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: Cellular and Molecular Life Sciences

Article Title: ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation

doi: 10.1007/s00018-022-04186-5

Figure Lengend Snippet: Upregulation of the SASP by ROS and Padi2 knockdown leads to cellular senescence and the inhibition of the SASP ameliorates cellular senescence and osteogenic dysfunction. A Representative images of SA-β-Gal staining. MC3T3-E1 cells were cultured in the conditioned medium (CM) from MC3T3-E1 transfected with siCont or siPadi2 for 8 days and SA-β-Gal staining was performed. Percentage of SA-β-Gal-positive cells in each group was calculated as the percentage of SA-β-Gal-positive cells relative to the total cells stained with DAPI in the same field (8–10 fields/group). Magnification ×20, Scale bars 100 μm. Bars, statistical significance was determined using one-way ANOVA for multiple comparisons, * P < 0.05, *** P < 0.001. Three independent experiments were performed. B MC3T3-E1 cells were cultivated under Padi2 KD-CM for 8 days or siCont-CM, followed by performing RT-qPCR of Cdkn1a. Data for RT-qPCR are presented as the mean of three replicates; bars, ± SD. Statistical significance was determined using one-way ANOVA for multiple comparisons, ** P < 0.01, *** P < 0.001; n.s., not significant. Two independent experiments were performed. C Analysis of the relative expression levels of the SASP genes by RNA-seq data (black bar) and RT-qPCR (gray bar). Data are the fold change of 4 day-H 2 O 2 -treated group relative to the non-treated control. The red and blue-dotted lines mean fold change 1 and − 1, respectively. Data for RT-qPCR are presented as the mean of three replicates; bars, ± SD. statistical significance was determined using two-tailed Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n.s., not significant. Three independent experiments were performed for RT-qPCR. D MC3T3-E1 cells were transfected with siCont, siPadi2 #2 or siPadi2 #3 and then incubated for additional 4 days in the osteogenic medium. The expression level of each gene was measured by RT-qPCR and the fold change in mRNA levels was calculated by normalization to Gapdh . Data are presented as the mean of three replicates; bars, ± SD; statistical significance was determined using one-way ANOVA for multiple comparisons, * P < 0.05, *** P < 0.001, **** P < 0.0001. Three independent experiments were performed. E Five days-cell culture soup from MC3T3-E1 cells transfected with siCont, siPadi2 #2, or siPadi2 #3 was collected and the levels of CCL2, CCL5, and CCL7 in each cell culture soup were measured by ELISA. Data are presented as the mean of three replicates; bars, ± SD; statistical significance was determined using one-way ANOVA for multiple comparisons, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Three independent experiments were performed. F Representative images of SA-β-Gal staining. MC3T3-E1 cells knocked down with siCont or siPadi2 were treated with IgG or each neutralizing antibody for 4 days and SA-β-Gal staining was performed. Quantity of SA-β-Gal-positive cells was expressed as the percentage of SA-β-Gal-positive cells relative to the total cells stained with DAPI in the same field (8–10 fields/group). Magnification ×20, Scale bars 100 μm. bars, mean ± SD. statistical significance was determined using two-way ANOVA for multiple comparisons, *** P < 0.001. Three independent experiments were performed. G When MC3T3-E1 cells transfected with siCont, siCcl2, siCcl5, or siCcl7 were fully confluent, cells were treated with 100 μM H 2 O 2 for 4 days in osteogenic medium and ALP staining was performed. Quantification of ALP staining was performed by ImageJ. Bars, mean ± SD; statistical significance was determined using one-way or two-way ANOVA for multiple comparisons, * P < 0.05, ** P < 0.01, **** P < 0.0001; ns, not significant. H When MC3T3-E1 cells knocked down with siPadi2 in combination with or without siCcl2, 5, or 7 were fully confluent, cells were changed with osteogenic medium and incubated for 4 days and ALP staining was performed. Quantification of ALP staining (right). Bars, mean ± SD; statistical significance was determined using one-way or two-way ANOVA for multiple comparisons, * P < 0.05, ** P < 0.01,; ns not significant. Three independent experiments were performed

Article Snippet: Antibodies against the following proteins were used in this study: Padi2 (66386–1-Ig; Proteintech, Rosemont, IL, USA), α-tubulin (sc-8035; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), β-actin (sc-47778; Santa Cruz Biotechnology.), LaminA/C (sc-376248; Santa Cruz Biotechnology), p21 (#2947; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse CCL2 antibody (AB-479-NA; R&D Systems, Inc, Minneapolis, MN, USA), mouse CCL5 antibody (AF478; R&D Systems), mouse CCL7 antibody (AF-456-NA; R&D Systems), and normal goat IgG control (AB-108-C; R&D Systems).

Techniques: Knockdown, Inhibition, Staining, Cell Culture, Transfection, Quantitative RT-PCR, Expressing, RNA Sequencing, Control, Two Tailed Test, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences

Article Title: ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation

doi: 10.1007/s00018-022-04186-5

Figure Lengend Snippet: Schematic diagram depicted in the mechanism of oxidative stress-accelerated senescence and dysfunction of osteoblasts. The reduction of PADI2 by oxidative stress induces upregulation of CCL2, 5, and 7 known as the SASP, through the activation of NFκB signaling, leading to making a senescent environment and propagating cellular senescence to surrounding cells. Targeting these regulatory processes may be an effective way to prevent or treat excessive ROS-promoted cellular senescence and aging-related bone diseases

Article Snippet: Antibodies against the following proteins were used in this study: Padi2 (66386–1-Ig; Proteintech, Rosemont, IL, USA), α-tubulin (sc-8035; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), β-actin (sc-47778; Santa Cruz Biotechnology.), LaminA/C (sc-376248; Santa Cruz Biotechnology), p21 (#2947; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse CCL2 antibody (AB-479-NA; R&D Systems, Inc, Minneapolis, MN, USA), mouse CCL5 antibody (AF478; R&D Systems), mouse CCL7 antibody (AF-456-NA; R&D Systems), and normal goat IgG control (AB-108-C; R&D Systems).

Techniques: Activation Assay

Journal: PLoS Pathogens

Article Title: A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

doi: 10.1371/journal.ppat.1005410

Figure Lengend Snippet: Intratracheal administration of 0.5μg TNF (A and B) or 50μg poly(I:C) (C and D) to A20 AEC-KO or wild type littermates (A20 WT ). Absolute numbers of neutrophils or monocytes in bronchoalveolar lavages (BAL) as determined by flow cytometry at 6h and 24h post-treatment for TNF (A) or 24h post-treatment for poly(I:C) (C). IL-6, CXCL1 (KC), CCL2 (MCP-1) and TNF [only for poly(I:C)] protein levels in BAL fluid detected by Multiplex immunoassay (B and D). Data represent mean ± SEM of at least 4 mice per group (*p < 0.05; Student’s t -test).

Article Snippet: Recombinant mouse CCL2 (R&D Systems, endotoxin levels <0.01 EU per μg of protein as measured by the LAL method) was administered intranasally at a dose of 50 μg/kg at day 6 post infection.

Techniques: Flow Cytometry, Multiplex Assay

Journal: PLoS Pathogens

Article Title: A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

doi: 10.1371/journal.ppat.1005410

Figure Lengend Snippet: (A) Absolute numbers of monocytes, neutrophils and alveolar macrophages in bronchoalveolar lavages (BAL) of A20 AEC-KO or A20 WT mice at 2, 5, 8 and 12 days post-infection (days p.i.) with 0.05 X LD 50 X-47. (B) Absolute numbers of resident CD11b - or recruited CD11b + macrophages in the lungs of A20 WT and A20 AEC-KO mice. (C) CCL2 (MCP-1) protein levels in BAL fluid measured by Multiplex immunoassay at indicated time points post-infection. (D) Weight loss of A20 AEC-KO and A20 WT mice infected with 0.05 X LD 50 X-47. At day 6 p.i. (indicated by an arrow) mice received intranasal treatment with 50 μg/kg recombinant CCL2 (rCCL2) or PBS. Data were analysed using Student’s t -test (A, B and C *p < 0.05) and 2-way ANOVA (D, *p < 0.05 for A20 AEC-KO PBS vs. A20 WT PBS and # p < 0.05 for A20 AEC-KO PBS vs A20 AEC-Cre rCCL2). Data represent mean ± SEM of at least 3 mice per group. Data are representative of at least 2 independent experiments.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, endotoxin levels <0.01 EU per μg of protein as measured by the LAL method) was administered intranasally at a dose of 50 μg/kg at day 6 post infection.

Techniques: Infection, Multiplex Assay, Recombinant