Journal: eLife

Article Title: DNL343 is an investigational CNS penetrant eukaryotic initiation factor 2B activator that prevents and reverses the effects of neurodegeneration caused by the integrated stress response

doi: 10.7554/eLife.92173

Figure Lengend Snippet: ( A–B ) Expression of Gdf15 mRNA and GDF-15 protein in the brain of eIF2B homozygous (HOM) or wild-type mice treated with DNL343 or vehicle. ( C–D ) GDF-15 protein levels in the CSF and plasma of VWMD patients and healthy controls. ( E–F ) Expression of brain Gfap mRNA and plasma GFAP protein in eIF2B HOM or wild-type mice treated with DNL343 or vehicle. ( G–H ) GFAP protein levels in the CSF and plasma of VWMD patients and healthy controls. ( I–J ) TIMP-1 protein levels in the brain and CSF of eIF2B HOM or wild-type mice treated with DNL343 or vehicle. ( K–L ) TIMP-1 protein levels in the CSF and plasma of VWMD patients and healthy controls. ( M–N ) Expression of the Ccl2 transcript, which encodes the MCP-1 protein, and levels of MCP-1 protein in the brain of eIF2B HOM or wild-type mice treated with DNL343 or vehicle. ( O–P ) MCP-1 protein levels in the CSF and plasma of VWMD patients and healthy controls. ( Q–R ) NfL protein levels in the CSF and plasma of VWMD patients and healthy controls. ( S ) Heatmap visualization of relative changes in NfL, GDF-15, GFAP, and MCP-1 in the CSF of VWMD patients vs healthy controls, presented in log 2 scale. VWMD patient ID#s correspond across (CSF) and (plasma). Statistical significance was set at p<0.05. For all animal model panels, DNL343 dose is indicated on the x-axis and data is presented as mean ± SEM of N=9–18 mice per group. Statistical significance for DNL343 effect in the mouse model was determined by a one-way ANOVA followed by Dunn’s multiple comparison tests against vehicle-dosed animals of the same genotype (*, p<0.05. **, p<0.01, ***, p<0.001, ****, p<0.0001). Data from VWMD patients and healthy controls is presented as mean ± SEM. Statistical significance for the difference between samples from VWMD patients and healthy controls was assessed on log 2 fold change data using Welch’s t test (*, p<0.05. **, p<0.01, ***, p<0.001, ****, p<0.0001).

Article Snippet: ELISA kits were used to determine the levels of GDF-15 (mouse/rat GDF-15 Quantikine ELISA Kit, R&D Systems, catalog # MGD150), TIMP-1 (mouse TIMP-1 Quantikine ELISA Kit, R&D Systems, catalog # MTM100), and MCP-1 (Mouse CCL2/JE/MCP-1 Quantikine ELISA Kit, R&D Systems, catalog # MJE00B) in the brain, plasma, or CSF samples.

Techniques: Expressing, Animal Model, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: In vitro citrullination of bacterially produced chemokines leads to their partial degradation. ( a ) Colloidal Coomassie stained SDS-PAGE analysis of the MCP-1/CCL2 products of in vitro citrullination. In vitro citrullination of commercially available recombinant MCP-1/CCL2 and self-made bacterially produced MCP-1/CCL2 performed with recombinant human PAD2 and PAD4 or rabbit PAD2, respectively. Colloidal Coomassie stained MCP-1 bands are shown while recombinant humanPAD2/4 or rabbit PAD2 (Sigma Aldrich/Merck, Waltham, MA, USA) were added to reactions into the quantities below the Colloidal Coomassie sensitivity limits and cannot be visualized. ( b ) Immunoblot analysis of bacterially produced MCP-1/CCL2 upon an in vitro citrullination reaction. Self-made full-length bacterially produced MCP-1/CCL2 was citrullinated in vitro with rabbit PAD2 and resolved on SDS-PAGE, stained with Ponceau S. detection of total MCP-1/CCL2 and modified citrullines with Senshu’s antibody that recognizes that modified citrullines were made according to Senshu’s protocol . Results shown are a representative of three or more repetitive experiments.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: In Vitro, Produced, Staining, SDS Page, Recombinant, Western Blot, Modification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Mass-spectrometry verification of successful in vitro citrullination of MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 (mature form of the protein spans residues 24–99). The first row shows 23 amino acid-long signaling peptide and the second row–mature protein of 76 residues. All arginine residues are highlighted with circles. The boxed areas show the peptide that contains the citrullinated arginine residues that were detected. ( b ) Annotated tandem mass spectrometry (MS/MS) fragmentation spectrum for the citrullinated MCP1/CCL2 peptide, showing the citrullinated arginine residue at position 3 of the studied peptide (R45). The MS/MS fragmentation data were annotated using Expert System (Max Planck Institute of Biochemistry). The precursor ion was observed with a mass error of 1 part per million, and the error for the fragment ions was 0.02 daltons.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: Mass Spectrometry, In Vitro, Sequencing, Tandem Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Detection of citrullinated recombinant human MCP-1/CCL2. Standard curves of citrullinated recombinant human MCP-1/CCL2 were set up using enzyme-linked immunosorbent assay in duplicate ( a ) standard curve of citrullinated E. coli produced recombinant human MCP-1/CCL2 chemokine (R&D Systems), ( b ) standard curve for citrullinated MCP-1/CCL2 chemokine produced by and purified from human HEK 293T cells. Absorbance at 450 nm is shown. Coefficients of determination R 2 are indicated in red on the relevant plots. Results shown are a representative of three or more repetitive experiments.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Produced, Purification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Site-directed mutagenesis of potential glycosylation site at asparagine-14 in mammalian cell-produced MCP-1 significantly destabilizes recombinant human MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 with indicated positions of predicted N-glycosylation and citrullination site confirmed by a mass-spectrometry. ( b ) Immunoblot analysis of bacterially versus mammalian cell-produced MCP-1/CCL2 upon an in vitro citrullination reaction. Bacterially produced wild-type MCP-1/CCL2 or HEK293T cell-produced wild-type and N14Q mutant version of chemokine were incubated with EDTA-free proteinase inhibitor cocktail supplemented cellular lysates prepared from the control (vehicle-transfected) or indicated hPAD enzyme-transfected HEK293T cells. Citrullination reactions that were performed directly within cellular lysates essentially as published before . Recombinant chemokine was concentrated with immunoprecipitation and resolved on 10–18% polyacrylamide gradient SDS-PAGE. Results shown are a representative of three experiments.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: Mutagenesis, Produced, Recombinant, Sequencing, Mass Spectrometry, Western Blot, In Vitro, Incubation, Transfection, Immunoprecipitation, SDS Page

Journal: Nature Communications

Article Title: Tumor microenvironmental cytokines bound to cancer exosomes determine uptake by cytokine receptor-expressing cells and biodistribution

doi: 10.1038/s41467-021-23946-8

Figure Lengend Snippet: a Cytokine arrays incubated with exosomes purified from plasma of healthy subject (HS exo) or breast cancer patients (BC exo). The respective CCL2 spot is highlighted (red box). b Heatmap representation of quantification of cytokine profile of plasma exosomes of BC patients (BC exo) relative to healthy subjects (HS exo) ( n = 2 biologically independent samples/group). c Quantification of CCL2 in exosomes purified from plasma of healthy subjects (HS exo; n = 3 biologically independent samples) or BC patients ( n = 5 biologically independent samples) by ELISA. d , e Cytokine arrays incubated with ( d ) cell culture-derived EO771 exosomes or ( e ) exosome-depleted TIF obtained from overnight cultures of minced EO771 tumors grown in C57Bl/6 mice. Representative images are shown. f Absolute amounts of CCL2, IL-6, CXCL1, and GM-CSF in exosome-depleted TIF as assessed by CBA ( n = 7 biologically independent TIF preparations). The respective spots of each cytokine are highlighted in ( e ) (red boxes). g Binding of TIF-derived cytokines to cell culture-derived EO771 exosomes (BC exo/TIF; black triangles) as assessed by CBA. Exosomes alone served as controls (BC exo; black squares) ( n = 5 independent experiments, except for IL-6 ( n = 3 independent experiments)). h – j DiD-labeled cell culture-derived EO771 exosomes previously incubated (BC exo/TIF) or not (BC exo) with TIF were injected into C57Bl/6 wild-type (WT) mice, and biodistribution assessed 24 h after injection. h Representative ex vivo images of DiD fluorescence in various organs (clockwise from top-left: liver, spleen, kidney, lung, heart and bone marrow). i Quantification of DiD fluorescence in the same organs as shown in ( h ) (BC exo, n = 8 animals; black squares) (BC exo/TIF, n = 6 animals; black triangles). j Frequency of DiD + population within CD45.2 + cells from liver (BC exo and BC exo/TIF, n = 6 and n = 5 animals, respectively), spleen (BC exo, and BC exo/TIF n = 8 animals and n = 7 animals, respectively) and lung (BC exo and BC exo/TIF, n = 8 and n = 7 animals, respectively) as assessed by flow cytometry. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 as analyzed by two-tailed Mann–Whitney U -test.

Article Snippet: The following ELISA kits were used: mouse CCL2 DuoSet (R&D Systems, cat. #DY479); mouse IL-6 DuoSet (R&D Systems, cat. #DY40605); human CCL2 DuoSet (R&D Systems, cat. #DY279); human IL-6 DuoSet (R&D Systems, cat. #DY20605); human CD44 DuoSet (R&D Systems, cat. #DY704505); human glypican-1 DuoSet (R&D Systems, cat. #DY451905); human syndecan-1 ELISA Set (abcam, cat. #ab47352); human HSPG ELISA Kit (Perlecan) (abcam, cat. #ab274393); human versican ELISA Kit (Novus Biologicals, cat. #NBP275353).

Techniques: Incubation, Purification, Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay, Binding Assay, Labeling, Injection, Ex Vivo, Fluorescence, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Protein array analysis of (A) IMO2B1-conditioned media and (B) control medium. An array was section in order to test unconditioned medium and negative control IMO3A1 at the same time (B). Control medium that was not exposed to IMO cells (top, B) and IMO3A1-conditioned medium that failed to promote neurite outgrowth from chick SAG (batch 11.23; bottom, B) only detected the manufacturer's positive controls (biotinylated HRP conjugate, +) and low levels of gamma interferon (G). All other cytokines were below the level of detection in these media. In contrast, soluble TNF receptor 1 (T), gamma interferon (G), MCP-1 (M), and low levels of RANTES were detected in IMO2B1-conditioned medium shown to promote neurite outgrowth (A, batch 9.13). (− indicates negative controls, + positive controls).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Protein Array, Negative Control

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Immunofluorescence (green) representing the secondary antibody binding to the hair cell marker myosin VI was detected in hair cells of dissociated E14 mouse inner ear cultures. The dissociated cells form aggregates of hair cells and supporting cells. MCP-1 was also prominently expressed in hair cells (red), as well as in some unidentified cells surrounding the aggregate. Supporting cells within the aggregate were negative for MCP-1. The third panel demonstrates the areas where myosin VI and MCP-1 immunofluorescence overlaps (yellow). These results demonstrate that hair cells express MCP-1. Magnification, ×630.

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Immunofluorescence, Binding Assay, Marker

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Western blot analysis demonstrated MCP-1-like protein in active IMO2B1-conditioned medium when concentrated by filtration column chromatography. Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated). Molecular weight standards (kDa) are indicated at the left.

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Western Blot, Filtration, Column Chromatography, Recombinant, Molecular Weight

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: E5 chick SAG treated with IMO2B1 produced neurite outgrowth (mean 2.6, SE 0.6, n = 5,batch 9.22). Addition of 150 ng/ml of anti-MCP-1 antibody to the IMO2B1 medium decreased outgrowth of SAG significantly (mean 0.6, SE 0.3, n = 5, p < 0.01, batch 9.22).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Produced

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: (A) IMO2B1-conditioned medium promoted neurite outgrowth of E5 chick SAG (batch 9.22; explant score = 4). (B) Addition of anti-MCP-1 antibody (150 ng/ml) decreased E5 chick SAG outgrowth (explant score = 1.5). The photo shows the area of maximal outgrowth. Magnification, ×100 (A), ×200 (B).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques:

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Monocyte migration was measured using a classic chemotactic assay. The number of migrating cells is depicted minus the background number of cells migrating in control conditions (mean 58 cells, SE 19, n = 7). In the presence of MCP-1 (20 ng/ml), monocytes migrated to the side of the chamber containing the protein (mean 299, SE, 40, n = 4). Addition of the function-blocking anti-MCP-1 antibody reduced the migration of the monocytes significantly (mean 46, SE 31, n = 3, p < 0.01). IMO2B1 alone also induced the migration of monocytes above background levels (mean 58, SE 14, n = 7, batch 2.05) and the function-blocking antibody decreased this migration significantly (mean 11, SE 8, n = 6, p < 0.01).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Migration, Chemotaxis Assay, Blocking Assay

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: IMO2B1-conditioned medium produced outgrowth from E5 chick SAG (IMO2B1 batch 9.13, mean 3.4, SE 0.35, n = 7). When anti-MCP-1 function-blocking antibody was added at a concentration of 10 or 25 ng/ml (n = 14 total) the amount of outgrowth was reduced significantly (mean 1.97, SE 0.42, n = 14, p < 0.01). Supplementing the anti-MCP-1-treated IMO2B1-conditioned medium with MCP-1 (“add back,” 20 ng/ml) led to a statistically significant increase in SAG neurite outgrowth (mean 3.72, SE 0.31, n = 11, p < 0.01) above that observed with anti-MCP-1 only, indicating a direct role for MCP-1 in promoting SAG outgrowth. No further increase was noted when 200 ng/ml of MCP-1 (mean 2.7, SE 0.42, n = 5; p > 0.01) was added to the antibody-treated cultures.

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Produced, Blocking Assay, Concentration 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: 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