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High glucose induces cAMP-responsive element-binding protein 3 Like 3 expression and the epithelial-mesenchymal transition in SV-HUC-1 cells. A: Cell Counting Kit-8 assay was employed to assess the viability of SV-HUC-1 cells following treatment with varying concentrations of glucose; B: Relative mRNA expression of cAMP-responsive element-binding protein 3 Like 3 <t>(CREB3</t> L3) in SV-HUC-1 cells after treating with different levels of glucose, as detected by quantitative PCR; C: Relative mRNA expression of CREB3 L3 in SV-HUC-1 cells with or without silencing of CREB3 L; D: SV-HUC-1 cells were treated with 5 mmol/L and 15 mmol/L glucose for 72 hours with and without CREB3 L3 knockdown (KD). Protein expression of CREB3 L3, C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, and occludin in SV-HUC-1 cells in the control (Con), high glucose (HG), HG + small interfering RNA negative control (si-NC), and HG + KD group. Each group contained three biological replicates. a P < 0.05 vs 5 mmol/L group, b P < 0.01 vs 5 mmol/L group; c P < 0.001 vs 5 mmol/L group, d P < 0.05 vs si-NC group, e P < 0.01 vs si-NC group, f P < 0.001 vs si-NC group, g P < 0.01 vs Con group, h P < 0.001 vs Con group, i P < 0.01 vs HG + si-NC group, j P < 0.001 vs HG + si-NC group.
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High glucose induces cAMP-responsive element-binding protein 3 Like 3 expression and the epithelial-mesenchymal transition in SV-HUC-1 cells. A: Cell Counting Kit-8 assay was employed to assess the viability of SV-HUC-1 cells following treatment with varying concentrations of glucose; B: Relative mRNA expression of cAMP-responsive element-binding protein 3 Like 3 <t>(CREB3</t> L3) in SV-HUC-1 cells after treating with different levels of glucose, as detected by quantitative PCR; C: Relative mRNA expression of CREB3 L3 in SV-HUC-1 cells with or without silencing of CREB3 L; D: SV-HUC-1 cells were treated with 5 mmol/L and 15 mmol/L glucose for 72 hours with and without CREB3 L3 knockdown (KD). Protein expression of CREB3 L3, C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, and occludin in SV-HUC-1 cells in the control (Con), high glucose (HG), HG + small interfering RNA negative control (si-NC), and HG + KD group. Each group contained three biological replicates. a P < 0.05 vs 5 mmol/L group, b P < 0.01 vs 5 mmol/L group; c P < 0.001 vs 5 mmol/L group, d P < 0.05 vs si-NC group, e P < 0.01 vs si-NC group, f P < 0.001 vs si-NC group, g P < 0.01 vs Con group, h P < 0.001 vs Con group, i P < 0.01 vs HG + si-NC group, j P < 0.001 vs HG + si-NC group.
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High glucose induces cAMP-responsive element-binding protein 3 Like 3 expression and the epithelial-mesenchymal transition in SV-HUC-1 cells. A: Cell Counting Kit-8 assay was employed to assess the viability of SV-HUC-1 cells following treatment with varying concentrations of glucose; B: Relative mRNA expression of cAMP-responsive element-binding protein 3 Like 3 <t>(CREB3</t> L3) in SV-HUC-1 cells after treating with different levels of glucose, as detected by quantitative PCR; C: Relative mRNA expression of CREB3 L3 in SV-HUC-1 cells with or without silencing of CREB3 L; D: SV-HUC-1 cells were treated with 5 mmol/L and 15 mmol/L glucose for 72 hours with and without CREB3 L3 knockdown (KD). Protein expression of CREB3 L3, C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, and occludin in SV-HUC-1 cells in the control (Con), high glucose (HG), HG + small interfering RNA negative control (si-NC), and HG + KD group. Each group contained three biological replicates. a P < 0.05 vs 5 mmol/L group, b P < 0.01 vs 5 mmol/L group; c P < 0.001 vs 5 mmol/L group, d P < 0.05 vs si-NC group, e P < 0.01 vs si-NC group, f P < 0.001 vs si-NC group, g P < 0.01 vs Con group, h P < 0.001 vs Con group, i P < 0.01 vs HG + si-NC group, j P < 0.001 vs HG + si-NC group.
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High glucose induces cAMP-responsive element-binding protein 3 Like 3 expression and the epithelial-mesenchymal transition in SV-HUC-1 cells. A: Cell Counting Kit-8 assay was employed to assess the viability of SV-HUC-1 cells following treatment with varying concentrations of glucose; B: Relative mRNA expression of cAMP-responsive element-binding protein 3 Like 3 <t>(CREB3</t> L3) in SV-HUC-1 cells after treating with different levels of glucose, as detected by quantitative PCR; C: Relative mRNA expression of CREB3 L3 in SV-HUC-1 cells with or without silencing of CREB3 L; D: SV-HUC-1 cells were treated with 5 mmol/L and 15 mmol/L glucose for 72 hours with and without CREB3 L3 knockdown (KD). Protein expression of CREB3 L3, C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, and occludin in SV-HUC-1 cells in the control (Con), high glucose (HG), HG + small interfering RNA negative control (si-NC), and HG + KD group. Each group contained three biological replicates. a P < 0.05 vs 5 mmol/L group, b P < 0.01 vs 5 mmol/L group; c P < 0.001 vs 5 mmol/L group, d P < 0.05 vs si-NC group, e P < 0.01 vs si-NC group, f P < 0.001 vs si-NC group, g P < 0.01 vs Con group, h P < 0.001 vs Con group, i P < 0.01 vs HG + si-NC group, j P < 0.001 vs HG + si-NC group.
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The Ferroptosis-Related Differentially Expressed Genes (FRDEGs) in Degenerated Annulus Fibrosus
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High glucose induces cAMP-responsive element-binding protein 3 Like 3 expression and the epithelial-mesenchymal transition in SV-HUC-1 cells. A: Cell Counting Kit-8 assay was employed to assess the viability of SV-HUC-1 cells following treatment with varying concentrations of glucose; B: Relative mRNA expression of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3) in SV-HUC-1 cells after treating with different levels of glucose, as detected by quantitative PCR; C: Relative mRNA expression of CREB3 L3 in SV-HUC-1 cells with or without silencing of CREB3 L; D: SV-HUC-1 cells were treated with 5 mmol/L and 15 mmol/L glucose for 72 hours with and without CREB3 L3 knockdown (KD). Protein expression of CREB3 L3, C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, and occludin in SV-HUC-1 cells in the control (Con), high glucose (HG), HG + small interfering RNA negative control (si-NC), and HG + KD group. Each group contained three biological replicates. a P < 0.05 vs 5 mmol/L group, b P < 0.01 vs 5 mmol/L group; c P < 0.001 vs 5 mmol/L group, d P < 0.05 vs si-NC group, e P < 0.01 vs si-NC group, f P < 0.001 vs si-NC group, g P < 0.01 vs Con group, h P < 0.001 vs Con group, i P < 0.01 vs HG + si-NC group, j P < 0.001 vs HG + si-NC group.

Journal: World Journal of Diabetes

Article Title: Hyperglycemia-induced overexpression of CREB3 L3 promotes the epithelial-to-mesenchymal transition in bladder urothelial cells in diabetes mellitus

doi: 10.4239/wjd.v16.i8.108101

Figure Lengend Snippet: High glucose induces cAMP-responsive element-binding protein 3 Like 3 expression and the epithelial-mesenchymal transition in SV-HUC-1 cells. A: Cell Counting Kit-8 assay was employed to assess the viability of SV-HUC-1 cells following treatment with varying concentrations of glucose; B: Relative mRNA expression of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3) in SV-HUC-1 cells after treating with different levels of glucose, as detected by quantitative PCR; C: Relative mRNA expression of CREB3 L3 in SV-HUC-1 cells with or without silencing of CREB3 L; D: SV-HUC-1 cells were treated with 5 mmol/L and 15 mmol/L glucose for 72 hours with and without CREB3 L3 knockdown (KD). Protein expression of CREB3 L3, C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, and occludin in SV-HUC-1 cells in the control (Con), high glucose (HG), HG + small interfering RNA negative control (si-NC), and HG + KD group. Each group contained three biological replicates. a P < 0.05 vs 5 mmol/L group, b P < 0.01 vs 5 mmol/L group; c P < 0.001 vs 5 mmol/L group, d P < 0.05 vs si-NC group, e P < 0.01 vs si-NC group, f P < 0.001 vs si-NC group, g P < 0.01 vs Con group, h P < 0.001 vs Con group, i P < 0.01 vs HG + si-NC group, j P < 0.001 vs HG + si-NC group.

Article Snippet: CREB3 L3 , Biorbyt , orb382910 , 49 , Rabbit , 1:500.

Techniques: Binding Assay, Expressing, Cell Counting, Real-time Polymerase Chain Reaction, Knockdown, Control, Small Interfering RNA, Negative Control

Immunofluorescence of cAMP-responsive element-binding protein 3 Like 3, C-reactive protein, E-cadherin, and occludin in urothelial cells under high glucose condition. SV-HUC-1 cells were treated with 5 mmol/L and 15 mmol/L glucose for 72 hours with and without cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3) knockdown (KD). Immunofluorescence assay was conducted to detect the expression levels of CREB3 L3, C-reactive protein (CRP), E-cadherin, and occludin protein in SV-HUC-1 cells in the control (Con), high glucose (HG), HG + small interfering RNA negative control (si-NC), and HG + KD groups. Scale bar indicates 50 μm, and each group contained three replicates. a P < 0.01 vs Con group, b P < 0.001 vs Con group, c P < 0.05 vs HG + si-NC group, d P < 0.01 vs HG + si-NC group, e P < 0.001 vs HG + si-NC group.

Journal: World Journal of Diabetes

Article Title: Hyperglycemia-induced overexpression of CREB3 L3 promotes the epithelial-to-mesenchymal transition in bladder urothelial cells in diabetes mellitus

doi: 10.4239/wjd.v16.i8.108101

Figure Lengend Snippet: Immunofluorescence of cAMP-responsive element-binding protein 3 Like 3, C-reactive protein, E-cadherin, and occludin in urothelial cells under high glucose condition. SV-HUC-1 cells were treated with 5 mmol/L and 15 mmol/L glucose for 72 hours with and without cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3) knockdown (KD). Immunofluorescence assay was conducted to detect the expression levels of CREB3 L3, C-reactive protein (CRP), E-cadherin, and occludin protein in SV-HUC-1 cells in the control (Con), high glucose (HG), HG + small interfering RNA negative control (si-NC), and HG + KD groups. Scale bar indicates 50 μm, and each group contained three replicates. a P < 0.01 vs Con group, b P < 0.001 vs Con group, c P < 0.05 vs HG + si-NC group, d P < 0.01 vs HG + si-NC group, e P < 0.001 vs HG + si-NC group.

Article Snippet: CREB3 L3 , Biorbyt , orb382910 , 49 , Rabbit , 1:500.

Techniques: Immunofluorescence, Binding Assay, Knockdown, Expressing, Control, Small Interfering RNA, Negative Control

cAMP-responsive element-binding protein 3 Like 3 upregulates C-reactive protein expression, induces the epithelial-mesenchymal transition, and impairs cellular tight junctions in the diabetic cystopathy rat model. A: Western blot analysis of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, claudin 1, and occludin in the bladder urothelial tissues from each group; B: Immunofluorescence analysis of the expression of CREB3 L3, N-cadherin, E-cadherin, claudin 1, and occludin proteins in the bladder urothelial tissues from each group. Each group contained three biological replicates. Scale bar indicates 50 μm. a P < 0.05 vs negative control (NC) group, b P < 0.01 vs NC group, c P < 0.001 vs NC group; d P < 0.05 vs diabetes mellitus (DM) group, e P < 0.01 vs DM group, f P < 0.001 vs DM group.

Journal: World Journal of Diabetes

Article Title: Hyperglycemia-induced overexpression of CREB3 L3 promotes the epithelial-to-mesenchymal transition in bladder urothelial cells in diabetes mellitus

doi: 10.4239/wjd.v16.i8.108101

Figure Lengend Snippet: cAMP-responsive element-binding protein 3 Like 3 upregulates C-reactive protein expression, induces the epithelial-mesenchymal transition, and impairs cellular tight junctions in the diabetic cystopathy rat model. A: Western blot analysis of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, claudin 1, and occludin in the bladder urothelial tissues from each group; B: Immunofluorescence analysis of the expression of CREB3 L3, N-cadherin, E-cadherin, claudin 1, and occludin proteins in the bladder urothelial tissues from each group. Each group contained three biological replicates. Scale bar indicates 50 μm. a P < 0.05 vs negative control (NC) group, b P < 0.01 vs NC group, c P < 0.001 vs NC group; d P < 0.05 vs diabetes mellitus (DM) group, e P < 0.01 vs DM group, f P < 0.001 vs DM group.

Article Snippet: CREB3 L3 , Biorbyt , orb382910 , 49 , Rabbit , 1:500.

Techniques: Binding Assay, Expressing, Western Blot, Immunofluorescence, Negative Control

Establishment of diabetic cystopathy rat models for 8 weeks. A: At the end of the 8 weeks, the body weight, blood glucose and bladder weight from rats were measured; B: Hematoxylin and eosin staining of bladder tissues of the rats at the end of the 8 weeks, and each group contained three replicates; C: Western blotting detected the expression of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, claudin 1, and occludin in the bladder urothelial tissues of the rats at the end of 8 weeks. Each group contained three replicates. a P < 0.05 vs negative control (NC) group, b P < 0.01 vs NC group, c P < 0.001 vs NC group; d P < 0.05 vs diabetes mellitus (DM) group, e P < 0.01 vs DM group, f P < 0.001 vs DM group.

Journal: World Journal of Diabetes

Article Title: Hyperglycemia-induced overexpression of CREB3 L3 promotes the epithelial-to-mesenchymal transition in bladder urothelial cells in diabetes mellitus

doi: 10.4239/wjd.v16.i8.108101

Figure Lengend Snippet: Establishment of diabetic cystopathy rat models for 8 weeks. A: At the end of the 8 weeks, the body weight, blood glucose and bladder weight from rats were measured; B: Hematoxylin and eosin staining of bladder tissues of the rats at the end of the 8 weeks, and each group contained three replicates; C: Western blotting detected the expression of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, claudin 1, and occludin in the bladder urothelial tissues of the rats at the end of 8 weeks. Each group contained three replicates. a P < 0.05 vs negative control (NC) group, b P < 0.01 vs NC group, c P < 0.001 vs NC group; d P < 0.05 vs diabetes mellitus (DM) group, e P < 0.01 vs DM group, f P < 0.001 vs DM group.

Article Snippet: CREB3 L3 , Biorbyt , orb382910 , 49 , Rabbit , 1:500.

Techniques: Staining, Western Blot, Expressing, Binding Assay, Negative Control

cAMP-responsive element-binding protein 3 Like 3 continuously induces the epithelial-mesenchymal transition and impairs cellular tight junctions with the progression of diabetic cystopathy. At the end of 8 weeks, immunofluorescence staining of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), N-cadherin, E-cadherin, claudin 1, and occludin proteins in the bladder urothelial tissues from each group. Each group contained three replicates. Scale bar indicates 50 μm. a P < 0.05 vs negative control (NC) group, b P < 0.01 vs NC group, c P < 0.001 vs NC group, d P < 0.0001 vs NC group; e P < 0.05 vs diabetes mellitus (DM) group, f P < 0.01 vs DM group, g P < 0.001 vs DM group.

Journal: World Journal of Diabetes

Article Title: Hyperglycemia-induced overexpression of CREB3 L3 promotes the epithelial-to-mesenchymal transition in bladder urothelial cells in diabetes mellitus

doi: 10.4239/wjd.v16.i8.108101

Figure Lengend Snippet: cAMP-responsive element-binding protein 3 Like 3 continuously induces the epithelial-mesenchymal transition and impairs cellular tight junctions with the progression of diabetic cystopathy. At the end of 8 weeks, immunofluorescence staining of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), N-cadherin, E-cadherin, claudin 1, and occludin proteins in the bladder urothelial tissues from each group. Each group contained three replicates. Scale bar indicates 50 μm. a P < 0.05 vs negative control (NC) group, b P < 0.01 vs NC group, c P < 0.001 vs NC group, d P < 0.0001 vs NC group; e P < 0.05 vs diabetes mellitus (DM) group, f P < 0.01 vs DM group, g P < 0.001 vs DM group.

Article Snippet: CREB3 L3 , Biorbyt , orb382910 , 49 , Rabbit , 1:500.

Techniques: Binding Assay, Immunofluorescence, Staining, Negative Control

Establishment of diabetic cystopathy models of rats for 12 weeks. A: At the end of the 12 weeks, the body weight, blood glucose and bladder weight from rats were measured; B: Hematoxylin and eosin staining of bladder tissues from rats at the end of the 8 and 12 weeks; C: Western blotting detected the expression of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, claudin 1, and occludin proteins in the bladder urothelial tissues from each group at the end of 12 weeks. Each group contained three replicates. a P < 0.01 vs negative control (NC) group, b P < 0.001 vs NC group; c P < 0.01 vs diabetes mellitus (DM) group, d P < 0.001 vs DM group.

Journal: World Journal of Diabetes

Article Title: Hyperglycemia-induced overexpression of CREB3 L3 promotes the epithelial-to-mesenchymal transition in bladder urothelial cells in diabetes mellitus

doi: 10.4239/wjd.v16.i8.108101

Figure Lengend Snippet: Establishment of diabetic cystopathy models of rats for 12 weeks. A: At the end of the 12 weeks, the body weight, blood glucose and bladder weight from rats were measured; B: Hematoxylin and eosin staining of bladder tissues from rats at the end of the 8 and 12 weeks; C: Western blotting detected the expression of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), C-reactive protein (CRP), vimentin, N-cadherin, E-cadherin, claudin 1, and occludin proteins in the bladder urothelial tissues from each group at the end of 12 weeks. Each group contained three replicates. a P < 0.01 vs negative control (NC) group, b P < 0.001 vs NC group; c P < 0.01 vs diabetes mellitus (DM) group, d P < 0.001 vs DM group.

Article Snippet: CREB3 L3 , Biorbyt , orb382910 , 49 , Rabbit , 1:500.

Techniques: Staining, Western Blot, Expressing, Binding Assay, Negative Control

cAMP-responsive element-binding protein 3 Like 3 results in the epithelial-mesenchymal transition and impairs cellular tight junctions in the late stage of diabetic cystopathy. At the end of 12 weeks, immunofluorescence staining of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), N-cadherin, E-cadherin, claudin 1, and occludin proteins in the bladder urothelial tissues from each group. Each group contained three biological replicates. Scale bar indicates 50 μm. a P < 0.01 vs negative control (NC) group, b P < 0.001 vs NC group, c P < 0.0001 vs NC group; d P < 0.05 vs diabetes mellitus (DM) group, e P < 0.01 vs DM group.

Journal: World Journal of Diabetes

Article Title: Hyperglycemia-induced overexpression of CREB3 L3 promotes the epithelial-to-mesenchymal transition in bladder urothelial cells in diabetes mellitus

doi: 10.4239/wjd.v16.i8.108101

Figure Lengend Snippet: cAMP-responsive element-binding protein 3 Like 3 results in the epithelial-mesenchymal transition and impairs cellular tight junctions in the late stage of diabetic cystopathy. At the end of 12 weeks, immunofluorescence staining of cAMP-responsive element-binding protein 3 Like 3 (CREB3 L3), N-cadherin, E-cadherin, claudin 1, and occludin proteins in the bladder urothelial tissues from each group. Each group contained three biological replicates. Scale bar indicates 50 μm. a P < 0.01 vs negative control (NC) group, b P < 0.001 vs NC group, c P < 0.0001 vs NC group; d P < 0.05 vs diabetes mellitus (DM) group, e P < 0.01 vs DM group.

Article Snippet: CREB3 L3 , Biorbyt , orb382910 , 49 , Rabbit , 1:500.

Techniques: Binding Assay, Immunofluorescence, Staining, Negative Control

The Ferroptosis-Related Differentially Expressed Genes (FRDEGs) in Degenerated Annulus Fibrosus

Journal: Journal of Inflammation Research

Article Title: MUC1 and CREB3 are Hub Ferroptosis Suppressors for Nucleus Pulposus and Annulus Fibrosus Degeneration by Integrated Bioinformatics and Experimental Verification

doi: 10.2147/JIR.S489052

Figure Lengend Snippet: The Ferroptosis-Related Differentially Expressed Genes (FRDEGs) in Degenerated Annulus Fibrosus

Article Snippet: Cells were fixed with 4% paraformaldehyde solution, then permeabilized with 0.1% Triton-X100 solution for 30 min. After blocking with 1% goat serum, the cells were incubated overnight at 4°C with rabbit-derived MUC1 (Abcam, ab109185) and CREB3 (Proteintech, 11275-1-AP) antibody (diluted in 1% BSA-PBS at a dilution ratio of 1:200).

Techniques: Expressing, Marker

The expression level of each hub gene in the validation set. ( A ) Principal component analysis (PCA) of all the nucleus pulposus (NP) samples in GSE167199, GSE186542, and GSE244889. The expression of AKR1C1 ( B ), AKR1C3 ( C ), MUC1 ( D ), and ENPP2 ( E ) in NP samples. The expression of SCP2 ( F ), ABCC1 ( G ), KLF2 ( H ), IDO1 ( I ), and CREB3 ( J ) in annulus fibrosus samples. *p < 0.05; ns, nonstatistical significance.

Journal: Journal of Inflammation Research

Article Title: MUC1 and CREB3 are Hub Ferroptosis Suppressors for Nucleus Pulposus and Annulus Fibrosus Degeneration by Integrated Bioinformatics and Experimental Verification

doi: 10.2147/JIR.S489052

Figure Lengend Snippet: The expression level of each hub gene in the validation set. ( A ) Principal component analysis (PCA) of all the nucleus pulposus (NP) samples in GSE167199, GSE186542, and GSE244889. The expression of AKR1C1 ( B ), AKR1C3 ( C ), MUC1 ( D ), and ENPP2 ( E ) in NP samples. The expression of SCP2 ( F ), ABCC1 ( G ), KLF2 ( H ), IDO1 ( I ), and CREB3 ( J ) in annulus fibrosus samples. *p < 0.05; ns, nonstatistical significance.

Article Snippet: Cells were fixed with 4% paraformaldehyde solution, then permeabilized with 0.1% Triton-X100 solution for 30 min. After blocking with 1% goat serum, the cells were incubated overnight at 4°C with rabbit-derived MUC1 (Abcam, ab109185) and CREB3 (Proteintech, 11275-1-AP) antibody (diluted in 1% BSA-PBS at a dilution ratio of 1:200).

Techniques: Expressing, Biomarker Discovery

Single-cell RNC-seq analysis of the annulus fibrosus (AF) samples. ( A ) Visualization of clustering by TSNE plot using 0.5 resolution. ( B ) Bubble plot showing the expression of the ACAN, SOX9, and COL1A1 related different clusters. ( C ) Six cell types were identified, including AF cell (AFC), fibroblast, macrophage, muscle stem cell (MSC), nucleus pulposus cell (NPC), and T cell. ( D ) Seven subclusters for the AF cells were identified using 0.4 resolution. ( E-G ) Monocle pseudotime trajectory showing the progression of seven subclusters of AF cells. ( H ) TSNE image of CREB3 in subcluster 3 and 5, showing the high expression in cluster 3.

Journal: Journal of Inflammation Research

Article Title: MUC1 and CREB3 are Hub Ferroptosis Suppressors for Nucleus Pulposus and Annulus Fibrosus Degeneration by Integrated Bioinformatics and Experimental Verification

doi: 10.2147/JIR.S489052

Figure Lengend Snippet: Single-cell RNC-seq analysis of the annulus fibrosus (AF) samples. ( A ) Visualization of clustering by TSNE plot using 0.5 resolution. ( B ) Bubble plot showing the expression of the ACAN, SOX9, and COL1A1 related different clusters. ( C ) Six cell types were identified, including AF cell (AFC), fibroblast, macrophage, muscle stem cell (MSC), nucleus pulposus cell (NPC), and T cell. ( D ) Seven subclusters for the AF cells were identified using 0.4 resolution. ( E-G ) Monocle pseudotime trajectory showing the progression of seven subclusters of AF cells. ( H ) TSNE image of CREB3 in subcluster 3 and 5, showing the high expression in cluster 3.

Article Snippet: Cells were fixed with 4% paraformaldehyde solution, then permeabilized with 0.1% Triton-X100 solution for 30 min. After blocking with 1% goat serum, the cells were incubated overnight at 4°C with rabbit-derived MUC1 (Abcam, ab109185) and CREB3 (Proteintech, 11275-1-AP) antibody (diluted in 1% BSA-PBS at a dilution ratio of 1:200).

Techniques: Expressing

Experimental validations of MUC1 and CREB3 in nucleus pulposus (NP) and annulus fibrosus (AF). ( A and B ) Immunohistochemical results demonstrated that the expression of MUC1 was decreased in degenerated NP tissues. ( C and D ) Immunohistochemical results showed that the expression of MUC1 decreased in degenerated rat NP tissues. ( E ) Immunofluorescence detection of protein expression of MUC1 in NP cells, scale bar=50 μm. ( F and G ) Immunohistochemical results of the expression of CREB3 in degenerated rat AF tissues. The Fe 2+ ( H and J , scale bar = 20μm) and ROS ( H and K , scale bar = 100μm) intensity was increased in TBHP-treated AF cells, and mitochondrial morphology detection by transmission electron microscopy (TEM) ( I , scale bar = 1000 nm, the red arrows indicate mitochondria). ( L ) Immunofluorescence detection of protein expression of CREB3 in AF cells, scale bar=50 μm. ***p < 0.001.

Journal: Journal of Inflammation Research

Article Title: MUC1 and CREB3 are Hub Ferroptosis Suppressors for Nucleus Pulposus and Annulus Fibrosus Degeneration by Integrated Bioinformatics and Experimental Verification

doi: 10.2147/JIR.S489052

Figure Lengend Snippet: Experimental validations of MUC1 and CREB3 in nucleus pulposus (NP) and annulus fibrosus (AF). ( A and B ) Immunohistochemical results demonstrated that the expression of MUC1 was decreased in degenerated NP tissues. ( C and D ) Immunohistochemical results showed that the expression of MUC1 decreased in degenerated rat NP tissues. ( E ) Immunofluorescence detection of protein expression of MUC1 in NP cells, scale bar=50 μm. ( F and G ) Immunohistochemical results of the expression of CREB3 in degenerated rat AF tissues. The Fe 2+ ( H and J , scale bar = 20μm) and ROS ( H and K , scale bar = 100μm) intensity was increased in TBHP-treated AF cells, and mitochondrial morphology detection by transmission electron microscopy (TEM) ( I , scale bar = 1000 nm, the red arrows indicate mitochondria). ( L ) Immunofluorescence detection of protein expression of CREB3 in AF cells, scale bar=50 μm. ***p < 0.001.

Article Snippet: Cells were fixed with 4% paraformaldehyde solution, then permeabilized with 0.1% Triton-X100 solution for 30 min. After blocking with 1% goat serum, the cells were incubated overnight at 4°C with rabbit-derived MUC1 (Abcam, ab109185) and CREB3 (Proteintech, 11275-1-AP) antibody (diluted in 1% BSA-PBS at a dilution ratio of 1:200).

Techniques: Immunohistochemical staining, Expressing, Immunofluorescence, Transmission Assay, Electron Microscopy