Journal: Scientific reports

Article Title: Protein profiling identified key chemokines that regulate the maintenance of human pluripotent stem cells.

doi: 10.1038/s41598-017-15081-6

Figure Lengend Snippet: Figure 2. Chemokine screening strategy for hPSCs. (a–d) Immunofluorescence of pluripotency markers TRA- 1-81 and Oct4 on hPSCs. KSR medium (a,b); mTeSR1 medium (c,d); hESCs (a,c); hiPSCs (d); Feeder cells (b); Scale bars, 50 μm (a–d; arrows) and 10 μm (insets). Cells were counterstained with DAPI. (e) Chemokine screening strategy for hPSCs cultured in KSR (red) or mTeSR1 (green) media.

Article Snippet: Recombinant human chemokine proteins IL-8, SDF-1α, and IP-10 were purchased from R&D Systems (USA).

Techniques: Immunofluorescence, Cell Culture

Journal: Scientific reports

Article Title: Protein profiling identified key chemokines that regulate the maintenance of human pluripotent stem cells.

doi: 10.1038/s41598-017-15081-6

Figure Lengend Snippet: Figure 4. Chemokine signaling functionally mediates the transmigration of hPSCs. Chemoattractant effect of exogenous IP-10, IL-8, and SDF-1α on hPSCs. hESCs (a,b,e,f,i,j); hiPSCs (c,d,g,h,k,l); IL-8 (a,c) and CXCR2 antagonist SB265610 (b,d); SDF-1α (e,g) and CXCR4 antagonist AMD3100 (f,h); IP-10 (i,k) and CXCR3 antagonist NBI74330 (j,l). Values on graphs represent means ± sem, n = 3 individual experiments. *P < 0.05, **P < 0.01.

Article Snippet: Recombinant human chemokine proteins IL-8, SDF-1α, and IP-10 were purchased from R&D Systems (USA).

Techniques: Transmigration Assay

Journal: Scientific reports

Article Title: Protein profiling identified key chemokines that regulate the maintenance of human pluripotent stem cells.

doi: 10.1038/s41598-017-15081-6

Figure Lengend Snippet: Figure 5. Chemokine IL-8 facilitates the differentiation of hPSCs. Stemness of hPSCs was evaluated by the expressions of three pluripotency genes (Oct4, Nanog, and Rex-1) after treatment with IL-8 and CXCR2 antagonists reparixin/SB265610. hESCs (a–c); hiPSCs (d–f); IL-8 (a,d); Reparixin (b,e); and SB265610 (c,f). Values on graphs represent means ± sem, n = 3 individual experiments. *P < 0.05, **P < 0.01, ***P < 0. 001.

Article Snippet: Recombinant human chemokine proteins IL-8, SDF-1α, and IP-10 were purchased from R&D Systems (USA).

Techniques:

Journal: Scientific reports

Article Title: Protein profiling identified key chemokines that regulate the maintenance of human pluripotent stem cells.

doi: 10.1038/s41598-017-15081-6

Figure Lengend Snippet: Figure 7. Mobility and maintenance of hPSCs require multiple chemokine signals. Schematic representation of predominant chemokines from feeder cells or hPSCs themselves. 1) Released chemokines uniformly mediate the migration of hPSCs; 2) Both differentiating and maintaining chemokines were secreted in the culture supernatant. However, the final effect of chemokines tended to maintain the stemness of hPSCs in vitro.

Article Snippet: Recombinant human chemokine proteins IL-8, SDF-1α, and IP-10 were purchased from R&D Systems (USA).

Techniques: Migration, In Vitro

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

doi: 10.4049/jimmunol.175.12.8296

Figure Lengend Snippet: FIGURE 5. sRAGE and anti-RAGE inhibit CXCL8- and TNF-- but not ATRA-induced type X collagen expression. Primary normal human knee articular chondrocytes (donor ages, 29–62) (5 105 cells/six-well dish) were stimulated with CXCL8 (A), ATRA (B), or TNF- (C) in the presence or absence of 1 g/ml sRAGE or 20 g/ml anti-RAGE. SDS- PAGE and Western blotting analysis for type X collagen were performed on cell lysates at 5 days in culture. These data are representative of results from six normal human donors.

Article Snippet: All-trans retinoic acid (ATRA), and human recombinant CXCL8 and TNF- were from R&D Systems, and rabbit polyclonal Abs to type X collagen were from Calbiochem. mAb to RAGE was from Chemicon International.

Techniques: Expressing, SDS Page, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

doi: 10.4049/jimmunol.175.12.8296

Figure Lengend Snippet: FIGURE 3. RAGE expression and inducible S100A11 secretion in cul- tured human articular chondrocytes. A, RAGE expression. First-passage human knee articular chondrocytes (5 105 cells/six-well dish) from six normal donors (age range, 29–62), were prepared as described in Mate- rials and Methods, were studied for RAGE expression by flow cytometric analysis. The panels, taken from studies of one normal donor representative of all the donors, depict binding of isotype control IgG (open histogram) and additionally of anti-RAGE IgG without stimulation or in response to CXCL8, TNF-, and ATRA for 24 h (solid histograms). In normal chon- drocytes, constitutive RAGE expression was detected in 59 9% of cells, and there were no significant changes in the proportions of RAGE-express- ing chondrocytes in response to CXCL8, TNF-, or ATRA, as seen in the representative results from the single donor demonstrated here. B, S100A11 expression and multimerization in chondrocytes. SDS-PAGE/ Western blotting analysis was performed on 1 g of human recombinant S100A11 isolated from transfected 293 cells as described in Materials and Methods (left side of figure), and on 30-g aliquots of protein precipitated from conditioned medium of first-passage normal human knee chondro- cytes (right side of figure) (donor ages, 29–62). The chondrocytes were prepared under the same conditions as for A above, and stimulated for 24 h by addition of S100A11, CXCL8, TNF-, or ATRA, at which time con- ditioned media were studied. Results are representative of those from six different normal human donors, as in A.

Article Snippet: All-trans retinoic acid (ATRA), and human recombinant CXCL8 and TNF- were from R&D Systems, and rabbit polyclonal Abs to type X collagen were from Calbiochem. mAb to RAGE was from Chemicon International.

Techniques: Expressing, Binding Assay, Control, SDS Page, Western Blot, Recombinant, Isolation, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

doi: 10.4049/jimmunol.175.12.8296

Figure Lengend Snippet: FIGURE 7. Schematic of RAGE signaling in the induction of chondro- cyte hypertrophy. This schematic depicts the induction by CXCL8 and TNF- of secretion of S100A11 (and likely other chondrocyte-expressed RAGE ligands not limited to calgranulins). The paradigm further depicts subsequent RAGE signaling critically transduced by p38 MAPK pathway activation as a central event in chondrocyte hypertrophy associated with low-grade inflammation in the OA joint articular cartilage.

Article Snippet: All-trans retinoic acid (ATRA), and human recombinant CXCL8 and TNF- were from R&D Systems, and rabbit polyclonal Abs to type X collagen were from Calbiochem. mAb to RAGE was from Chemicon International.

Techniques: Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Aging changes the mechanism that underlies JAK2 modulation of neutrophil function

doi: 10.1093/jimmun/vkaf323

Figure Lengend Snippet: Age-specific changes in JAK2 regulation of neutrophil migration. (A) Functional heatmap of select pathways in secretome of neutrophils from young and aged mice across conditions (normalized to control). Heatmap show z -scores generated from P values and log fold/ratio changes. JAK inhibition (baricitinib [BAR], pan-JAK; AZD1480 [AZD], JAK2) increased regulation of IGF and chemokine signaling pathways compared to PMA treatment in neutrophils from young mice. In neutrophils from aged mice, both AZD and BAR treatment only increased regulation of IGF compared to PMA treatment. (B) Functional heatmap of migration-associated pathways in the cell fraction of samples. AZD treatment (JAK2 inhibition) led to an increase in integrin signaling, IL-1 signaling, and actin cytoskeleton signaling, and a decrease in actin nucleation by ARP-WASP compared to PMA treatment in neutrophils isolated from young mice. In neutrophils from aged mice, JAK2 inhibition increased all listed functions compared to PMA treatment, except for actin nucleation by ARP-WASP. BAR treatment (pan-JAK inhibition) led to an increase in integrin signaling and a decrease in the rest of the listed functions compared to PMA treatment in neutrophils from young mice. BAR-treated neutrophils from aged mice increased integrin signaling and decreased actin cytoskeleton signaling and actin nucleation by ARP-WASP, with no changes in IL-1 signaling compared to PMA-treated cells. (C) Heatmap depicting abundance of protein (log ratio normalized to control), associated with neutrophil motility. Overall, JAK regulation on neutrophil motility proteins is age dependent. AZD-treated neutrophils from young and aged mice showed a decrease in ARPC2 and an increase in myristoylated alanine-rich c-kinase substrate. AZD-treated neutrophils from aged mice had a decrease in I-κB kinase. BAR-treated neutrophils from young and aged mice showed increased VCAM1. BAR-treated neutrophils from young mice display decreased STAT3 and NF-κB. Those from aged mice showed a decrease in ARPC2 and I-κB kinase. (D) Bar graphs showing the relative abundance of 2 cytoskeletal proteins in each condition. ARPC2 and ARPC3 levels were decreased in neutrophils from young and aged mice in the presence of AZD (JAK2 inhibition). In neutrophils from young mice, BAR (pan-JAK inhibition) also decreased ARPC2 levels. (E) Proposed model of molecular mechanism of JAK2 modulation of neutrophil migration. We propose that JAK2 promotes the assembly of the actin nucleation complex by increasing the levels of ARPC2/3. This leads to actin branching and formation of pseudopodia to facilitate movement. Age-dependent regulation of migration by JAK2 occurs through its differential modulation of neutrophil chemokine release (increased CXCL2 in neutrophils from aged mice, and decreased SDF-1α in neutrophils from young mice). (F) Functional assessment of migration showed that overall, fewer neutrophils from age mice migrate toward IL-8 compared to those from young mice. Only BAR significantly decreased neutrophil migration of neutrophils from young mice compared to PMA treatment. (G, H) Migration pattern between neutrophils from young and aged mice after pan-JAK or JAK2 inhibition (young: n =13/condition; aged: n =11/condition). * P <0.05; bar graphs represent mean±SEM. (D, F) Individual points correspond to individual mice (pink, females; green, males). Diagram generated with BioRender.

Article Snippet: These transwell chambers were placed on a 96-well plate; the lower chamber of the well contained 150μL of media and the chemoattractant IL-8 (20 ng/mL; Kingfisher BioTech, catalog #RP0357H-005).

Techniques: Migration, Functional Assay, Control, Generated, Inhibition, Protein-Protein interactions, Isolation