-5T were coincubated with the C5a-stimulated supernatant of platelets isolated from WT, C5ar1 −/− , and C5ar1 −/− Cxcl4 −/− mice. Furthermore, the supernatant of C5ar1 −/− platelets was supplemented with CXL4 (2 µg/ml). C5a-stimulated WT platelet supernatant inhibited endothelial tube formation, which was not detectable in C5ar1 −/− and C5ar1 −/− Cxcl4 −/− platelets. Reconstitution with CXCL4 in the C5ar1 −/− group led to a similar level of tube formation as in the WT group. Data are displayed as the mean ± SEM ( n = 5 independent experiments). The total tube length after treatment with vehicle-stimulated platelet supernatant represents 100%. * p < 0.05. b CXCL4 was immunodepleted from C5a-stimulated WT as well as C5ar1 −/− platelet supernatant, and the impact of this supernatant on endothelial tube formation was assessed. Data are shown as the mean ± SEM ( n = 5 independent experiments). The total tube length in the group with control-depleted supernatant from C5a-stimulated C5ar1 −/− platelets represents 100%. * p < 0.05. c Freshly isolated washed murine WT platelets were preincubated with vehicle control or kinase inhibitors SH-6, Wortmannin, and Gö976 before stimulation with C5a. The supernatant was then coincubated with MHEC-5T and endothelial tube formation was quantified. Preincubation of platelets with SH-6, Wortmannin, and Gö976 led to a level of tube formation not significantly different from vehicle control-stimulated WT platelet supernatant. Data are shown as the mean ± SEM ( n = 4–8 independent experiments). The total tube length in the group with control-preincubated vehicle control-stimulated WT platelet supernatant represents 100%. * p < 0.05. d Primary murine lung endothelial cells were isolated from Cxcr3 −/− mice (for endothelial cell quality control refer to Supplementary Fig. 3). After coincubation with C5a-stimulated platelet supernatant, WT endothelial cells displayed decreased endothelial migration in the scratched wound assay. Cxcr3 −/− endothelial cells exhibited a significantly smaller decrease in migration compared with WT cells. Data are displayed as the mean ± SEM ( n = 6 independent experiments). Migration in the WT endothelial cell group treated with vehicle-stimulated WT platelet supernatant subtracted represents 100%. * p < 0.05. e Similarly, the decrease in endothelial tube formation was significantly smaller in Cxcr3 −/− endothelial cells following coincubation with C5a-stimulated platelet supernatant compared with control supernatant and comparable to the level C5ar1 −/− platelet supernatant stimulated with C5a. Data are shown as the mean ± SEM ( n = 5 independent experiments). The total tube length in the group treated with vehicle-stimulated WT platelet supernatant represents 100%. * p < 0.05. f Similar to the hindlimb ischemia model (Fig. ), complement activation (C3b, red) colocalized with vascular structures (IB4, green) within the Matrigel plug 7 days after implantation. ×400 magnification. Scale bars represent 100 µm. Displayed is a representative image of four independent experiments. g To study the paracrine effect of platelets after C5a stimulation in vivo, we used the Matrigel model to analyze vascularization after the addition of platelets and soluble mediators. Matrigel was supplemented with bFGF and injected into WT or C5ar1 −/− mice. Quantification of neovascularization after 7 days yielded a significantly higher level of growth factor-induced angiogenesis in C5ar1 −/− animals than in WT controls. Data are shown as the mean ± SEM ( n = 6–8 Matrigels per group). The neovascularization area fraction within Matrigels from WT mice represents 100%. * p < 0.05. h Freshly isolated WT or C5ar1 −/− platelets were resuspended in Matrigel, the Matrigel was implanted into C5ar1 −/− animals, and vascularization was assessed after 7 days. Data are presented as the mean ± SEM ( n = 7–8 Matrigels per group). The neovascularization area fraction in Matrigels from C5ar1 −/− mice supplemented with C5ar1 −/− platelets represents 100% expressed as the area fraction of nuclear staining. * p < 0.05. i WT platelets or Cxcl4 −/− platelets were coinjected with Matrigel into C5ar1 −/− animals. Injection of Cxcl4 −/− platelets or vehicle control yielded similar levels of growth factor-induced vessel formation. However, injection of WT platelets significantly reversed the phenotype of increased neovascularization in C5ar1 −/− mice. Data are shown as the mean ± SEM ( n = 6 Matrigels per group). The neovascularization area fraction in Matrigels from C5ar1 −/− mice re-transfused with vehicle control represents the 100% value expressed as the area fraction of nuclear staining. * p < 0.05, WT platelets versus Cxcl4 −/− platelets. j WT platelets were coinjected with Matrigel into C5ar1 −/− mice, and animals were treated systemically with an anti-CXCL4 antibody or IgG control. While the animals that received WT platelets and IgG control showed a reduced level of growth factor-induced angiogenesis, the animals that received WT platelets and anti-CXCL4 antibody did not exhibit significantly different vessel formation levels compared with control animals that did not receive platelet retransfusion. Data are shown as the mean ± SEM ( n = 7–9 Matrigels per group). The neovascularization area fraction in Matrigels from WT mice without platelet injection and vehicle control treatment represents 100% expressed as the area fraction of nuclear staining. * p < 0.001, anti-CXCL4 antibody versus IgG control. k Matrigel was supplemented with bFGF and additionally PMX53 or PMX control and injected into WT mice. Quantification of neovascularization after 7 days yielded a significantly higher level of growth factor-induced angiogenesis in the PMX53 group. Data are shown as the mean ± SEM ( n = 7 Matrigels per group). The neovascularization area fraction within Matrigels from WT mice supplemented with PMX control represents 100%. * p < 0.05. l Representative immunofluorescent stainings of Matrigel plug sections at ×400 magnification shows Matrigel neovascularization (IB4 in green, nuclei in blue) is increased after supplementation of Matrigel with PMX53. Scale bars represent 100 µm. Image is representative of at least four analyzed plugs. m Ischemic hindlimb muscle tissue from WT or C5ar1 −/− mice was stained for the presence of CXCL4 deposition (red) 1 week after induction of ischemia. IB4 staining (green) depicts vascular structures, DAPI (blue) nuclei. ×630 magnification, scale bars represent 2 µm. n Quantification of CXCL4 abundance by measuring the area fraction of CXCL4-positive staining in whole-muscle sections acquired by tile scanning at ×400 magnification. Data are shown as the mean ± SEM ( n = 10 whole-muscle sections per group) and are displayed as the percentage of control. The area fraction of CXCL4 staining in hindlimb muscle sections from WT mice represents 100%. * p < 0.01. o Ischemic muscle tissue from the hindlimb ischemia experiments was homogenized, and samples of equal protein content were probed for CXCL4 concentration using ELISA. Homogenates from C5ar1 −/− mice yielded significantly lower CXCL4 levels compared with those from WT mice. Data are shown as the mean ± SEM ( n = 6–8 muscles processed). The CXCL4 concentration in WT muscle homogenates measured by ELISA represents 100%. * p < 0.05. p Freshly isolated washed murine WT platelets were isolated and preincubated with PMX205 or vehicle control. C5a-induced CXCL4 secretion was quantified by ELISA. PMX205 inhibited C5a-induced CXCL4 secretion. Data are shown as the mean ± SEM ( n = 5–25 independent experiments). The CXCL4 concentration in the supernatant of WT platelets stimulated with vehicle control represents 100%. * p < 0.05. q WT mice treated with the C5aR1 inhibitor PMX205 or control were subjected to hindlimb ischemia and analyzed after 2 weeks. We observed increased revascularization in PMX205-treated animals. Data are shown as the mean ± SEM ( n = 7 animals per group) and are displayed as the percentage of the perfusion in the contralateral control limb. * p < 0.05 Two-sided Student’s t test in g – k , n , o . One-way ANOVA with Bonferroni’s post hoc test in a – e , p . Two-way ANOVA with Bonferroni’s post hoc test in q .

Article Snippet: For human samples, we used a human CXCL4/PF4 Quantikine ELISA Kit (R&D), a human thrombospondin-1 Quantikine ELISA Kit (R&D), a human PDGF BB ELISA Kit (ab100624, Abcam), and a human Endostatin ELISA Kit (RayBiotech, Peachtree Corners, GA, USA).

Techniques: Isolation, Migration, Activation Assay, In Vivo, Injection, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A) Analysis of transforming growth factor β1 (TGFβ1) levels in bone marrow fluid derived from C57BL/6N, thrombopoietin (TPO)-deficient ( Tpo −/- ) or MPL-deficient mice ( Mpl −/- ). n=6-10 mice. One-way ANOVA with Dunnett’s test for multiple comparisons. (B) Visualization of latency-associated peptide (LAP)/TGFβ1, von Willebrand Factor (vWF) and the megakaryocyte (MK) marker CD42b in femoral cryosections. Nuclei were counterstained using DAPI. Scale bars: 20 µm. (C, D) Correlation analysis between CD42b and vWF (C) and CD42b and LAP/TGFβ1 (D) . Each dot represents one MK. n=2 mice. (E, F) Analysis of TGFβ1 and PF4 levels in cell culture supernatant after incubation of HSPCs in the absence (Control) or presence of TPO. Values were normalized to TPO values. n=8 mice. Unpaired, two-tailed Student’s t-test. (G) Visualization of LAP/TGFβ1, vWF and proplatelet basic protein (PPBP) in round and proplatelet-forming MKs. Goat (gt) and rabbit (rb) isotype antibodies served as staining controls. Nuclei were counterstained using DAPI. Scale bars: left: 10 µm; right: 5 µm. (H) Pearson colocalization coefficient analysis using Costes threshold of proplatelet-forming MKs imaged using super-resolution microscopy (Airyscan Leica LSM880, 63x objective). Each dot represents one MK. LAP/TGFβ1 served as a positive control. (I) Visualization of LC3B, LAP/TGFβ1 and CD42b in native bone marrow MKs. Nuclei were counterstained using DAPI. Scale bars: 15 µm. (J) Pearson colocalization coefficient analysis using Costes threshold for LC3B and LAP/TGFβ1. Each dot represents one MK. n=2 mice. (K) Correlation analysis between LAP/TGFβ1 and LC3B in cultured, bone marrow-derived MKs. Each dot represents one MK. n=3 mice. (L, M) Visualization of vWF and LAP/TGFβ1 (L) as well as LC3B and LAP/TGFβ1 (M) in platelets adhered on a fibrinogen-coated surface. Platelets were stained for α-tubulin to highlight the cytoskeleton. Scale bars: 5 µm. All data are presented as mean ± SD.

Article Snippet: Cytokine levels in bone marrow fluid or cell culture supernatants were determined using a mouse TGF-beta 1 DuoSet ELISA Kit (DY1679, R&D Systems), a mouse CXCL4/PF4 DuoSet ELISA Kit (DY595, R&D Systems) or a mouse IL-1beta DuoSet ELISA Kit (DY401, R&D Systems).

Techniques: Derivative Assay, Marker, Cell Culture, Incubation, Control, Two Tailed Test, Staining, Super-Resolution Microscopy, Positive Control

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A) Schematic visualizing how hydroxychloroquine (HQ) and verteporfin (VP) interfere with autophagy. (B) Percentage of CD41 + /CD42d + -positive cells after treatment of HSPCs with DMSO, 5 µM HQ or 5 µM VP for 72h was assessed using flow cytometry. Values were normalized to the DMSO control. n=3-4 mice. One-way ANOVA with Dunnett’s test for multiple comparisons. (C-E) Analysis of TGFβ1 (C) , IL1β (D) and PF4 levels (E) in cell culture supernatant after treatment of HSPCs with DMSO, 5 µM HQ or 5 µM VP for 72h. Supernatant derived from cells cultured in the absence of TPO were included as a control. Values were normalized to DMSO values. n=4-6 mice. One-way ANOVA with Dunnett’s test for multiple comparisons. (F) Visualization of LC3B, LAP/TGFβ1 and CD42b in enriched bone marrow-derived MKs treated with DMSO, HQ or VP for 72h. Nuclei were counterstained using DAPI. Scale bars: 20 µm. (G-I) MK area (G) , LAP/TGFβ1 (H) and LC3B MFI (I) were analyzed in cultured MKs. At least 70 MKs were analyzed per condition. n=3. One-way ANOVA with Dunnett’s test for multiple comparisons. (J, K) Immunoblot (J) and densitometric analysis (K) of enriched bone marrow-derived MKs treated with DMSO or HQ for 72h. n=6. Unpaired, two-tailed Student’s t-test. (L) Schematic on the inhibitory function of CCG1423 (CCG) and Y27632 (Y) on RhoA and Rho kinases (ROCKs). (M) Percentage of CD41 + /CD42d + -positive cells after treatment of HSPCs with DMSO, 5 µM CCG or 500 nM Y for 72h was assessed using flow cytometry. Values were normalized to the DMSO control. n=3 mice. One-way ANOVA with Dunnett’s test for multiple comparisons. (N-P) Analysis of TGFβ1 (N) , IL1β (O) and PF4 levels (P) in cell culture supernatant after treatment of HSPCs with DMSO, CCG or Y for 72h. Supernatant derived from cells cultured in the absence of TPO were included as a control. Values were normalized to DMSO values. n=3-6 mice. One-way ANOVA with Dunnett’s test for multiple comparisons. (Q, R) MK area was assessed (Q) and LC3B, LAP/TGFβ1 and CD42b were visualized (R) in enriched bone marrow-derived MKs treated with DMSO, CCG or Y for 72h. Nuclei were counterstained using DAPI. Scale bars: 20 µm. (S, T) , LAP/TGFβ1 (S) and LC3B MFI (T) were analyzed in cultured MKs. At least 70 MKs were analyzed per condition. n=3. One-way ANOVA with Dunnett’s test for multiple comparisons. Schematics were generated using Biorender.com.

Article Snippet: Cytokine levels in bone marrow fluid or cell culture supernatants were determined using a mouse TGF-beta 1 DuoSet ELISA Kit (DY1679, R&D Systems), a mouse CXCL4/PF4 DuoSet ELISA Kit (DY595, R&D Systems) or a mouse IL-1beta DuoSet ELISA Kit (DY401, R&D Systems).

Techniques: Flow Cytometry, Control, Cell Culture, Derivative Assay, Western Blot, Two Tailed Test, Generated

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A) White blood cells (WBC) count of mice transplanted with CD117 + MSCV-EGFP (Control)- or MSCV-IRES-EGFP-MPL W515L (MPL W515L )-transduced cells three weeks after transplantation. n=4 mice. Unpaired, two-tailed Student’s t-test. (B-D) Platelet count (B) , mean platelet volume (MPV) (C) and immature platelet fraction (IPF) (D) of mice transplanted with CD117 + Control or MPL W515L -transduced cells. n=4 mice. Unpaired, two-tailed Student’s t-test. (E, F) Percentage of EGFP + platelets (E) and CD45 + cells (F) of mice transplanted with Control or MPL W515L -transduced cells three weeks after transplantation. n=4. Unpaired, two-tailed Student’s t-test. (G) Spleen to body weight of mice transplanted with Control or MPL W515L -transduced cells. n=4 mice. Unpaired, two-tailed Student’s t-test. (H-J) Visualization (H) and quantification of MFI of CD41 (I) and TGFβ1/LAP (J) in femoral cryosections of mice transplanted with Control or MPL W515L -transduced cells. At least 50 MKs were analyzed per mouse. n=3 mice. Unpaired, two-tailed Student’s t-test. All data are presented as mean ± SD. (K-M) Visualization (K) and quantification of collagen I (L) and collagen IV deposition (M) in femoral cryosections of mice transplanted with Control or MPL W515L -transduced cells. n=3 mice. Each dot represents one field of view (FOV). Three FOVs were analyzed per mouse. Unpaired, two-tailed Student’s t-test. All data are presented as mean ± SD. (N, O) Analysis of TGFβ1 (N) and PF4 levels (O) in cell culture supernatant of CD117 + Control or MPL W515L -transduced cells after culture in the presence of TPO for 72h. n=4 mice. Unpaired, two-tailed Student’s t-test. (P, Q) LC3B immunoblot (P) and densitometric analysis (Q) of platelets derived from mice transplanted with CD117 + Control or MPL W515L -transduced cells. n=6 mice. Unpaired, two-tailed Student’s t-test. (R, S) RhoA immunoblot (R) and densitometric analysis (S) of platelets derived from mice transplanted with CD117 + Control or MPL W515L -transduced cells. n=6 mice. Unpaired, two-tailed Student’s t-test. All data are presented as mean ± SD.

Article Snippet: Cytokine levels in bone marrow fluid or cell culture supernatants were determined using a mouse TGF-beta 1 DuoSet ELISA Kit (DY1679, R&D Systems), a mouse CXCL4/PF4 DuoSet ELISA Kit (DY595, R&D Systems) or a mouse IL-1beta DuoSet ELISA Kit (DY401, R&D Systems).

Techniques: Control, Transplantation Assay, Two Tailed Test, Cell Culture, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A) Schematic of autophagy-related gene 5 (ATG5) in autophagosome formation. (B) Schematic of the MPL W515L transplant model in mice. (C) Transduction efficiency of CD117 + cells derived from WT and Atg5 HSC-KO mice transduced with MPL W515L one or four days after transduction. n=4. Multiple, unpaired Student’s t-tests. (D, E) Percentage of EGFP + CD45 + cells (D) and platelets (E) of WT and Atg5 HSC-KO mice transplanted with Control or MPL W515L -transduced cells four weeks after transplantation. n=5-6 mice. Two-way ANOVA with Sidak’s correction for multiple comparisons. (F) Spleen to body weight of WT and Atg5 HSC-KO mice transplanted with Control or MPL W515L -transduced cells. n=5-6 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (G, H) White blood cell (WBC) (G) and red blood cell (RBC) counts (H) of WT and Atg5 HSC-KO mice. n=5-6 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (I, J) Platelet counts (I) and immature platelet fraction (IPF) (J) of WT and Atg5 HSC-KO mice. n=5-6 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (K, L) Visualization (K) and quantification (L) of splenic MKs of transplanted WT and Atg5 HSC-KO mice. n=5-6 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. Scale bars: 500 µm. (M) Quantification of MKs and megakaryocyte progenitors (MkP) in the bone marrow of MPL W515L mice transplanted with WT or Atg5 HSC-KO cells by flow cytometry. n=5-6 mice. Two-way ANOVA with Sidak’s correction for multiple comparisons. (N) Quantification of MK numbers in femoral cryosections of MPL W515L mice transplanted with WT or Atg5 HSC-KO cells. n=5 mice. Unpaired, two-tailed Student’s t-test. (O-Q) Visualization (O) and quantification of intracellular LAP/TGFβ1 (P) and collagen I (Q) in MPL W515L mice transplanted with WT or Atg5 HSC-KO cells. n=5-6 mice. Unpaired, two-tailed Student’s t-test. Scale bars: 50 µm. (R-T) Analysis of TGFβ1 (R) , IL1β (S) and PF4 levels (T ) in bone marrow fluid of MPL W515L mice transplanted with WT or Atg5 HSC-KO cells. n=5-6 mice. Unpaired, two-tailed Student’s t-test. All data are presented as mean ± SD. Schematics were generated using Biorender.com.

Article Snippet: Cytokine levels in bone marrow fluid or cell culture supernatants were determined using a mouse TGF-beta 1 DuoSet ELISA Kit (DY1679, R&D Systems), a mouse CXCL4/PF4 DuoSet ELISA Kit (DY595, R&D Systems) or a mouse IL-1beta DuoSet ELISA Kit (DY401, R&D Systems).

Techniques: Transduction, Derivative Assay, Control, Transplantation Assay, Flow Cytometry, Two Tailed Test, Generated

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A) Transduction efficiency of CD117 + cells derived from WT and RhoA MK-KO mice transduced with MPL W515L one day after transduction. n=3 mice. Multiple unpaired Student’s t-tests. (B, C) Percentage of EGFP + CD45 + cells (B) and platelets (C) of WT and RhoA MK-KO mice transplanted with Control or MPL W515L -transduced cells four weeks after transplantation. n=5-6 mice. Two-way ANOVA with Sidak’s correction for multiple comparisons and unpaired, two-tailed Student’s t-test. (D) Spleen to body weight of WT and RhoA MK-KO mice transplanted with Control or MPL W515L -transduced cells. n=5-6 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (E, F) White blood cell (WBC) (E) and red blood cell (RBC) counts (F) of transplanted WT and RhoA MK-KO mice. n=5-6 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (G) Platelet counts of transplanted WT and RhoA MK-KO mice. n=5-6 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (H, I) Visualization (H) and quantification (I) of splenic MKs of MPL W515L -transplanted WT and RhoA MK-KO mice. n=6 mice. Scale bars: 500 µm. Unpaired, two-tailed Student’s t-test. (J) Quantification of MK numbers in femoral cryosections of WT and RhoA MK-KO mice transplanted with MPL W515L -transduced cells. n=6 mice. Unpaired, two-tailed Student’s t-test. (K-M) Visualization (K) and quantification of collagen IV (L) and intracellular LAP/TGFβ1 (M) and in transplanted WT and RhoA MK-KO mice. n=6. Unpaired, two-tailed Student’s t-test. Scale bars: 50 µm. (N-P) Analysis of TGFβ1 (N) , IL1β (O) and PF4 levels (P ) in bone marrow fluid of MPL W515L -transplanted WT and RhoA MK-KO mice. n=6 mice. Unpaired, two-tailed Student’s t-test. All data are presented as mean ± SD.

Article Snippet: Cytokine levels in bone marrow fluid or cell culture supernatants were determined using a mouse TGF-beta 1 DuoSet ELISA Kit (DY1679, R&D Systems), a mouse CXCL4/PF4 DuoSet ELISA Kit (DY595, R&D Systems) or a mouse IL-1beta DuoSet ELISA Kit (DY401, R&D Systems).

Techniques: Transduction, Derivative Assay, Control, Transplantation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A, B) Percentage of EGFP + CD45 + cells (A) and platelets (B) of PBS- or Y27632 (Y)-treated mice transplanted with MPL W515L -transduced cells three weeks after transplantation. n=5 mice. Two-way ANOVA with Sidak’s correction for multiple comparisons and unpaired, two-tailed Student’s t-test. (C) Spleen to body weight of transplanted PBS- and Y-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. (D, E) White blood cell (WBC) (D) and red blood cell (RBC) counts (E) of transplanted PBS- and Y-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. (F-H) Platelet count (F), mean platelet volume (MPV) (G) and immature platelet fraction (IPF) (H) in transplanted PBS- and HQ-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. All data are presented as mean ± SD. (I-K) Visualization (I) and quantification of intracellular LAP/TGFβ1 (J) and collagen IV (K) in transplanted PBS- and Y-treated mice. n=5. Unpaired, two-tailed Student’s t-test. Scale bars: 50 µm. (L) Quantification of MK numbers in femoral cryosections in transplanted PBS- and Y-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. (M-O) Analysis of TGFβ1 (M) , IL1β (N) and PF4 levels (O ) in bone marrow fluid of transplanted PBS- and Y-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. All data are presented as mean ± SD. Schematic was generated using Biorender.com.

Article Snippet: Cytokine levels in bone marrow fluid or cell culture supernatants were determined using a mouse TGF-beta 1 DuoSet ELISA Kit (DY1679, R&D Systems), a mouse CXCL4/PF4 DuoSet ELISA Kit (DY595, R&D Systems) or a mouse IL-1beta DuoSet ELISA Kit (DY401, R&D Systems).

Techniques: Transplantation Assay, Two Tailed Test, Generated

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A, B) Percentage of EGFP + CD45 + cells (A) and platelets (B) of vehicle (Veh; carboxymethylcellulose), ruxolitinib (R)-, ruxolitinib and hydroxychloroquine (HQ) (R+HQ)- and ruxolitinib and Y27632 (R+Y)-treated mice transplanted with MPL W515L -transduced cells three weeks after transplantation. n=5 mice. Two-way ANOVA with Sidak’s correction for multiple comparisons and one-way ANOVA with Sidak’s correction for multiple comparisons. (C) Spleen to body weight of transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (D, E) White blood cell (WBC) (D) and red blood cell (RBC) counts (E) of transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (F, G) Platelet count (F) and immature platelet fraction (IPF) (G) in transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (H-J) Visualization (H) and quantification of MK numbers (I) and collagen IV deposition (J) in transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. Scale bars: 50 µm. (K-M) Analysis of TGFβ1 (K) , IL1β (L) and PF4 levels (M ) in bone marrow fluid of transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. All data are presented as mean ± SD.

Article Snippet: Cytokine levels in bone marrow fluid or cell culture supernatants were determined using a mouse TGF-beta 1 DuoSet ELISA Kit (DY1679, R&D Systems), a mouse CXCL4/PF4 DuoSet ELISA Kit (DY595, R&D Systems) or a mouse IL-1beta DuoSet ELISA Kit (DY401, R&D Systems).

Techniques: Transplantation Assay

Journal: PLoS ONE

Article Title: Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles

doi: 10.1371/journal.pone.0159580

Figure Lengend Snippet: List of used enzyme-linked immunosorbent assay (ELISA) kits. All kits were purchased as indicated and used according to the manufacture’s guide.

Article Snippet: CXCL4 (PF4) , Platelet factor 4 (PF4) ELISA kit , Cloud-Clone Corp., Houston, USA , 128 pg/mL.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles

doi: 10.1371/journal.pone.0159580

Figure Lengend Snippet: Samples were analyzed by ELISA. Values are mean ± SD. IL-6, interleukin-6; CXCL2, chemokine (C-X-C motif) ligand 2; CXCL7, chemokine (C-X-C motif) ligand 7; CXCL3, chemokine (C-X-C motif) ligand 3; CXCL1, chemokine (C-X-C motif) ligand 1; CXCL4, chemokine (C-X-C motif) ligand 4; IL-10, inteleukin-10; TGF-β1, transforming growth factor-β1; SDF-1, stromal-cell derived factor-1. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: CXCL4 (PF4) , Platelet factor 4 (PF4) ELISA kit , Cloud-Clone Corp., Houston, USA , 128 pg/mL.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles

doi: 10.1371/journal.pone.0159580

Figure Lengend Snippet: Identified proteins clustered to immune modulators. Identified proteins from proteomic analysis at 12 h after injury. Gene symbols and protein names correspond to Uniprot Knowledgebase. Quantitative values are given as Log10-transformed fold changes BD/STW. Significances are indicated as asterisks with * p-value < 0.05, ** p-value < 0.01 and *** p-value < 0.001. n = 4 for each defect scenario.

Article Snippet: CXCL4 (PF4) , Platelet factor 4 (PF4) ELISA kit , Cloud-Clone Corp., Houston, USA , 128 pg/mL.

Techniques: Significance Assay

Journal: Molecular Medicine

Article Title: CXCL4 deficiency limits M4 macrophage infiltration and attenuates hyperoxia-induced lung injury

doi: 10.1186/s10020-024-01043-y

Figure Lengend Snippet: M4 macrophages are activated and CXCL4 is upregulated in BPD. ( A ) Representative images of H&E staining (arrow represented the pathological structural change in the lung tissues of mice) and IF for F4/80 (a macrophage marker), S100A8, MMP7, and DAPI in lung tissues from NOX (21% O2) and HYX (95% O2) groups ( n = 6). ( B ) Flow cytometric analysis showing the percentage of F4/80 + S100A8 + MMP7 + cells in NOX and HYX groups ( n = 6). ( C - D ) qRT-PCR analysis and ELISA quantification of CXCL4 expression levels ( n = 6). ( E ) IF staining for F4/80 and CXCL4 in lung tissues ( n = 6). ( F - G ) Western blot analysis of CXCL4, TNF-α, IL-6, MMP7, S100A8, and GAPDH in lung tissues from NOX and HYX groups ( n = 6)

Article Snippet: CXCL4 concentrations were quantified with a mouse CXCL4/PF-4 ELISA kit (MCX400, R&D Systems, USA).

Techniques: Staining, Marker, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Molecular Medicine

Article Title: CXCL4 deficiency limits M4 macrophage infiltration and attenuates hyperoxia-induced lung injury

doi: 10.1186/s10020-024-01043-y

Figure Lengend Snippet: Loss of CXCL 4 alleviates hyperoxia-induced alveolar epithelial type 2 cells (AT2) injury and suppresses S1P metabolism. ( A ) H&E staining of lung tissues from WT and CXCL4 KO mice exposed to NOX (21% O 2 ) and HYX (95% O2) conditions for 7 days ( n = 6). ( B ) Quantification of the average surface area of a single alveolus (µm²) ( n = 6). ( C ) Radial Alveolar Count (RAC) analysis ( n = 6). ( D ) Representative images of TUNEL staining (green), SFTPC (red), and DAPI (blue) in lung sections, indicating apoptosis and AT2 cell distribution ( n = 6). ( E ) Quantification of TUNEL-positive cells ( n = 6). ( F ) Quantification of SFTPC + cells per field of view ( n = 6). ( G - H ) Western blot analysis of SPHK1, SPHK2, SPT2, and S1PL, genes associated with sphingosine-1-phosphate metabolism ( n = 6)

Article Snippet: CXCL4 concentrations were quantified with a mouse CXCL4/PF-4 ELISA kit (MCX400, R&D Systems, USA).

Techniques: Staining, TUNEL Assay, Western Blot

Journal: Molecular Medicine

Article Title: CXCL4 deficiency limits M4 macrophage infiltration and attenuates hyperoxia-induced lung injury

doi: 10.1186/s10020-024-01043-y

Figure Lengend Snippet: Deletion of CXCL4 weakens fibrotic lung remodeling. ( A ) Representative images of lung tissue sections from WT and CXCL4 KO mice under NOX (21% O 2 ) and HYX (95% O 2 ) conditions ( n = 6). ( B ) Measurement of septal thickness (µm) in the NOX_WT, NOX_CXCL4 KO, HYX_WT, and HYX_CXCL4 KO groups ( n = 6). ( C ) Analysis of elastic fibers relative to lung tissue ( n = 6). ( D ) Sirius Red staining showing changes in collagen content ( n = 6). ( E ) Quantification of collagen amount relative to lung tissue ( n = 6). ( F ) Western blot analysis of phosphorylated SMAD2 ( n = 6). ( G ) Relative expression levels of p-SMAD2/SMAD2 protein ratio ( n = 6)

Article Snippet: CXCL4 concentrations were quantified with a mouse CXCL4/PF-4 ELISA kit (MCX400, R&D Systems, USA).

Techniques: Staining, Western Blot, Expressing

Journal: Molecular Medicine

Article Title: CXCL4 deficiency limits M4 macrophage infiltration and attenuates hyperoxia-induced lung injury

doi: 10.1186/s10020-024-01043-y

Figure Lengend Snippet: Deletion of CXCL4 prevents the progression of M4 macrophages in the lung. ( A ) IF staining for F4/80, S100A8, and DAPI in lung tissues from WT and CXCL4 KO mice under NOX (21% O 2 ) and HYX (95% O 2 ) conditions ( n = 6). ( B ) IF staining for F4/80, MMP7, and DAPI ( n = 6). ( C ) Flow cytometry analysis quantifying F4/80 + S100A8 + MMP7 + cells (%) ( n = 6). ( D - E ) Western blot analysis of CXCL4, TNF-α, IL-6, MMP7, S100A8, and GAPDH ( n = 6)

Article Snippet: CXCL4 concentrations were quantified with a mouse CXCL4/PF-4 ELISA kit (MCX400, R&D Systems, USA).

Techniques: Staining, Flow Cytometry, Western Blot

Journal: Molecular Medicine

Article Title: CXCL4 deficiency limits M4 macrophage infiltration and attenuates hyperoxia-induced lung injury

doi: 10.1186/s10020-024-01043-y

Figure Lengend Snippet: CXCL4 induces M4 macrophages in vitro and drives macrophage migration via CCR 1. ( A - B ) Expression levels of S100A8 and MMP7 in macrophages treated with M-CSF (M0) and CXCL4 (M4) ( n = 3). ( C - D ) Dose-dependent expression of MMP7 and S100A8 in response to varying concentrations of CXCL4 (4, 2, 1, 0.5 µM) ( n = 3). ( E ) IF staining of S100A8 and DAPI ( n = 3). ( F ) IF staining of MMP7 and DAPI ( n = 3). ( G - H ) ELISA results for MMP7 and S100A8 ( n = 3). ( I - J ) Macrophage migration assay comparing control, CXCL4, CCR1 inhibitor (CCR1 Inhi), and CXCL4 + CCR1 Inhi groups. Arrows represented the type of migrated cells ( n = 3)

Article Snippet: CXCL4 concentrations were quantified with a mouse CXCL4/PF-4 ELISA kit (MCX400, R&D Systems, USA).

Techniques: In Vitro, Migration, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecular Medicine

Article Title: CXCL4 deficiency limits M4 macrophage infiltration and attenuates hyperoxia-induced lung injury

doi: 10.1186/s10020-024-01043-y

Figure Lengend Snippet: CXCL4 deficiency promotes lung matrix remodeling during regeneration. ( A ) H&E staining of lung tissues from WT and CXCL4 KO mice exposed to HYX followed by a recovery period under NOX conditions ( n = 6). ( B ) Measurement of the average surface area of a single alveolus (µm²) ( n = 6). ( C ) Radial Alveolar Count (RAC) analysis ( n = 6). ( D ) Measurement of septal thickness (µm) ( n = 6). ( E - F ) Quantification of collagen amount relative to lung tissue ( n = 6)

Article Snippet: CXCL4 concentrations were quantified with a mouse CXCL4/PF-4 ELISA kit (MCX400, R&D Systems, USA).

Techniques: Staining