Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Senescence-associated secretory phenotype promotes chronic ocular graft-vs-host disease in mice and humans.

doi: 10.1096/fj.201900218R

Figure Lengend Snippet: FIGURE 2 SASP-related molecules are elevated in cGVHD lacrimal glands. A, B, Lacrimal gland (LG) sections from cGVHD and control mice 3 and 8 weeks after bone marrow transplantation (BMT) were stained for ɤ-H2A.X and 53BP1 (DNA damage response, green, A), Ki-67 (proliferation, green, A) and p21 (senescence, green, B). B, Lacrimal gland (LG) sections from cGVHD and control mice 4 weeks after bone marrow transplantation (BMT) were stained for p16 (senescence) and CD68 (macrophage). Colocalization of p16 (green) and CD68 (red) (arrows) shown by quadrant image (B, right). C, Flow cytometric analysis of IL-6 and p16-expressing CD68+ cells. *P<.05. D, Caspase-1 (inflammasome, green), IL-1β and IL-8 (upstream of SASP induction, green), and CXCL1 and CXCL9 (SASP components). Colocalization of CD68 (red) and CXCL1 or CXCL9 (green). IL-1β+ cells on the ducts are indicated by white arrows. E, The leukocyte marker CD45 (green) and the fibrosis markers HSP47 and α-SMA (green); colocalization with senescent T cell markers (PD1, white; CD153, red) and a SASP marker (OPN, green). The number of PD1+CD153+OPN+ cells per field of the LG 4 weeks after BMT. Data are representative of three (8 weeks after BMT) or one (3 and 4 weeks after BMT) independent experiment (n = 3 or 4 per group;> 5 fields). A-E, Data are presented as the mean ± SEM. *P < .05, ** P < .01, *** P < .001, unpaired Student’s t test. DAPI stains nuclei blue. Scale bar: 50 µm

Article Snippet: After being washed with perm/wash buffer (554714, BD Pharmingen) twice, the cells were then incubated with PElabeled anti-mouse IL-6 antibody (554401, MP5-20F3, BD Pharmingen) and p16 antibody (sc-1207, M-156, Santa Cruz Biotechnology) for 30 minutes on ice.

Techniques: Control, Transplantation Assay, Staining, Expressing, Marker

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Senescence-associated secretory phenotype promotes chronic ocular graft-vs-host disease in mice and humans.

doi: 10.1096/fj.201900218R

Figure Lengend Snippet: FIGURE 4 Effects of ABT-263 in the cGVHD lacrimal gland. A-C, Lacrimal gland (LG) sections from ABT263- and vehicle-treated mice 4 weeks after bone marrow transplantation (BMT) were stained for 53BP1 (DNA damage response, A upper low), p16 and p21 (senescence, A upper low), Ki-67 (proliferation, A upper low), caspase-1 (inflammasome component, A middle low), IL-1β and IL-8 (upstream indicators of SASP induction, A middle low), IL-6 (SASP component, A middle low), CD68 (macrophages, A lower low), CD45 (leukocytes, A lower low), osteopontin (OPN) (proinflammatory cytokine and SASP, A lower low), and HSP47 and α-SMA (fibrosis, A lower low) (green), and CD4 and CD153 (T cell and senescent T cell, C). The number of target (except OPN)-positive cells per field in the stroma and of OPN+ cells per field on acini from the ABT-263-treated mice (n = 5) and the controls (n = 8) (> 5 fields per sample) 4 weeks after BMT B, Representative images of CXCL9, a SASP molecule, in LGs from ABT-263- and vehicle-treated mice 4 weeks after BMT. Green areas indicate CXCL9+ secretion from vascular endothelia measured by Image J software (n = 3-4 per group;> 5 fields per sample). A - B Scale bar, 50 µm. C, Senescent CD4+ (green) and CD153+ T cells (green) among inflammatory cells in the lobules and stroma in LGs from ABT-263-treated mice (n = 7) and controls (n = 9) 4 weeks after BMT. The number of CD4+ and CD153+ cells per field from ABT-263-treated and control mice (> 5 fields per sample). Representative images of CD153+ cells in LGs from control mice (white arrowheads) are shown. Scale bars: 50 µm (left and middle) and 20 µm (right). A-C, DAPI stains nuclei in blue. Data are representative of two independent experiments and presented as the mean ± SEM. *P < .05, ** P < .01, unpaired Student’s t test. ABT-263, a potent senolytic, is a specific inhibitor of the antiapoptotic proteins BCL-2 and BCL-xL

Article Snippet: After being washed with perm/wash buffer (554714, BD Pharmingen) twice, the cells were then incubated with PElabeled anti-mouse IL-6 antibody (554401, MP5-20F3, BD Pharmingen) and p16 antibody (sc-1207, M-156, Santa Cruz Biotechnology) for 30 minutes on ice.

Techniques: Transplantation Assay, Staining, Software, Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Senescence-associated secretory phenotype promotes chronic ocular graft-vs-host disease in mice and humans.

doi: 10.1096/fj.201900218R

Figure Lengend Snippet: FIGURE 6 MR16-1 reduces the protein expression of senescence biomarkers and SASP-related molecules in cGVHD lacrimal glands. A-B, Lacrimal gland (LG) sections from MR16-1- and vehicle-treated mice 4 weeks after bone marrow transplantation (BMT) were stained for ɤ-H2A.X and 53BP1 (DNA damage response), p16 and p21 (senescence), Ki-67 (proliferation), caspase-1 (inflammasome), IL-8 (upstream indicator of SASP induction), IL-6 (SASP component), CD68 (macrophages), D45 (leukocytes), and HSP47 and α-SMA (fibrosis) (green). The number of target (except OPN)-positive cells per field in the stroma and of OPN+ cells per field in acini from MR16-1-treated and vehicle-treated mice (n = 5 per group; >5 fields). Data are presented as the mean ± SEM. *P < .05, ** P < .01, unpaired Student’s t test. Scale bar: 50 µm. C, Representative images of CXCL9 (major SASP component) and OPN (proinflammatory cytokine) in the LGs of MR16-1- and vehicle-treated mice 4 weeks after BMT. Green areas indicating secretion (CXCL9 or OPN) from vascular endothelia were measured by ImageJ software (n = 5 per group;> 5 fields). Data are presented as the mean ± SEM. **P < .01, unpaired Student’s t test. Scale bar: 50 µm. D, Representative images of CXCL9 or OPN inside endothelia (white arrows) in the stroma of cGVHD LGs from vehicle-treated control mice 4 weeks after BMT. BV, blood vessel. Scale bar: 20 µm. A-D, DAPI stains nuclei blue. MR16-1, anti-mouse IL-6 receptor monoclonal antibody

Article Snippet: After being washed with perm/wash buffer (554714, BD Pharmingen) twice, the cells were then incubated with PElabeled anti-mouse IL-6 antibody (554401, MP5-20F3, BD Pharmingen) and p16 antibody (sc-1207, M-156, Santa Cruz Biotechnology) for 30 minutes on ice.

Techniques: Expressing, Transplantation Assay, Staining, Software, Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Senescence-associated secretory phenotype promotes chronic ocular graft-vs-host disease in mice and humans.

doi: 10.1096/fj.201900218R

Figure Lengend Snippet: FIGURE 7 Cocultures of macrophages and T cells from various spleen sources. A, Cellular source of macrophages and T cells from spleen for cocultures. Macrophages and T cells from syngeneic controls (blue), cGVHD mice (gray), wild-type B10.D2 mice (red), and wild-type BALB/c mice (green). B, Flow cytometry analyses after coculture of macrophages and T cells from various sources. WT, wild-type. C, Percentages of IL- 6+p16+ (white bar), IL-6+ cells (black bar), and p16+ cells (gray bar) among cocultured CD68+ macrophages . Syngeneic controls (n = 4), cGVHD mice (n = 4), wild-type B10.D2. mice (n = 4), and wild-type BALB/c mice (n = 4). Data analysed at 4 weeks after BMT for syngeneic and cGVHD mice.

Article Snippet: After being washed with perm/wash buffer (554714, BD Pharmingen) twice, the cells were then incubated with PElabeled anti-mouse IL-6 antibody (554401, MP5-20F3, BD Pharmingen) and p16 antibody (sc-1207, M-156, Santa Cruz Biotechnology) for 30 minutes on ice.

Techniques: Flow Cytometry