Journal: PLoS ONE

Article Title: Increased Cellular Senescence and Vascular Rarefaction Exacerbate the Progression of Kidney Fibrosis in Aged Mice Following Transient Ischemic Injury

doi: 10.1371/journal.pone.0070464

Figure Lengend Snippet: Representative micrographs of F4/80 (A) and CD4 (B) immunostained sections of the outer medulla of young and aged kidneys. Quantitation of pixels per field indicate significantly increased numbers of cells in the aged kidneys 6 weeks post-reperfusion (n = 4–5/group). There are equivalent low levels in young and age matched normals. *p≤0.05 indicates a significant difference vs respective normal. **p≤0.05 indicates a significant difference between young and aged ischemia. Bar = 100 microns. C. Gene expression of MCP-1 and TNF-α indicate increased mRNA levels in the aged kidneys 6 weeks post-reperfusion (n = 7 for young and 3 for aged mice). *p≤0.05 indicates a significant difference between young and aged ischemia.

Article Snippet: Primers specific for mouse included MCP-1 (Mm99999056_m1), TNF-α (Mm00443258_m1) and 18S (Hs99999901_s1).

Techniques: Quantitation Assay, Gene Expression

Journal: Experimental eye research

Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

doi: 10.1016/j.exer.2017.01.002

Figure Lengend Snippet: Photoreceptor cell line 661W was treated with 0.1% DMSO (veh) or 0.01 μM thapsigargin (TG). (A–G) Cells were collected for mRNA extraction at indicated time points. The mRNA levels of ER stress-related genes (GRP78, ATF4, CHOP, XBP1s and ATF6), CXCL10 and CCL2 were analyze by qPCR. *p<0.05 vs 0h; n=3. (H, I) Conditioned medium was collected at 24 hours after treatment and subjected to ELISA to measure CXCL10 and CCL2 production which was normalized to total protein of cell lysates. Cells treated with vehicle were used as reference. *p<0.05 compared with vehicle; n=4.

Article Snippet: The concentrations of CXCL10 and CCL2 in cell-free supernatants were measured with Mouse CXCL10/IP-10/CRG-2 or CCL2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and normalized to total protein of the cell lysates.

Techniques: Extraction, Enzyme-linked Immunosorbent Assay

Journal: Experimental eye research

Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

doi: 10.1016/j.exer.2017.01.002

Figure Lengend Snippet: Photoreceptor cells were treated with 0.01 μM TG in the presence or absence of ER stress blocker 4-phenylbutyrate (4-PBA, 4mM) for 6 hours. RNA was extracted and qPCR was performed to assess the expression of CXCL10, CCL2 and ER stress markers (GRP78, ATF4, CHOP, XBP1s and ATF6). Cells treated with 0.1% DMSO (vehicle) were used as reference *p<0.05 vs vehicle control; #p<0.05 vs TG treatment; n=4.

Article Snippet: The concentrations of CXCL10 and CCL2 in cell-free supernatants were measured with Mouse CXCL10/IP-10/CRG-2 or CCL2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and normalized to total protein of the cell lysates.

Techniques: Expressing, Control

Journal: Experimental eye research

Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

doi: 10.1016/j.exer.2017.01.002

Figure Lengend Snippet: (A) Photoreceptor cells were treated with TG for 1 hour and 3 hours, and total and phosphorylated PERK were assessed by Western blot. Equal loading of protein was confirmed with antibody against α-Tubulin. (B) Photoreceptor cells were stably expressed with control shRNA (sh-Con) or PERK shRNA (sh-PERK) and the knockdown efficiency of PERK was evaluated by Western blot and quantified by densitometry. Cells transfected with control shRNA were used as reference. *p<0.05 vs control shRNA. (C–H) Photoreceptor cells stably expressing control or PERK shRNA were treated with 0.1% DMSO (vehicle) or TG for 6 hours. The levels of CXCL10, CCL2, GRP78, ATF6, CHOP and ATF4 mRNA were analyzed by qPCR. Cells transfected with control shRNA and treated with vehicle were used as reference. *p<0.05 vs vehicle-treated cells with control shRNA; #p<0.05 vs TG-treated cells with control shRNA; n=3.

Article Snippet: The concentrations of CXCL10 and CCL2 in cell-free supernatants were measured with Mouse CXCL10/IP-10/CRG-2 or CCL2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and normalized to total protein of the cell lysates.

Techniques: Western Blot, Stable Transfection, Control, shRNA, Knockdown, Transfection, Expressing

Journal: Experimental eye research

Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

doi: 10.1016/j.exer.2017.01.002

Figure Lengend Snippet: Photoreceptor cells stably expressing control shRNA (sh-Con) or XBP1 shRNA (sh-XBP1) were treated with vehicle (veh) or 0.01 μM TG. (A) 3 hours after TG treatment, the level of XBP1 protein in nucleus was evaluated by Western blot and quantified by densitometry. Lamin B1 served as loading control. Cells transfected with control shRNA and treated with vehicle were used as reference. (B–G) 6 hours after TG treatment, CXCL10, CCL2, GRP78, ATF4, ATF6 and CHOP mRNA levels were analyzed by qPCR. Cells transfected with sh-Con and treated with vehicle were used as reference. *p<0.05 vs vehicle-treated cells with control shRNA; #p<0.05 vs TG-treated cells with control sh-RNA; n=4.

Article Snippet: The concentrations of CXCL10 and CCL2 in cell-free supernatants were measured with Mouse CXCL10/IP-10/CRG-2 or CCL2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and normalized to total protein of the cell lysates.

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Transfection

Journal: Experimental eye research

Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

doi: 10.1016/j.exer.2017.01.002

Figure Lengend Snippet: (A, B) Photoreceptor cells were stably expressed with control shRNA (sh-Con), PERK shRNA (sh-PERK) or XBP1 shRNA (sh-XBP1) and treated with TG for 3 hours. Phosphorylated and total RelA and IkBα were evaluated by Western blot and quantified by densitometry. Equal loading of protein was confirmed with antibody against total RelA or α-Tubulin. Cells transfected with control shRNA and treated with vehicle were used as reference. *p<0.05 vs vehicle-treated cells with sh-Con. #p<0.05 vs TG-treated cells with sh-Con; n=3. (C, D, E) Photoreceptors were pretreated with NF-κB inhibitor PDTC (10 μM) for 30 minutes and followed with TG treatment for 6 hours. The mRNA levels of CXCL10, CCL2 and GRP78 were analyzed by qPCR. Cells treated with vehicle were used as reference. *p<0.05 vs vehicle treatment; #p<0.05 vs TG treatment; n=3.

Article Snippet: The concentrations of CXCL10 and CCL2 in cell-free supernatants were measured with Mouse CXCL10/IP-10/CRG-2 or CCL2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and normalized to total protein of the cell lysates.

Techniques: Stable Transfection, Control, shRNA, Western Blot, Transfection

Journal: Experimental eye research

Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

doi: 10.1016/j.exer.2017.01.002

Figure Lengend Snippet: (A–B) Photoreceptor cells were stably expressed with control shRNA (sh-Con), PERK shRNA (sh-PERK) or XBP1 shRNA (sh-XBP1) and treated with TG for 30 minutes. Phosphorylated and total STAT3 and JAK1 were evaluated by Western blot and quantified by densitometry. Cells transfected with control shRNA and treated with 0.1% DMSO (vehicle) were used as reference. *p<0.05 vs vehicle-treated cells with sh-Con. #p<0.05 vs TG-treated cells with sh-Con; n=3. (C, D, E) Photoreceptors were pretreated with STAT3 inhibitor Stattic (3 μM) for 30 minutes and followed with TG treatment for 6 hours. The mRNA levels of CXCL10, CCL2 and GRP78 were analyzed by qPCR. Cells treated with vehicle were used as reference. *p<0.05 vs vehicle treatment; #p<0.05 vs TG treatment; n=3.

Article Snippet: The concentrations of CXCL10 and CCL2 in cell-free supernatants were measured with Mouse CXCL10/IP-10/CRG-2 or CCL2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and normalized to total protein of the cell lysates.

Techniques: Stable Transfection, Control, shRNA, Western Blot, Transfection

Journal: Experimental eye research

Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

doi: 10.1016/j.exer.2017.01.002

Figure Lengend Snippet: (A-D) Photoreceptor cells were treated with 200 μg/ml AGE for the indicated time periods. CXCL10 and CCL2 mRNA levels were analyzed by qPCR (A, B). Phosphorylated and total PERK were evaluated by Western blot and quantified by densitometry (C). XBP1s gene expression was analyzed by qPCR (D). Cells treated with vehicle (BSA) were used as reference. *p<0.05 vs 0h; n=3. (E–H) Photoreceptor cells stably expressing control or PERK or XBP1 shRNA were treated with vehicle (BSA) or AGE. The levels of CXCL10 and CCL2 mRNA were analyzed by qPCR at 12 hours after treatment (n=4) (E, F). CXCL10 and CCL2 protein levels in conditioned medium were measured by ELISA at 24 hours after treatment, and normalized to total protein of cell lysates (n=3) (G–H). Cells transfected with control shRNA and treated with vehicle were used as reference. *p<0.05 vs vehicle-treated cells with control sh-RNA; #p<0.05 vs AGE-treated cells with control shRNA.

Article Snippet: The concentrations of CXCL10 and CCL2 in cell-free supernatants were measured with Mouse CXCL10/IP-10/CRG-2 or CCL2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and normalized to total protein of the cell lysates.

Techniques: Western Blot, Gene Expression, Stable Transfection, Expressing, Control, shRNA, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Experimental eye research

Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

doi: 10.1016/j.exer.2017.01.002

Figure Lengend Snippet: Photoreceptors stably expressing sh-Con, sh-PERK or sh-XBP1 were cultured in normal glucose (NG, 5.5 mM) or high glucose (HG, 30 mM) for 48 hours and conditioned medium was harvested. CXCL10 and CCL2 protein levels were measured by ELISA and normalized to total protein of cell lysates. *p<0.05 vs NG-treated cells with control shRNA; #p<0.05 vs HG-treated cells with control shRNA; n=3.

Article Snippet: The concentrations of CXCL10 and CCL2 in cell-free supernatants were measured with Mouse CXCL10/IP-10/CRG-2 or CCL2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and normalized to total protein of the cell lysates.

Techniques: Stable Transfection, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, shRNA

Journal: Experimental eye research

Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

doi: 10.1016/j.exer.2017.01.002

Figure Lengend Snippet: Proposed signaling pathways in ER stress-regulated expression of CXCL10 and CCL2 in photoreceptor cells.

Article Snippet: The concentrations of CXCL10 and CCL2 in cell-free supernatants were measured with Mouse CXCL10/IP-10/CRG-2 or CCL2 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions and normalized to total protein of the cell lysates.

Techniques: Protein-Protein interactions, Expressing

Journal: Digestive Diseases and Sciences

Article Title: Reduced IL-37 Production Increases Spontaneous Chemokine Expressions in Colon Epithelial Cells

doi: 10.1007/s10620-016-4422-9

Figure Lengend Snippet: mRNA ( a ) and protein ( b ) levels of CCL2, CCL5, CXCL8, CXCL10, and CXCL11 ( n = 5). The filled bars represent cells stimulated with 100 ng/ml flagellin for 12 h, whereas empty bars denote unstimulated cells. First, unstimulated cells with reduced IL-37 levels (IL-37sgRNA) were compared to unstimulated cells transfected with an empty plasmid (TFneg), whereas the IL-37sgRNA cells stimulated with flagellin are compared to TFneg cells stimulated with flagellin. Second, unstimulated cells are compared to flagellin-stimulated cells, within the same cell type (WT, TFneg or IL-37sgRNA). Statistically significant differences are marked with *, **, or *** depicting p < 0.05, < 0.01, or < 0.001. All data are shown as mean ± SEM. WT wild-type T84 cells, N.D not detected

Article Snippet: For gene expression analysis using qRT-PCR, a TaqMan Fast Universal Master Mix was used with the following TaqMan primer–probe sets [ , , ]: IL-37 (Hs00367201_m1), CCL2/MCP-1 (Hs00234140_m1), CCL3/MIP-1α (Hs00234142_m1), CCL4/MIP-1β (Hs99999148_m1), CCL5/RANTES (Hs00174575_m1), CCL20/MIP3A (Hs00355476_m1), CCL22/MDC (Hs01574247_m1), CXCL8/IL-8 (Hs00174103_m1), CXCL9/MIG (Hs00171065_m1), CXCL10/IP-10 (Hs01124251_g1), CXCL11/I-TAC (Hs04187682_g1), and CX 3 CL1/fractalkine (Hs00171086_m1).

Techniques: Transfection, Plasmid Preparation