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Absolute Biotech Inc rabbit polyclonal anti-zbp1

<t>ZBP1</t> is upregulated during the osteogenic differentiation but downregulated during the adipogenic differentiation of mBMSCs. a RT-qPCR analysis and western blot analysis of ZBP1 expression during the osteogenic differentiation of mBMSCs. b RT-qPCR analysis and western blot analysis of ZBP1 expression during the adipogenic differentiation of mBMSCs. c IHC analysis of ZBP1 expression in the mouse femur. Macrophages (stars); osteoblasts on the trabecular bone surface (black arrows). n = 6. Bars indicate 30 μm. BM, bone marrow; TB, trabecular bone; MA, marrow adipocytes. d Semiquantitative quantification of ZBP1 immunostaining intensities in osteoblasts, osteocytes, marrow adipocytes, macrophages, and other marrow cells. * P < 0.05; ** P < 0.01; *** P < 0.001

Rabbit Polyclonal Anti Zbp1, supplied by Absolute Biotech Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews

rabbit polyclonal anti-zbp1 - by Bioz Stars, 2026-03

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1) Product Images from "ZBP1 (DAI/DLM-1) promotes osteogenic differentiation while inhibiting adipogenic differentiation in mesenchymal stem cells through a positive feedback loop of Wnt/β-catenin signaling"

Article Title: ZBP1 (DAI/DLM-1) promotes osteogenic differentiation while inhibiting adipogenic differentiation in mesenchymal stem cells through a positive feedback loop of Wnt/β-catenin signaling

Journal: Bone Research

doi: 10.1038/s41413-020-0085-4

ZBP1 is upregulated during the osteogenic differentiation but downregulated during the adipogenic differentiation of mBMSCs. a RT-qPCR analysis and western blot analysis of ZBP1 expression during the osteogenic differentiation of mBMSCs. b RT-qPCR analysis and western blot analysis of ZBP1 expression during the adipogenic differentiation of mBMSCs. c IHC analysis of ZBP1 expression in the mouse femur. Macrophages (stars); osteoblasts on the trabecular bone surface (black arrows). n = 6. Bars indicate 30 μm. BM, bone marrow; TB, trabecular bone; MA, marrow adipocytes. d Semiquantitative quantification of ZBP1 immunostaining intensities in osteoblasts, osteocytes, marrow adipocytes, macrophages, and other marrow cells. * P < 0.05; ** P < 0.01; *** P < 0.001

Figure Legend Snippet: ZBP1 is upregulated during the osteogenic differentiation but downregulated during the adipogenic differentiation of mBMSCs. a RT-qPCR analysis and western blot analysis of ZBP1 expression during the osteogenic differentiation of mBMSCs. b RT-qPCR analysis and western blot analysis of ZBP1 expression during the adipogenic differentiation of mBMSCs. c IHC analysis of ZBP1 expression in the mouse femur. Macrophages (stars); osteoblasts on the trabecular bone surface (black arrows). n = 6. Bars indicate 30 μm. BM, bone marrow; TB, trabecular bone; MA, marrow adipocytes. d Semiquantitative quantification of ZBP1 immunostaining intensities in osteoblasts, osteocytes, marrow adipocytes, macrophages, and other marrow cells. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques Used: Quantitative RT-PCR, Western Blot, Expressing, Immunostaining

Depletion of ZBP1 suppresses the osteogenic differentiation but promotes the adipogenic differentiation of mBMSCs. a The siRNA-mediated depletion of ZBP1 was assessed by RT-qPCR and western blot analysis. b ALP activity assays of ZBP1-depleted mBMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-depleted mBMSCs and control cells after 2 weeks of osteogenic induction. d RT-qPCR analysis of osteogenesis-related genes ( Runx2 , sp7, Ibsp , and Bglap ) in ZBP1-depleted mBMSCs and control cells. e H&E staining of transplant sections generated from ZBP1-depleted mBMSCs and control cells in HA/TCP scaffolds. Bar, 60 μm. n = 6. f , g Oil Red O staining of ZBP1-depleted mBMSCs and control cells after 3 weeks of adipogenic induction. Bar, 100 μm. h RT-qPCR analysis of adipogenic markers ( CD36, Cebpa, Lpl , and Pparg ) in ZBP1-depleted mBMSCs and control cells after 7 days of adipogenic induction. * P < 0.05; ** P < 0.01; *** P < 0.001

Figure Legend Snippet: Depletion of ZBP1 suppresses the osteogenic differentiation but promotes the adipogenic differentiation of mBMSCs. a The siRNA-mediated depletion of ZBP1 was assessed by RT-qPCR and western blot analysis. b ALP activity assays of ZBP1-depleted mBMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-depleted mBMSCs and control cells after 2 weeks of osteogenic induction. d RT-qPCR analysis of osteogenesis-related genes ( Runx2 , sp7, Ibsp , and Bglap ) in ZBP1-depleted mBMSCs and control cells. e H&E staining of transplant sections generated from ZBP1-depleted mBMSCs and control cells in HA/TCP scaffolds. Bar, 60 μm. n = 6. f , g Oil Red O staining of ZBP1-depleted mBMSCs and control cells after 3 weeks of adipogenic induction. Bar, 100 μm. h RT-qPCR analysis of adipogenic markers ( CD36, Cebpa, Lpl , and Pparg ) in ZBP1-depleted mBMSCs and control cells after 7 days of adipogenic induction. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques Used: Quantitative RT-PCR, Western Blot, Activity Assay, Staining, Generated

Overexpression of ZBP1 enhances the osteogenic differentiation but inhibits the adipogenic differentiation of mBMSCs. a The overexpression of ZBP1 was determined by RT-qPCR and western blot analysis. b ALP activity assays of ZBP1-overexpressing mBMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-overexpressing mBMSCs and control cells after 2 weeks of osteogenic stimulation. d RT-qPCR analysis of osteogenesis-related genes ( Runx2 , sp7, Ibsp , and Bglap ) in ZBP1-overexpressing mBMSCs and control cells. e H&E staining of transplant sections generated from ZBP1-overexpressing mBMSCs and control cells in HA/TCP scaffolds. Bar, 60 μm. n = 6. f Oil Red O staining of ZBP1-overexpressing mBMSCs and control cells after 3 weeks of adipogenic stimulation. Bar, 100 μm. g RT-qPCR analysis of adipogenic markers ( CD36, Cebpa, Lpl , and Pparg ) in ZBP1-overexpressing mBMSCs and control cells after 7 days of adipogenic induction. * P < 0.05; ** P < 0.01; *** P < 0.001

Figure Legend Snippet: Overexpression of ZBP1 enhances the osteogenic differentiation but inhibits the adipogenic differentiation of mBMSCs. a The overexpression of ZBP1 was determined by RT-qPCR and western blot analysis. b ALP activity assays of ZBP1-overexpressing mBMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-overexpressing mBMSCs and control cells after 2 weeks of osteogenic stimulation. d RT-qPCR analysis of osteogenesis-related genes ( Runx2 , sp7, Ibsp , and Bglap ) in ZBP1-overexpressing mBMSCs and control cells. e H&E staining of transplant sections generated from ZBP1-overexpressing mBMSCs and control cells in HA/TCP scaffolds. Bar, 60 μm. n = 6. f Oil Red O staining of ZBP1-overexpressing mBMSCs and control cells after 3 weeks of adipogenic stimulation. Bar, 100 μm. g RT-qPCR analysis of adipogenic markers ( CD36, Cebpa, Lpl , and Pparg ) in ZBP1-overexpressing mBMSCs and control cells after 7 days of adipogenic induction. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques Used: Over Expression, Quantitative RT-PCR, Western Blot, Activity Assay, Staining, Generated

Overexpression of ZBP1 enhances the osteogenic differentiation but inhibits the adipogenic differentiation of hMSCs. a The overexpression of ZBP1 was determined by RT-qPCR and western blot analysis in hMSCs. b ALP activity assays of ZBP1-overexpressing hMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-overexpressing hMSCs and control cells after 3 weeks of osteogenic induction. d RT-qPCR analysis of osteogenic markers ( RUNX2 , SP7, COL1A1 , and SPP1 ) in ZBP1-overexpressing hMSCs and control cells. e Oil Red O staining of ZBP1-overexpressing hMSCs and control cells after 4 weeks of adipogenic induction. Bar, 100 μm. f RT-qPCR analysis of adipogenic markers ( CD36, CEBPA, LPL , and PPARG ) in ZBP1-overexpressing hMSCs and control cells after 7 days of adipogenic induction. * P < 0.05; ** P < 0.01; *** P < 0.001

Figure Legend Snippet: Overexpression of ZBP1 enhances the osteogenic differentiation but inhibits the adipogenic differentiation of hMSCs. a The overexpression of ZBP1 was determined by RT-qPCR and western blot analysis in hMSCs. b ALP activity assays of ZBP1-overexpressing hMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-overexpressing hMSCs and control cells after 3 weeks of osteogenic induction. d RT-qPCR analysis of osteogenic markers ( RUNX2 , SP7, COL1A1 , and SPP1 ) in ZBP1-overexpressing hMSCs and control cells. e Oil Red O staining of ZBP1-overexpressing hMSCs and control cells after 4 weeks of adipogenic induction. Bar, 100 μm. f RT-qPCR analysis of adipogenic markers ( CD36, CEBPA, LPL , and PPARG ) in ZBP1-overexpressing hMSCs and control cells after 7 days of adipogenic induction. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques Used: Over Expression, Quantitative RT-PCR, Western Blot, Activity Assay, Staining

Restoring ZBP1 expression rescues the osteogenic potential of ZBP1-depleted mBMSCs. a RT-qPCR and western blot analysis of the restoration of ZBP1 in ZBP-depleted mBMSCs. ALP activity ( b ) and ECM mineralization ( c ) were rescued by restoring ZBP1 expression in ZBP-depleted mBMSCs. d The expression of osteogenic markers ( sp7, Ibsp , and Bglap ) was rescued by restoring ZBP1 expression in ZBP1-depleted mBMSCs. * P < 0.05; ** P < 0.01; *** P < 0.001

Figure Legend Snippet: Restoring ZBP1 expression rescues the osteogenic potential of ZBP1-depleted mBMSCs. a RT-qPCR and western blot analysis of the restoration of ZBP1 in ZBP-depleted mBMSCs. ALP activity ( b ) and ECM mineralization ( c ) were rescued by restoring ZBP1 expression in ZBP-depleted mBMSCs. d The expression of osteogenic markers ( sp7, Ibsp , and Bglap ) was rescued by restoring ZBP1 expression in ZBP1-depleted mBMSCs. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay

ZBP1 is required for Wnt/β-catenin signaling. a GO analysis of genes downregulated (over 1.5-fold) in ZBP1-depleted mBMSCs. The top 8 ontology terms are shown. b GSEA of Wnt signaling-related genes in ZBP1-depleted mBMSCs compared with controls. The normalized enrichment score (NES) = −1.41, P < 0.05, and false discovery rate (FDR) < 0.05. c Topflash luciferase reporter assays of Wnt signaling activity in ZBP1-depleted mBMSCs and control cells after treatment with 100 ng·mL −1 Wnt3a for 24 h. RT-qPCR analysis of the expression of Axin2 ( d ) and Ccnd1 ( e ) in ZBP1-depleted mBMSCs and control cells after treatment with 100 ng·mL −1 Wnt3a for 4 h. f , g Endogenous IP reveals the interaction between ZBP1 and β-catenin in mBMSCs treated with Wnt3a (100 ng·mL −1 ) for 2 h. h Western blot analysis of the nuclear extract (NE) and cytoplasmic extract (CE) from ZBP1-depleted mBMSCs and control cells treated with 100 ng·mL −1 Wnt3a for 2 h. ChIP assay of the occupancy of β-catenin at the promoters of Runx2 ( i ) and Sp7 ( j ) in ZBP1-knockdown mBMSCs and control cells after treatment with 100 ng·mL −1 Wnt3a for 4 h. Arrows indicate primer annealing sites. * P < 0.05; ** P < 0.01; *** P < 0.001

Figure Legend Snippet: ZBP1 is required for Wnt/β-catenin signaling. a GO analysis of genes downregulated (over 1.5-fold) in ZBP1-depleted mBMSCs. The top 8 ontology terms are shown. b GSEA of Wnt signaling-related genes in ZBP1-depleted mBMSCs compared with controls. The normalized enrichment score (NES) = −1.41, P < 0.05, and false discovery rate (FDR) < 0.05. c Topflash luciferase reporter assays of Wnt signaling activity in ZBP1-depleted mBMSCs and control cells after treatment with 100 ng·mL −1 Wnt3a for 24 h. RT-qPCR analysis of the expression of Axin2 ( d ) and Ccnd1 ( e ) in ZBP1-depleted mBMSCs and control cells after treatment with 100 ng·mL −1 Wnt3a for 4 h. f , g Endogenous IP reveals the interaction between ZBP1 and β-catenin in mBMSCs treated with Wnt3a (100 ng·mL −1 ) for 2 h. h Western blot analysis of the nuclear extract (NE) and cytoplasmic extract (CE) from ZBP1-depleted mBMSCs and control cells treated with 100 ng·mL −1 Wnt3a for 2 h. ChIP assay of the occupancy of β-catenin at the promoters of Runx2 ( i ) and Sp7 ( j ) in ZBP1-knockdown mBMSCs and control cells after treatment with 100 ng·mL −1 Wnt3a for 4 h. Arrows indicate primer annealing sites. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques Used: Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Western Blot

Zbp1 is a target of Wnt/β-catenin signaling. a RT-qPCR analysis of Zbp1 expression in mBMSCs treated with 100 ng·mL −1 Wnt3a for 4 h. b Western blot analysis of ZBP1 expression in mBMSCs treated with 100 ng·mL −1 Wnt3a for 24 h. c RT-qPCR analysis of Zbp1 expression in mBMSCs that were pretreated with 100 ng·mL −1 DKK1 for 2 h followed by 100 ng·mL −1 Wnt3a treatment for 2 h. d ChIP assay analysis of the occupancy of β-catenin at the promoter of Zbp1 after treatment with 100 ng·mL −1 Wnt3a for 4 h in mBMSCs. Arrows indicate primer annealing sites. WRE, Wnt/β-catenin response element. * P < 0.05; ** P < 0.01; *** P < 0.001

Figure Legend Snippet: Zbp1 is a target of Wnt/β-catenin signaling. a RT-qPCR analysis of Zbp1 expression in mBMSCs treated with 100 ng·mL −1 Wnt3a for 4 h. b Western blot analysis of ZBP1 expression in mBMSCs treated with 100 ng·mL −1 Wnt3a for 24 h. c RT-qPCR analysis of Zbp1 expression in mBMSCs that were pretreated with 100 ng·mL −1 DKK1 for 2 h followed by 100 ng·mL −1 Wnt3a treatment for 2 h. d ChIP assay analysis of the occupancy of β-catenin at the promoter of Zbp1 after treatment with 100 ng·mL −1 Wnt3a for 4 h in mBMSCs. Arrows indicate primer annealing sites. WRE, Wnt/β-catenin response element. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques Used: Quantitative RT-PCR, Expressing, Western Blot

Image Search Results

a Time-lapse microscopy of PI-stained (red) hBMECs infected with RS218 (MOI = 10), showing 8 min interval images taken at 60 × magnification. The 10 frames demonstrated the progression of cell death within 72 min before membrane rupture. White dashed lines indicate the boundaries of the cell membrane. Scale bar, 20 μm. b , c hBMECs stimulated with RS218 (MOI = 1 or 10) were used to determine cytotoxicity by LDH release ( b , n = 3 biologically independent samples) and kinetics of cell death of PI penetration ( c , n = 5 biologically independent samples). d , e hBMECs were pretreated with NSA or zVAD alone or in combination for 0.5 h, and cytotoxicity ( d , n = 6 biologically independent samples) and cell death kinetics of PI penetration ( e , n = 3 biologically independent samples) were assayed. f Flow cytometry of Annexin V-FITC and PI staining in hBMECs infected with RS218 (MOI = 1). Representative images of immunofluorescence represented morphological analysis of Annexin V-FITC and PI-labeled cells for the indicated times postinfection. Scale bar, 20 μm. g Immunoblot analysis of MLKL phosphorylation, GSDMD, caspase-3, and PARP1 cleavage in hBMECs infected with RS218 (MOI = 1) at the indicated times. Blots for GAPDH served as loading controls. +, indicated pharmacologically-induced death of hBMECs. FL, full-length. h Pro caspase-8 was co-immunoprecipitated with RIPK1, RIPK3, ZBP1 and ASC in uninfected or RS218-infected hBMECs (MOI = 1) for 3 h. i Immunofluorescence representative images of 2-day-old mice brain tissue sections 18 h after injection. The nucleus was labeled in blue, pMLKL or GSDMD-N or caspase-3 p17 in green, and endothelial cell marker CD31 in red. Scale bar, 20 μm. All experiments were representative of at least three independent experiments with similar results. Bars indicated the mean plus SD. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test, with P- values denoted as follows: **** P < 0.0001, *** P < 0.0005, ** P < 0.005, * P < 0.05, n.s., no significant difference. Exact P- values were provided in the source data. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: RIPK1 kinase drove brain microvascular endothelial cells death and blood-brain barrier disruption in neonatal Escherichia coli meningitis

doi: 10.1038/s41467-025-62760-4

Figure Lengend Snippet: a Time-lapse microscopy of PI-stained (red) hBMECs infected with RS218 (MOI = 10), showing 8 min interval images taken at 60 × magnification. The 10 frames demonstrated the progression of cell death within 72 min before membrane rupture. White dashed lines indicate the boundaries of the cell membrane. Scale bar, 20 μm. b , c hBMECs stimulated with RS218 (MOI = 1 or 10) were used to determine cytotoxicity by LDH release ( b , n = 3 biologically independent samples) and kinetics of cell death of PI penetration ( c , n = 5 biologically independent samples). d , e hBMECs were pretreated with NSA or zVAD alone or in combination for 0.5 h, and cytotoxicity ( d , n = 6 biologically independent samples) and cell death kinetics of PI penetration ( e , n = 3 biologically independent samples) were assayed. f Flow cytometry of Annexin V-FITC and PI staining in hBMECs infected with RS218 (MOI = 1). Representative images of immunofluorescence represented morphological analysis of Annexin V-FITC and PI-labeled cells for the indicated times postinfection. Scale bar, 20 μm. g Immunoblot analysis of MLKL phosphorylation, GSDMD, caspase-3, and PARP1 cleavage in hBMECs infected with RS218 (MOI = 1) at the indicated times. Blots for GAPDH served as loading controls. +, indicated pharmacologically-induced death of hBMECs. FL, full-length. h Pro caspase-8 was co-immunoprecipitated with RIPK1, RIPK3, ZBP1 and ASC in uninfected or RS218-infected hBMECs (MOI = 1) for 3 h. i Immunofluorescence representative images of 2-day-old mice brain tissue sections 18 h after injection. The nucleus was labeled in blue, pMLKL or GSDMD-N or caspase-3 p17 in green, and endothelial cell marker CD31 in red. Scale bar, 20 μm. All experiments were representative of at least three independent experiments with similar results. Bars indicated the mean plus SD. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test, with P- values denoted as follows: **** P < 0.0001, *** P < 0.0005, ** P < 0.005, * P < 0.05, n.s., no significant difference. Exact P- values were provided in the source data. Source data are provided as a Source Data file.

Article Snippet: The PVDF membranes then blotted with rabbit anti-caspase 3/p17 antibody (Proteintech, 19677-1-AP, 1:2000), mouse anti-PARP1 Monoclonal antibody (Proteintech, 66520-1-Ig, 1:20000), rabbit anti-GSDMD-NT polyclonal antibody (Bioworld, BS67358, 1:2000), rabbit anti-MLKL Antibody (ABclonal, A19685, 1:2000), rabbit anti-Phospho-MLKL (Ser358) Antibody (Affinity, AF7420, 1:2000), rabbit anti-RIPK1-Specific Polyclonal antibody (ABclonal, A19580, 1:2000), rabbit anti-Phospho-RIPK1 (Ser161) Antibody (Abmart, TA7377, 1:2000), rabbit anti-Phospho-RIPK1 (Ser166) Antibody (Abmart, TA2398S, 1:2000), rabbit anti-RIP3 Polyclonal antibody (Proteintech, 17563-1-AP, 1:2000), rabbit anti-RIP3 (phospho S227) antibody (abcam, ab209384, 1:2000), mouse anti-caspase-1/p10 antibody (Santa Cruz Biotechnology, sc-56036, 1:500), rabbit anti-caspase 8/p43/p18 Polyclonal antibody (Proteintech, 13423-1-AP, 1:1000), rabbit anti-ASC/TMS1 Polyclonal antibody (Proteintech, 10500-1-AP, 1:20000) or rabbit anti-TRIF (TICAM1) antibody (abcam, ab302562, 1:1000) or rabbit anti-ZBP1 Polyclonal antibody (Proteintech, 13285-1-AP, 1:5000) or rabbit anti-TLR4 Polyclonal antibody (Proteintech, 19811-1-AP, 1:2000) or rabbit anti-TNF Receptor I antibody (abcam, ab223352, 1:1000) or rabbit anti-GAPDH polyclonal antibody (Bioworld, AP0063, 1:10000), followed by incubation overnight at 4 °C.

Techniques: Time-lapse Microscopy, Staining, Infection, Membrane, Flow Cytometry, Immunofluorescence, Labeling, Western Blot, Phospho-proteomics, Immunoprecipitation, Injection, Marker

a Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in WT, RIPK1 KO, or ZBP1 KO cells infected with RS218 (MOI = 1). b Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in hBMECs expressing RIPK1 WT or D138N under RS218 infection (MOI = 1). c Immunoblot of RIPK1 total protein and phosphorylation levels at S161 and S166 in WT, TNFR1 KO, TLR4 KO, or double KO cells post RS218 infection (MOI = 1). Blots for GAPDH served as loading controls. d Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in hBMECs expressing RIPK1 WT, S161N, or S166N cells (MOI = 1). e , f In WT, TNFR1 KO, TLR4 KO, or double KO cells, the cytotoxicity was assessed by LDH release ( e , n = 3 biologically independent samples), and cell death kinetics were measured by PI uptake ( f , n = 3 biologically independent samples). g Co-IP analysis of RIPK1 with TNFR1 and TLR4 interactions in WT and ZBP1 KO cells. h Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in WT, TNFR1 KO, TLR4 KO or double KO cells (MOI = 1). i Immunoblot of RIPK1 total protein and phosphorylation levels at S161 in WT or ZBP1 KO cells. Blots for GAPDH served as loading controls. All experiments were representative of at least three independent experiments with similar results. Bars indicated the mean plus SD. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test, with P- values denoted as follows: **** P < 0.0001, *** P < 0.0005, ** P < 0.005, * P < 0.05, n.s., no significant difference. Exact P- values were provided in the source data. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: RIPK1 kinase drove brain microvascular endothelial cells death and blood-brain barrier disruption in neonatal Escherichia coli meningitis

doi: 10.1038/s41467-025-62760-4

Figure Lengend Snippet: a Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in WT, RIPK1 KO, or ZBP1 KO cells infected with RS218 (MOI = 1). b Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in hBMECs expressing RIPK1 WT or D138N under RS218 infection (MOI = 1). c Immunoblot of RIPK1 total protein and phosphorylation levels at S161 and S166 in WT, TNFR1 KO, TLR4 KO, or double KO cells post RS218 infection (MOI = 1). Blots for GAPDH served as loading controls. d Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in hBMECs expressing RIPK1 WT, S161N, or S166N cells (MOI = 1). e , f In WT, TNFR1 KO, TLR4 KO, or double KO cells, the cytotoxicity was assessed by LDH release ( e , n = 3 biologically independent samples), and cell death kinetics were measured by PI uptake ( f , n = 3 biologically independent samples). g Co-IP analysis of RIPK1 with TNFR1 and TLR4 interactions in WT and ZBP1 KO cells. h Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in WT, TNFR1 KO, TLR4 KO or double KO cells (MOI = 1). i Immunoblot of RIPK1 total protein and phosphorylation levels at S161 in WT or ZBP1 KO cells. Blots for GAPDH served as loading controls. All experiments were representative of at least three independent experiments with similar results. Bars indicated the mean plus SD. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test, with P- values denoted as follows: **** P < 0.0001, *** P < 0.0005, ** P < 0.005, * P < 0.05, n.s., no significant difference. Exact P- values were provided in the source data. Source data are provided as a Source Data file.

Techniques: Co-Immunoprecipitation Assay, Infection, Expressing, Western Blot, Phospho-proteomics

Journal: Bone Research

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: ZBP1 is upregulated during the osteogenic differentiation but downregulated during the adipogenic differentiation of mBMSCs. a RT-qPCR analysis and western blot analysis of ZBP1 expression during the osteogenic differentiation of mBMSCs. b RT-qPCR analysis and western blot analysis of ZBP1 expression during the adipogenic differentiation of mBMSCs. c IHC analysis of ZBP1 expression in the mouse femur. Macrophages (stars); osteoblasts on the trabecular bone surface (black arrows). n = 6. Bars indicate 30 μm. BM, bone marrow; TB, trabecular bone; MA, marrow adipocytes. d Semiquantitative quantification of ZBP1 immunostaining intensities in osteoblasts, osteocytes, marrow adipocytes, macrophages, and other marrow cells. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The antibodies used for the IP experiments were as follows: mouse monoclonal anti-β-catenin (BD Biosciences), polyclonal anti-mouse normal IgG (Millipore), rabbit polyclonal anti-ZBP1 (LifeSpan BioSciences), and polyclonal anti-rabbit normal IgG (Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunostaining

Journal: Bone Research

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Depletion of ZBP1 suppresses the osteogenic differentiation but promotes the adipogenic differentiation of mBMSCs. a The siRNA-mediated depletion of ZBP1 was assessed by RT-qPCR and western blot analysis. b ALP activity assays of ZBP1-depleted mBMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-depleted mBMSCs and control cells after 2 weeks of osteogenic induction. d RT-qPCR analysis of osteogenesis-related genes ( Runx2 , sp7, Ibsp , and Bglap ) in ZBP1-depleted mBMSCs and control cells. e H&E staining of transplant sections generated from ZBP1-depleted mBMSCs and control cells in HA/TCP scaffolds. Bar, 60 μm. n = 6. f , g Oil Red O staining of ZBP1-depleted mBMSCs and control cells after 3 weeks of adipogenic induction. Bar, 100 μm. h RT-qPCR analysis of adipogenic markers ( CD36, Cebpa, Lpl , and Pparg ) in ZBP1-depleted mBMSCs and control cells after 7 days of adipogenic induction. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques: Quantitative RT-PCR, Western Blot, Activity Assay, Staining, Generated

Journal: Bone Research

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Overexpression of ZBP1 enhances the osteogenic differentiation but inhibits the adipogenic differentiation of mBMSCs. a The overexpression of ZBP1 was determined by RT-qPCR and western blot analysis. b ALP activity assays of ZBP1-overexpressing mBMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-overexpressing mBMSCs and control cells after 2 weeks of osteogenic stimulation. d RT-qPCR analysis of osteogenesis-related genes ( Runx2 , sp7, Ibsp , and Bglap ) in ZBP1-overexpressing mBMSCs and control cells. e H&E staining of transplant sections generated from ZBP1-overexpressing mBMSCs and control cells in HA/TCP scaffolds. Bar, 60 μm. n = 6. f Oil Red O staining of ZBP1-overexpressing mBMSCs and control cells after 3 weeks of adipogenic stimulation. Bar, 100 μm. g RT-qPCR analysis of adipogenic markers ( CD36, Cebpa, Lpl , and Pparg ) in ZBP1-overexpressing mBMSCs and control cells after 7 days of adipogenic induction. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Activity Assay, Staining, Generated

Journal: Bone Research

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Overexpression of ZBP1 enhances the osteogenic differentiation but inhibits the adipogenic differentiation of hMSCs. a The overexpression of ZBP1 was determined by RT-qPCR and western blot analysis in hMSCs. b ALP activity assays of ZBP1-overexpressing hMSCs and control cells after 7 days of osteogenic induction. c ARS staining of ZBP1-overexpressing hMSCs and control cells after 3 weeks of osteogenic induction. d RT-qPCR analysis of osteogenic markers ( RUNX2 , SP7, COL1A1 , and SPP1 ) in ZBP1-overexpressing hMSCs and control cells. e Oil Red O staining of ZBP1-overexpressing hMSCs and control cells after 4 weeks of adipogenic induction. Bar, 100 μm. f RT-qPCR analysis of adipogenic markers ( CD36, CEBPA, LPL , and PPARG ) in ZBP1-overexpressing hMSCs and control cells after 7 days of adipogenic induction. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Activity Assay, Staining

Journal: Bone Research

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Restoring ZBP1 expression rescues the osteogenic potential of ZBP1-depleted mBMSCs. a RT-qPCR and western blot analysis of the restoration of ZBP1 in ZBP-depleted mBMSCs. ALP activity ( b ) and ECM mineralization ( c ) were rescued by restoring ZBP1 expression in ZBP-depleted mBMSCs. d The expression of osteogenic markers ( sp7, Ibsp , and Bglap ) was rescued by restoring ZBP1 expression in ZBP1-depleted mBMSCs. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay

Journal: Bone Research

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: ZBP1 is required for Wnt/β-catenin signaling. a GO analysis of genes downregulated (over 1.5-fold) in ZBP1-depleted mBMSCs. The top 8 ontology terms are shown. b GSEA of Wnt signaling-related genes in ZBP1-depleted mBMSCs compared with controls. The normalized enrichment score (NES) = −1.41, P < 0.05, and false discovery rate (FDR) < 0.05. c Topflash luciferase reporter assays of Wnt signaling activity in ZBP1-depleted mBMSCs and control cells after treatment with 100 ng·mL −1 Wnt3a for 24 h. RT-qPCR analysis of the expression of Axin2 ( d ) and Ccnd1 ( e ) in ZBP1-depleted mBMSCs and control cells after treatment with 100 ng·mL −1 Wnt3a for 4 h. f , g Endogenous IP reveals the interaction between ZBP1 and β-catenin in mBMSCs treated with Wnt3a (100 ng·mL −1 ) for 2 h. h Western blot analysis of the nuclear extract (NE) and cytoplasmic extract (CE) from ZBP1-depleted mBMSCs and control cells treated with 100 ng·mL −1 Wnt3a for 2 h. ChIP assay of the occupancy of β-catenin at the promoters of Runx2 ( i ) and Sp7 ( j ) in ZBP1-knockdown mBMSCs and control cells after treatment with 100 ng·mL −1 Wnt3a for 4 h. Arrows indicate primer annealing sites. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques: Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Bone Research

doi: 10.1038/s41413-020-0085-4

Figure Lengend Snippet: Zbp1 is a target of Wnt/β-catenin signaling. a RT-qPCR analysis of Zbp1 expression in mBMSCs treated with 100 ng·mL −1 Wnt3a for 4 h. b Western blot analysis of ZBP1 expression in mBMSCs treated with 100 ng·mL −1 Wnt3a for 24 h. c RT-qPCR analysis of Zbp1 expression in mBMSCs that were pretreated with 100 ng·mL −1 DKK1 for 2 h followed by 100 ng·mL −1 Wnt3a treatment for 2 h. d ChIP assay analysis of the occupancy of β-catenin at the promoter of Zbp1 after treatment with 100 ng·mL −1 Wnt3a for 4 h in mBMSCs. Arrows indicate primer annealing sites. WRE, Wnt/β-catenin response element. * P < 0.05; ** P < 0.01; *** P < 0.001

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Interaction of microRNA-222 (miR-222) with the zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) mRNAs. A: miR-222 and U6 RNA levels in HCT-116 cells transfected with biotinylated miR-222 for 24 h. Values are means ± SE from 3 independent experiments (n = 4). *P < 0.01 compared with cells transfected with control scramble oligomer as analyzed by one-way ANOVA followed by Duncan’s test. B: levels of ZBP1, PLCγ1, claudin-1 (CCND1), and FZD7 mRNAs in the materials pulled down by biotin-miR-222 (left) and total input mRNAs (right) in cells described in A. Fzd7 served as a positive control.

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: Interaction of microRNA-222 (miR-222) with the zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) mRNAs. A: miR-222 and U6 RNA levels in HCT-116 cells transfected with biotinylated miR-222 for 24 h. Values are means ± SE from 3 independent experiments (n = 4). *P < 0.01 compared with cells transfected with control scramble oligomer as analyzed by one-way ANOVA followed by Duncan’s test. B: levels of ZBP1, PLCγ1, claudin-1 (CCND1), and FZD7 mRNAs in the materials pulled down by biotin-miR-222 (left) and total input mRNAs (right) in cells described in A. Fzd7 served as a positive control.

Article Snippet: The affinity-purified rabbit polyclonal antibodies against ZBP1 (cat. no. 8482), PCNA (cat. no. 13110), and PLCγ1 (cat. no. 5690) were purchased from Cell Signaling Technologies (Danvers, MA), and the antibody against GAPDH (cat. no. sc-25778) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Transfection, Positive Control

Ectopically expressed microRNA-222 (miR-222) represses the expression of zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1). A: levels of miR-222 and U6 RNA 48 h after transfection with pre-miR-222 as measured by quantitative PCR analysis. Values are means ± SE from independent experiments (n = 4). *P < 0.05, compared with cells transfected with control scrambled oligomer analyzed by one-way ANOVA followed by Duncan’s test. B: immunoblots of ZBP1, PLCγ1, and PCNA proteins in HCT-116 cells described in A. Whole cell lysates were harvested and prepared for Western blotting; equal loading was monitored by assessing GAPDH levels. C: quantitative analysis of ZBP1 and PLCγ1 immunoblotting signals as measured by densitometry using Bio-Rad-XRS system equipped with Image laboratory software (version 4.1) and used “Quantity tool” to determine the band intensity volume. The values were normalized with internal loading control GAPDH. Values are means ± SE of data from 3 independent experiments (n = 3). D and E: changes in ZBP1, PLCγ1, and PCNA proteins in IEC-Cdx2L1 cells 48 h after transfection with pre-miR-222. Values are means ± SE (n = 3). Statistical test: means are compared with the scramble (cells exposed to pre-miR-222) by nonparametric comparison (*P < 0.0495, Kruskal-Wallis test).

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: Ectopically expressed microRNA-222 (miR-222) represses the expression of zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1). A: levels of miR-222 and U6 RNA 48 h after transfection with pre-miR-222 as measured by quantitative PCR analysis. Values are means ± SE from independent experiments (n = 4). *P < 0.05, compared with cells transfected with control scrambled oligomer analyzed by one-way ANOVA followed by Duncan’s test. B: immunoblots of ZBP1, PLCγ1, and PCNA proteins in HCT-116 cells described in A. Whole cell lysates were harvested and prepared for Western blotting; equal loading was monitored by assessing GAPDH levels. C: quantitative analysis of ZBP1 and PLCγ1 immunoblotting signals as measured by densitometry using Bio-Rad-XRS system equipped with Image laboratory software (version 4.1) and used “Quantity tool” to determine the band intensity volume. The values were normalized with internal loading control GAPDH. Values are means ± SE of data from 3 independent experiments (n = 3). D and E: changes in ZBP1, PLCγ1, and PCNA proteins in IEC-Cdx2L1 cells 48 h after transfection with pre-miR-222. Values are means ± SE (n = 3). Statistical test: means are compared with the scramble (cells exposed to pre-miR-222) by nonparametric comparison (*P < 0.0495, Kruskal-Wallis test).

Techniques: Expressing, Binding Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Software

microRNA-222 (miR-222) overexpression destabilizes the zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) mRNAs. A: levels of the ZBP1 and PLCγ1 mRNAs in cells transfected with pre-miR-222 for 48 h. JunD served as a negative control. Values are the means ± SE from independent experiments (n = 3). *P < 0.01, compared with cells transfected with control scramble oligomer as analyzed by one-way ANOVA followed by Duncan’s test. B–D: half-lives of the ZBP1, PLCγ1, and GAPDH mRNA in cells described in A. Total cellular RNA was isolated at indicated times after administration of actinomycin D (5 μg/ml), and the levels of ZBP1, PLCγ1, and GAPDH mRNAs were measured by quantitative PCR analysis. GAPDH mRNA served as a control. *P < 0.05, compared with cells transfected with C-oligo as analyzed by one-way ANOVA followed by Duncan’s test.

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: microRNA-222 (miR-222) overexpression destabilizes the zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) mRNAs. A: levels of the ZBP1 and PLCγ1 mRNAs in cells transfected with pre-miR-222 for 48 h. JunD served as a negative control. Values are the means ± SE from independent experiments (n = 3). *P < 0.01, compared with cells transfected with control scramble oligomer as analyzed by one-way ANOVA followed by Duncan’s test. B–D: half-lives of the ZBP1, PLCγ1, and GAPDH mRNA in cells described in A. Total cellular RNA was isolated at indicated times after administration of actinomycin D (5 μg/ml), and the levels of ZBP1, PLCγ1, and GAPDH mRNAs were measured by quantitative PCR analysis. GAPDH mRNA served as a control. *P < 0.05, compared with cells transfected with C-oligo as analyzed by one-way ANOVA followed by Duncan’s test.

Techniques: Over Expression, Binding Assay, Transfection, Negative Control, Isolation, Real-time Polymerase Chain Reaction

microRNA-222 (miR-222) silencing enhances the expression of zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1). A: levels of miR-222 and U6 RNA 48 h after transfection with anti-miR-222. Values are the means ± SE from 3 independent experiments (n = 4). *P < 0.05, compared with cells transfected with control oligomer (C-oligo). B: immunoblots of ZBP1, PLCγ, and PCNA proteins in cells described in A. C: quantitative analysis of ZBP1 and PLCγ1 immunoblotting signals by densitometry using Bio-Rad-XRS system equipped with Image laboratory software (version 4.1) and used “Quantity tool” to determine the band intensity volume. The values were normalized with internal loading control GAPDH. Values are means ± SE of data from 3 independent experiments (n = 3). Statistical test: means are compared with the scramble (cells exposed to Anti-miR-222) by nonparametric comparison (*P < 0.0495, Kruskal-Wallis test).

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: microRNA-222 (miR-222) silencing enhances the expression of zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1). A: levels of miR-222 and U6 RNA 48 h after transfection with anti-miR-222. Values are the means ± SE from 3 independent experiments (n = 4). *P < 0.05, compared with cells transfected with control oligomer (C-oligo). B: immunoblots of ZBP1, PLCγ, and PCNA proteins in cells described in A. C: quantitative analysis of ZBP1 and PLCγ1 immunoblotting signals by densitometry using Bio-Rad-XRS system equipped with Image laboratory software (version 4.1) and used “Quantity tool” to determine the band intensity volume. The values were normalized with internal loading control GAPDH. Values are means ± SE of data from 3 independent experiments (n = 3). Statistical test: means are compared with the scramble (cells exposed to Anti-miR-222) by nonparametric comparison (*P < 0.0495, Kruskal-Wallis test).

Techniques: Expressing, Binding Assay, Transfection, Western Blot, Software

microRNA-222 (miR-222) silencing increases the stability of the zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) mRNAs. A: levels of the ZBP1 and PLCγ1 mRNAs in cells transfected with anti-miR-222 for 48 h. JunD served as a negative control. Values are the means ± SE from independent experiments (n = 4). *P < 0.01, compared with cells transfected with C-oligo. B–D: half-lives of the ZBP1, PLCγ1, and GAPDH mRNA in cells described in A. Total cellular RNA was isolated at indicated times after administration of actinomycin D (5 μg/ml), and the levels of ZBP1, PLCγ1, and GAPDH mRNAs were measured by quantitative PCR analysis. GAPDH mRNA served as a control. *P < 0.05, compared with cells transfected with C-oligo as analyzed by one-way ANOVA followed by Duncan’s test.

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: microRNA-222 (miR-222) silencing increases the stability of the zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) mRNAs. A: levels of the ZBP1 and PLCγ1 mRNAs in cells transfected with anti-miR-222 for 48 h. JunD served as a negative control. Values are the means ± SE from independent experiments (n = 4). *P < 0.01, compared with cells transfected with C-oligo. B–D: half-lives of the ZBP1, PLCγ1, and GAPDH mRNA in cells described in A. Total cellular RNA was isolated at indicated times after administration of actinomycin D (5 μg/ml), and the levels of ZBP1, PLCγ1, and GAPDH mRNAs were measured by quantitative PCR analysis. GAPDH mRNA served as a control. *P < 0.05, compared with cells transfected with C-oligo as analyzed by one-way ANOVA followed by Duncan’s test.

Techniques: Binding Assay, Transfection, Negative Control, Isolation, Real-time Polymerase Chain Reaction

microRNA-222 (miR-222)-regulated expression of zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) modulates rapid epithelial restitution after wounding. A: images of cell migration: a, 0 h after wounding in control cells (Con-0 h); b, 6 h after wounding in control cells (Con-6 h); c, 6 h after wounding in cells transfected with scramble oligo (C-oligo-6 h); d, 6 h after wounding in cells transfected with pre-miR-222 alone for 48 h (Pre-miR-222–6 h); e, 6 h after wounding in cells cotransfected with pre-miR-222 and the expression vector encoding ZBP1 (Pre-miR222 + ZBP1); f, 6 h after wounding in cells cotransfected with pre-miR-222 and the expression vector encoding PLCγ1 (Pre-miR222+ PLCγ1). Scale bar, 100 μm; magnification, ×100. B: summarized data showing rates of cell migration 6 h after wounding in cells described in A. Values are the means ± SE of data from 6 dishes and repeated 4 times independently (n = 4). *,+P < 0.05, compared with cells transfected with scramble and cells transfected with pre-miR-222, respectively as analyzed by one-way ANOVA followed by Duncan’s test. C: images of cell migration: a: 0 h after wounding in control cells; b: 6 h after wounding in control cells; c, 6 h after wounding in cells transfected with scramble oligo; d: 6 h after wounding in cells transfected with anti-miR-222 alone for 48 h; e: 6 h after wounding in cells cotransfected with anti-miR-222 and siZBP1 (Anti-miR222 + siZBP1); f: 6 h after wounding in cells cotransfected with anti-miR-222 and siPLCγ1 (Anti-miR222 + siPLCγ1); and g: 6 h after wounding in cells cotransfected with anti-miR-222, siZBP1 and siPLCγ1 (anti-miR222 + siZBP1 + siPLCγ1). Scale bar, 100 μm; magnification, ×100. D: summarized data showing rates of cell migration in cells described in C. Values are the means ± SE of data from 6 dishes and repeated four times independently (n = 4). *,+P < 0.05, compared with cells transfected with scramble and cells transfected with Anti-miR-222, respectively as analyzed by one-way ANOVA followed by Duncan’s test. E: immunoblot of PCNA protein in nonwounding and 0 h and 6 h after wounding (#1 and #2). Whole cell lysates were harvested and prepared for Western blotting; equal loading was monitored by assessing GAPDH levels.

Journal: American Journal of Physiology - Cell Physiology

Article Title: miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells

doi: 10.1152/ajpcell.00165.2018

Figure Lengend Snippet: microRNA-222 (miR-222)-regulated expression of zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) modulates rapid epithelial restitution after wounding. A: images of cell migration: a, 0 h after wounding in control cells (Con-0 h); b, 6 h after wounding in control cells (Con-6 h); c, 6 h after wounding in cells transfected with scramble oligo (C-oligo-6 h); d, 6 h after wounding in cells transfected with pre-miR-222 alone for 48 h (Pre-miR-222–6 h); e, 6 h after wounding in cells cotransfected with pre-miR-222 and the expression vector encoding ZBP1 (Pre-miR222 + ZBP1); f, 6 h after wounding in cells cotransfected with pre-miR-222 and the expression vector encoding PLCγ1 (Pre-miR222+ PLCγ1). Scale bar, 100 μm; magnification, ×100. B: summarized data showing rates of cell migration 6 h after wounding in cells described in A. Values are the means ± SE of data from 6 dishes and repeated 4 times independently (n = 4). *,+P < 0.05, compared with cells transfected with scramble and cells transfected with pre-miR-222, respectively as analyzed by one-way ANOVA followed by Duncan’s test. C: images of cell migration: a: 0 h after wounding in control cells; b: 6 h after wounding in control cells; c, 6 h after wounding in cells transfected with scramble oligo; d: 6 h after wounding in cells transfected with anti-miR-222 alone for 48 h; e: 6 h after wounding in cells cotransfected with anti-miR-222 and siZBP1 (Anti-miR222 + siZBP1); f: 6 h after wounding in cells cotransfected with anti-miR-222 and siPLCγ1 (Anti-miR222 + siPLCγ1); and g: 6 h after wounding in cells cotransfected with anti-miR-222, siZBP1 and siPLCγ1 (anti-miR222 + siZBP1 + siPLCγ1). Scale bar, 100 μm; magnification, ×100. D: summarized data showing rates of cell migration in cells described in C. Values are the means ± SE of data from 6 dishes and repeated four times independently (n = 4). *,+P < 0.05, compared with cells transfected with scramble and cells transfected with Anti-miR-222, respectively as analyzed by one-way ANOVA followed by Duncan’s test. E: immunoblot of PCNA protein in nonwounding and 0 h and 6 h after wounding (#1 and #2). Whole cell lysates were harvested and prepared for Western blotting; equal loading was monitored by assessing GAPDH levels.

Techniques: Expressing, Binding Assay, Migration, Transfection, Plasmid Preparation, Western Blot

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