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: Neuroscience

Article Title: Prenatal exposure to ethanol stimulates hypothalamic CCR2 chemokine receptor system: Possible relation to increased density of orexigenic peptide neurons and ethanol drinking in adolescent offspring

doi: 10.1016/j.neuroscience.2015.09.020

Figure Lengend Snippet: Antibodies used in single immunofluorescence histochemistry. Vendor for secondary antibodies: JacksonImmunoResearch Laboratories, Inc, PA

Article Snippet: Goat anti-CCL2 , 1/100 , Santa Cruz, CA , Cy3-Donkey anti-Goat IgG , 1/100.

Techniques: Immunofluorescence, Concentration Assay

Journal: Current Issues in Molecular Biology

Article Title: Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer

doi: 10.3390/cimb43030122

Figure Lengend Snippet: A product of 4T1 cells upregulates MCP-1 production by fibroblasts. ( A ) Ten thousand 3T3 cells, 1 × 10 4 , 2 × 10 4 or 4 × 10 4 4T1 cells, or 1 × 10 4 3T3 cells with different numbers (1 × 10 4 , 2 × 10 4 , or 4 × 10 4 ) of 4T1 cells were seeded into 24-well plates. After overnight incubation at 37 °C, the medium was replaced by 0.5 mL fresh medium. After a 4-day incubation at 37 °C, cell-free culture supernatants were collected, and the concentration of MCP-1 was measured by ELISA. Data are presented as mean ± SD. *** p < 0.0001, n = 3. Representative of three independent experiments with similar results. ( B ) Fifty thousand 3T3 cells were seeded into 24-well plates. After overnight incubation at 37 °C, the medium was replaced by 0.5 mL fresh medium containing different concentrations of 4T1-sup and incubated for 24 h at 37 °C. Cell-free culture supernatants were collected, and the concentration of MCP-1 was measured by ELISA. Data are presented as mean ± SD. ** p < 0.01, **** p < 0.00001. n = 3. Representative of three independent experiments with similar results. ( C ) Fifty thousand lung fibroblasts isolated from two normal BALB/c mice were seeded into 24-well plates. After overnight incubation at 37 °C, the medium was replaced by a fresh medium containing different concentrations of 4T1-sup. After a 24-h incubation, cell-free culture supernatants were collected, and the concentration of MCP-1 was measured by ELISA. Data are presented as mean ± SD. **** p < 0.00001, n = 3.

Article Snippet: The concentration of MCP-1 was measured using an ELISA kit specific for mouse MCP-1 (BioLegend) or by sandwich ELISA using anti-mouse MCP-1 antibodies (R&D Systems, Minneapolis, MN, USA).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Current Issues in Molecular Biology

Article Title: Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer

doi: 10.3390/cimb43030122

Figure Lengend Snippet: Increased MCP-1 production by fibroblasts in response to 4T1-sup was due to PDGFs. ( A ) Fifty thousand 3T3 cells were seeded into 24-well plates. After overnight incubation at 37 °C, the medium was replaced by 0.5 mL fresh medium containing 50% ( v / v ) of 4T1-sup and incubated for 1, 3, 6 or 12 h at 37 °C. Total RNA was isolated, and the expression of Mcp-1 or Kc/Cxcl1 mRNA was evaluated by qRT-PCR. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3. Representative of two independent experiments with similar results. ( B ) Total RNA was isolated from 4T1 cells, and the expression of Pdgfa , b , c , and d mRNA was evaluated by RT-PCR. Representative of two independent experiments with similar results. ( C ) Ten μg of 4T1 cell lysate was loaded onto a polyacrylamide gel, and SDS-PAGE was performed. Proteins were transferred onto a membrane, and the presence of PDGF-A was examined by Western blotting. Representative of three independent experiments with similar results. ( D ) Fifty thousand 3T3 cells were seeded into 24-well plates. After overnight incubation at 37 °C, the medium was replaced by 0.5 mL fresh medium containing 100 ng/mL recombinant mouse PDGF-AA or PDGF-BB. After a 24-h incubation at 37 °C, cell-free culture supernatants were collected, and the concentration of MCP-1 was measured by ELISA. Data are presented as mean ± SD. ** p < 0.01, **** p < 0.00001, n = 3. Representative of four independent experiments with similar results.

Article Snippet: The concentration of MCP-1 was measured using an ELISA kit specific for mouse MCP-1 (BioLegend) or by sandwich ELISA using anti-mouse MCP-1 antibodies (R&D Systems, Minneapolis, MN, USA).

Techniques: Incubation, Isolation, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, SDS Page, Membrane, Western Blot, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Current Issues in Molecular Biology

Article Title: Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer

doi: 10.3390/cimb43030122

Figure Lengend Snippet: Pretreatment of 3T3 cells with crenolanib inhibited increased MCP-1 production induced by 4T1-sup. ( A ) Fifty thousand 3T3 cells were seeded into 24-well plates. After overnight incubation at 37 °C, the medium was replaced by a fresh medium containing DMSO or 0.2 or 2 μM crenolanib. After a 30-min incubation at 37 °C, the same volume of medium or 4T1-sup was added and incubated for an additional 24 h. Cell-free culture supernatants were collected, and the concentration of MCP-1 was measured by ELISA. Data are presented as mean ± SD. **** p < 0.00001, n = 3. Representative of three independent experiments with similar results. ( B ) Fifty thousand 3T3 cells were seeded into 24-well plates. After overnight incubation at 37 °C, the medium was replaced by a fresh medium containing DMSO or 0.2 or 2 μM crenolanib. After a 30-min incubation at 37 °C, the same volume of medium or 4T1-sup was added and incubated for an additional 24 h. Total RNA was isolated, and the expression of MCP-1 mRNA was evaluated by qRT-PCR. Data are presented as mean ± SD. * p < 0.05, n = 3. Representative of three experiments with similar results.

Article Snippet: The concentration of MCP-1 was measured using an ELISA kit specific for mouse MCP-1 (BioLegend) or by sandwich ELISA using anti-mouse MCP-1 antibodies (R&D Systems, Minneapolis, MN, USA).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Quantitative RT-PCR

Journal: Current Issues in Molecular Biology

Article Title: Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer

doi: 10.3390/cimb43030122

Figure Lengend Snippet: Treatment with trapidil or crenolanib had no effect on MCP-1 production or tumor growth. ( A ) One hundred thousand 4T1 cells were injected into a mammary pad of WT mice. Five days later, when tumors were clearly detectable, 100 μL of 10 μM crenolanib in PBS was intratumorally injected five times every 12 h. Mice were euthanized 4 h after the final injection, and the weight of the tumor (left panel) and the expression of Mcp-1 mRNA in tumors (middle panel) and serum MCP-1 concentration (right panel) were measured. Data are presented as mean ± SD. n = 3 for each group. Representative of two independent experiments with similar results. ( B ) One hundred thousand 4T1 cells were injected into a mammary pad of WT BALB/c mice. Five days later, when tumors were clearly detectable, trapidil (20 mg/kg) in 100 μL PBS was intraperitoneally injected twice a day for 7 days. Mice were euthanized on the next day, and tumor weight and serum MCP-1 concentration were measured. Data are presented as mean ± SD. n = 3 for each group. Representative of two independent experiments with similar results. ( C ) One hundred thousand A8 cells were injected into a mammary pad of WT mice. Five days later, when tumors were clearly detectable, 500 μL of 10 μM crenolanib in PBS was injected i.p. twice a day for 3 days. To directly block PDGFRs in a tumor, 100 μL of 10 μM crenolanib in PBS was injected i.t. three times every 12 h. Mice were euthanized 12 h after the final injection, and the weight of the tumor ( left panel), the expression of Mcp-1 mRNA in tumors ( middle panel) and serum MCP-1 concentration ( right panel) were measured. Data are presented as mean ± SD. n = 3 for each group. Representative of two independent experiments with similar results.

Article Snippet: The concentration of MCP-1 was measured using an ELISA kit specific for mouse MCP-1 (BioLegend) or by sandwich ELISA using anti-mouse MCP-1 antibodies (R&D Systems, Minneapolis, MN, USA).

Techniques: Injection, Expressing, Concentration Assay, Blocking Assay

Journal: Current Issues in Molecular Biology

Article Title: Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer

doi: 10.3390/cimb43030122

Figure Lengend Snippet: Histological examination and the detection of cells expressing Mcp-1 mRNA in 5-day 4T1 tumors. One hundred thousand 4T1 cells in 100 μL PBS were injected into a mammary pad of WT female BALB/c mice. Five days after the injection, the mice were euthanized, and tumors were harvested. Consecutive paraffin sections from three individual tumors were examined by different staining and in situ hybridization. Representative photos are presented. ( A ) H&E staining, scale bar = 100 μm. ( B ) IHC with anti-Ly6G Ab, scale bar = 100 μm. ( C ) IHC with anti-F4/80 Ab, scale bar = 100 μm. ( D ) IHC with anti-αSMA, scale bar = 100 μm. ( E ) ISH. Green, Mcp-1 mRNA; Red; Act2 mRNA. Peritumoral area. Scale bar = 50 μm. ( F ) ISH. Green, Mcp-1 mRNA; Red; Act2 mRNA. Intratumoral area. Scale bar = 50 μm. ( G ) ISH. Green, Mcp-1 mRNA; Red; Adgre1 mRNA. Peritumoral area. Scale bar = 50 μm. ( H ) ISH. Green, Mcp-1 mRNA; Red; Adgre1 mRNA. Intratumoral area. Scale bar = 50 μm.

Article Snippet: The concentration of MCP-1 was measured using an ELISA kit specific for mouse MCP-1 (BioLegend) or by sandwich ELISA using anti-mouse MCP-1 antibodies (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Injection, Staining, In Situ Hybridization

Journal: Current Issues in Molecular Biology

Article Title: Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer

doi: 10.3390/cimb43030122

Figure Lengend Snippet: Detection of cells expressing Mcp-1 mRNA in 14-day 4T1 tumors. One hundred thousand 4T1 cells in 100 μL PBS were injected into a mammary pad of WT female BALB/c mice. Fourteen days after the injection, the mice were euthanized, and tumors were harvested. ( A ) Consecutive paraffin sections from three individual tumors were examined for the expression of Mcp-1 (green) and Act2 (upper panels) or Adgre1 (red) mRNA (lower panels) by ISH. Representative photos are presented. Scale bar = 50 μm. ( B ) One million peritoneal exudate macrophages were incubated for 24 h in the absence or presence of necrotic 4T1 cells, and the concentration of MCP-1 in the cell-free supernatants was measured by ELISA. Data are presented as mean ± SD. n = 3, ** p < 0.01. ( C ) One million peritoneal exudate macrophages were incubated for 24 h in the absence or presence of 50 or 100 μL of necrotic GM-CSF-deficient A8 cells, and the concentration of MCP-1 in the cell-free supernatants was measured by ELISA. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, n = 3. Representative of three independent experiments with similar results.

Article Snippet: The concentration of MCP-1 was measured using an ELISA kit specific for mouse MCP-1 (BioLegend) or by sandwich ELISA using anti-mouse MCP-1 antibodies (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Injection, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay