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: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: Effect of monosodium urate (MSU) crystals on CCL2 production in cultured fibroblast-like synoviocytes (FLS) . (a) FLS were treated with increasing concentrations of MSU crystals for 24 hours. (b) Synoviocytes were incubated in the presence (black circles) or absence (white circles) of 50 μg/ml MSU crystals for the indicated time. CCL2 was measured in culture supernatants with ELISA. Results are presented as mean ± SD of three separate experiments. (c) FLS were stimulated (grey columns) or not (white columns) for 24 hours with 50 μg/ml MSU crystals in the presence or absence of 250 ng/ml IL-1Ra. IL-1Ra was either added with MSU crystals (MSU + IL-1Ra) or added to FLS 1 hour before activation by MSU crystals (IL-1Ra + MSU). (d) FLS were stimulated (black columns) or not (white columns) for 24 hours with 125 pg/ml IL-1β in the presence or absence of 250 ng/ml of IL-1Ra. IL-1Ra was either added with IL-1β (IL-1β + IL-1Ra) or added to FLS 1 hour before activation by IL-1β (IL-1Ra + IL-1β). (c, d) Culture supernatants were analyzed for the production of CCL2. Results are presented as mean ± SD of three separate experiments.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: CCL2 is stored in cytoplasmic vesicles in fibroblast-like synoviocytes (FLS) . FLS were cultured for 24 hours in the absence (a, c) or presence (b) of 50 μg/ml monosodium urate (MSU) crystals. Cells were subjected to immunostaining with anti-CCL2 antibodies (a, b) or incubated with the second antibody only as a negative control (c) , and then analyzed with confocal microscopy, as described in Materials and Methods. Original magnification, ×1,000.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Cell Culture, Immunostaining, Incubation, Negative Control, Confocal Microscopy

Journal: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: High-density lipoproteins (HDL) inhibit CCL2 production induced by monosodium urate (MSU) crystals in fibroblast-like synoviocytes (FLS) . (a, b) FLS were stimulated (grey columns) or not (white columns) for 24 hours with 50 μg/ml MSU crystals in the presence or absence of the indicated concentration of HDL. Alternatively, FLS were pretreated (Ptt.) with the indicated concentration of HDL, washed, and then stimulated for 24 hours with 50 μg/ml MSU crystals. Culture supernatants were analyzed for the production of CCL2 (a) and IL-8 (b). Results are presented as mean ± SD of duplicate determinations and are representative of independent experiments carried out with FLS isolated from three different patients. (c) FLS were stimulated (black columns) or not (white columns) with 10 pg/ml IL-1β for 24 hours. Culture supernatants were analyzed for the production of CCL2. (d) Culture supernatants of FLS, activated as in (a) and (b), were analyzed for their ability to induce mononuclear cell migration, as described in Materials and Methods. Migration induced by culture medium (white column), 10 ng/ml CCL2 (black column), and culture supernatants of FLS activated as in (a) and (b) (grey columns). Four fields were counted for the number of migrated cells. Results represent the mean ± SD of the number of cells/field in four fields. A representative experiment of three is presented.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Concentration Assay, Isolation, Migration

Journal: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: Fibroblast-like synoviocytes (FLS) stimulated by monosodium urate (MSU) crystals in the presence of high-density lipoproteins (HDL) retain intracellular CCL2 . FLS were stimulated or not with MSU crystals (50 μg/ml) in the presence or absence of the indicated concentration of HDL. After 24 hours, cells were fixed and subjected to immunostaining with anti-CCL2 antibodies. (a) Resting FLS; (b, c) FLS stimulated with MSU crystals in the absence (b) or presence of HDL (c) . Original magnification × 600. (d) FLS were treated (white columns) or not (grey columns) with 10 μg/ml cycloheximide (CHX) for 30 minutes and then activated for the indicated time with 50 μg/ml MSU crystals. Results represent mean ± SD of three independent experiments.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Concentration Assay, Immunostaining

Journal: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: High-density lipoproteins (HDL) diminish monosodium urate (MSU) crystal-induced CCL2 transcript levels . Fibroblast-like synoviocytes (FLS) were cultured for 18 hours alone or with MSU crystals (50 μg/ml) in the presence or absence of HDL (100 μg/ml), as indicated. Total RNA was prepared as described in Materials and Methods. CCL2 mRNA levels were determined with duplex quantitative real-time polymerase chain reaction (PCR) analysis of triplicates normalized to the levels of the 18S mRNA. The relative expression levels of CCL2 mRNA are presented as mean ± SD of the percentage of relative CCL2 mRNA expression. The value of mRNA levels in MSU crystal-stimulated FLS (MSU) is arbitrarily considered as 1.0. Results are representative of three distinct experiments.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Expressing