Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Proprotein convertase subtilisin/kexin type 9 contributes to cisplatin-induced acute kidney injury by interacting with cyclase-associated protein 1 to promote megalin lysosomal degradation.

doi: 10.1016/j.bbamcr.2025.119984

Figure Lengend Snippet: Fig. 5. PCSK9 binds CAP1 to promote megalin lysosomal degradation. Co-immunoprecipitation of (A) PCSK9, (B) megalin, and (C) CAP1 from HK2 cells lysate. (D) CAP1 mRNA expression measured by qRT-PCR in HK2 cells (n = 4). (E) Western blot and statistical analyses of CAP1 protein expression in HK2 cells (n = 4). (F) Western blot and statistical analyses of megalin protein expression in HK2 cells (n = 4). (G) Representative images of co-immunofluorescence staining of LAMP2 and megalin in HK2 cells (scale bar = 10 μm). The colocalization of megalin and lysosomal marker LAMP2 was quantified using Pearson's colocalization coefficient (n = 4). (H) HLA-C mRNA expression measured by qRT-PCR in HK2 cells (n = 6). (I) Western blot and statistical analyses of megalin protein expression in HK2 cells (n = 4). Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Renal proximal tubular cell-specific CAP1 inactivation was achieved by crossing CAP1 flox/flox mice (C57BL/6J background, Cyagen Biosciences, Guangzhou, China) with kidney androgen-regulated protein (KAP)-Cre transgenic mice (C57BL/6J background, GemPharmatech, Nanjing, China).

Techniques: Immunoprecipitation, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Marker

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Proprotein convertase subtilisin/kexin type 9 contributes to cisplatin-induced acute kidney injury by interacting with cyclase-associated protein 1 to promote megalin lysosomal degradation.

doi: 10.1016/j.bbamcr.2025.119984

Figure Lengend Snippet: Fig. 6. Cisplatin enhances the binding of PCSK9 and CAP1, induce megalin downregulation and endocytosis dysfunction. (A) PCSK9 mRNA expression measured by qRT-PCR in HK2 cells (n = 6). (B) CAP1 mRNA expression measured by qRT-PCR in HK2 cells (n = 6). (C) Western blot and statistical analyses of PCSK9 and CAP1 protein expression in HK2 cells culture medium (n = 3). (D) Co-immunoprecipitation of PCSK9 from HK2 cells lysate. (E) Western blot and statistical analyses of KIM1 and NGAL protein expression in HK2 cells (n = 4). (F) Western blot and statistical analyses of megalin protein expression in HK2 cells (n = 4). (G) Repre sentative confocal images of FITC-BSA uptake in HK2 cells (scale bar = 25 μm). Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Renal proximal tubular cell-specific CAP1 inactivation was achieved by crossing CAP1 flox/flox mice (C57BL/6J background, Cyagen Biosciences, Guangzhou, China) with kidney androgen-regulated protein (KAP)-Cre transgenic mice (C57BL/6J background, GemPharmatech, Nanjing, China).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Proprotein convertase subtilisin/kexin type 9 contributes to cisplatin-induced acute kidney injury by interacting with cyclase-associated protein 1 to promote megalin lysosomal degradation.

doi: 10.1016/j.bbamcr.2025.119984

Figure Lengend Snippet: Fig. 7. CAP1 knockout increases megalin levels in mouse overexpressing PCSK9 or in a mouse model of cisplatin-induced AKI. (A) CAP1 mRNA expression measured by qRT-PCR in mouse renal cortex (n = 6). (B) Western blot and statistical analyses of CAP1 protein expression in mouse renal cortex (n = 6). (C, D) Western blot and statistical analyses of megalin protein expression in mouse renal cortex (n = 4). (E) Coomassie blue staining of polyacrylamide gels after urine protein electro phoresis. (F) Western blot and statistical analyses of KIM1 and NGAL protein expression in mouse renal cortex (n = 4). (G) Scr and (H) BUN levels in mouse plasma (n = 4). (I) Representative images of PAS staining in kidney tissue sections and tubular injury score (n = 4, ×200, scale bar = 50 μm; ×400, scale bar = 20 μm). Data are represented as the mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Renal proximal tubular cell-specific CAP1 inactivation was achieved by crossing CAP1 flox/flox mice (C57BL/6J background, Cyagen Biosciences, Guangzhou, China) with kidney androgen-regulated protein (KAP)-Cre transgenic mice (C57BL/6J background, GemPharmatech, Nanjing, China).

Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Western Blot, Staining, Clinical Proteomics

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Proprotein convertase subtilisin/kexin type 9 contributes to cisplatin-induced acute kidney injury by interacting with cyclase-associated protein 1 to promote megalin lysosomal degradation.

doi: 10.1016/j.bbamcr.2025.119984

Figure Lengend Snippet: Fig. 8. Mechanism of PCSK9-mediated megalin downregulation. With cisplatin stimulation, renal tubular epithelial cells secrete more PCSK9 and CAP1, thereby raising the level of the megalin-PCSK9-CAP1 complex. The complex is targeted to the lysosome under the action of HLA-C and subsequently degraded by the lysosome, resulting in the downregulation of megalin.

Article Snippet: Renal proximal tubular cell-specific CAP1 inactivation was achieved by crossing CAP1 flox/flox mice (C57BL/6J background, Cyagen Biosciences, Guangzhou, China) with kidney androgen-regulated protein (KAP)-Cre transgenic mice (C57BL/6J background, GemPharmatech, Nanjing, China).

Techniques:

Journal: Journal of integrative neuroscience

Article Title: Molecular expression and functional analysis of genes in children with temporal lobe epilepsy.

doi: 10.31083/j.jin.2019.01.13

Figure Lengend Snippet: Figure 1. miR-135a-5p expression is increased in temporal lobe epilepsy. A: Quantitative qRT-PCR analyses of miR-135a-5p levels in hippocampus of children. B: Prediction of miR-135a-5p binding site in Caap1 miRNA 3’-UTR by microRNA website (www.microrna.org/microrna/home.do). C: mRNA level of Caap1 in hippocampus of children. D: Protein abundance of Caap1 in hip-

Article Snippet: CAAP1 antibody (NBP1-86644) was obtained from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Quantitative Proteomics

Journal: Journal of integrative neuroscience

Article Title: Molecular expression and functional analysis of genes in children with temporal lobe epilepsy.

doi: 10.31083/j.jin.2019.01.13

Figure Lengend Snippet: Figure 4. miR-135a-5p regulates Caap1 expression negatively in TLE model. A: mRNA level of Caap1 in primary rat hippocampus neurons from newborn rats treated with or without miR-135a-5p inhibitor. B: Protein abundance of Caap1 in hippocampus neurons from newborn rats.

Article Snippet: CAAP1 antibody (NBP1-86644) was obtained from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Quantitative Proteomics

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 1 Expression of miR-32 increases with Tat C treatment in a dose-dependent manner. (a) CHME3 cells were treated with an increasing dose of HIV-1 Tat C protein. After 24 hours, cells were harvested for RNA isolation and protein lysate preparation. miR-32 assays were performed by quantitative PCR with TaqMan probes and primers specific for human miR-32. Data was normalized to the expression level of the small RNA, RNU24, and results are shown as fold change compared with untreated control. Changes in miR-32 expression level were significant (P ≤0.05). (b), Western blot analysis for tumor necrosis factor receptor-associated factor 3(TRAF3) of the same samples treated with increasing concentrations of Tat C, showing a gradual reduction in TRAF3 protein expression. (c) Western blot image intensity was normalized to β-tubulin. All experiments were performed three times and are presented as mean ± SE. Changes in the level of expression of TRAF3 in response to increasing dose of Tat C were significant (**P ≤0.005, *P ≤0.05) compared with the untreated group.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 2 HIV-1 Tat C protein downregulates tumor necrosis factor receptor-associated factor 3 (TRAF3) protein expression. (a) Western blot analysis of TRAF3 in CHME3 cells exposed to HIV-1 Tat C protein. Treating CHME3 cells with 500 ng/ml Tat C significantly reduced the cellular TRAF3 protein level. (b) Densitometry analysis of TRAF3, normalized to β-tubulin image density. The change in TRAF3 expression level in the treated group versus the untreated control group was significant (*P ≤0.05). (c) Quantitative PCR analysis of TRAF3 in CHME3 cells exposed to HIV Tat C protein. The graph is representative of three independent experiments. All experiments were performed at least three times and data are presented as mean ± SE. **P ≤0.005.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 4 Overexpresssion of miR-32 suppresses tumor necrosis factor receptor-associated factor 3(TRAF3) protein expression. (a) Western blot analysis for TRAF3 in CHME3 cells after miR-32 overexpression. Plasmid pCMV-miR-32 was transfected into CHME3 cells. The empty vector was used as the negative control. Cell lysates were prepared after 24 hours of transfection, and western blot analysis was performed using anti-TRAF3 antibody. miR-32 overexpression significantly reduced both mRNA and protein levels of TRAF3 (P ≤0.05) (indicated by * in the transfected group) compared with empty vector. (b) Quantitative (q)PCR analysis of miR-32 overexpression in CHME3 cells, using TaqMan miR-32 assay. miR-32 expression was found to be 7.5-fold higher in miR-32-overexpressed cells. (c) Densitometry quantification of TRAF3 normalized to β- tubulin. (d) qPCR analysis for detection of changes in transcript level of TRAF3 after miR-32 overexpression in CHME3 cells. All experiments were performed at least three times and data are presented as mean ± SE.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Expressing, Western Blot, Over Expression, Plasmid Preparation, Transfection, Negative Control

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 5 Anti-miR-32 transfection rescues tumor necrosis factor receptor-associated factor 3 (TRAF3) protein expression in CHME3 cells. (a) Transfection efficiency of anti-miR, by using Cy3-labeled anti-miR as negative control. (b) Quantitative (q)PCR analysis of cellular miR-32 level after anti-miR-32 transfection, to confirm the suppression of miR-32. The expression level of miR-32 decreased by 40% in cells transfected with anti-miR-32; compared to cells transfected with scrambled anti-miR negative control (*P ≤0.05). (c) Western blot analysis of TRAF3 in CHME3 cells after anti-miR-32 transfection, showing the recovery of TRAF3 expression level in cells treated with anti-miR-32 and anti-miR-32 plus Tat. Anti-miR-32 transfection was performed at a concentration of 100 pmol/l. After 24 hours of anti-miR-32 transfection, a set of transfected cells were treated with 500 ng/ml Tat C protein to augment the cellular expression level of miR-32. (d) Densitometry analysis of TRAF3 normalized to β-tubulin. There was a significant (**P ≤0.005) recovery of TRAF3 expression level.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Transfection, Expressing, Labeling, Negative Control, Western Blot, Concentration Assay

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 6 miR-32 directly targets the 3′-UTR of tumor necrosis factor receptor-associated factor 3 (TRAF3). (a) Seed sequence in miR-32 and complementary sequence in the 3′ UTR of TRAF3 mRNA showing seven-mer binding in wild-type (WT) TRAF3 3′ UTR. A deletion mutation of 4 base pairs in the 3′ UTR of TRAF3 was generated by site-directed mutagenesis. This alteration in the 3′ UTR sequence of TRAF3 abrogated the interaction of miR-32 and the 3′ UTR of TRAF3, resulting in translational derepression. (b) Luciferase assays were performed by transfecting HeLa cells with pCMV-β-gal (normalization control), WT TRAF3 3′ UTR and mutated (MUT) TRAF3 3′ UTR plasmids, along with pCMV-miR-32 plasmids. Normalized luciferase light units of control cells are presented as 100 units, and relative light units (RLU) of other treatments are shown accordingly. All experiments were performed three times and data are presented as mean ± SE (error bars). ***P ≤0.0005.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Sequencing, Binding Assay, Mutagenesis, Generated, Luciferase, Control

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 8 Recovery of tumor necrosis factor receptor-associated factor 3 (TRAF3) expression by anti-miR-32 transfection suppresses expression levels of total interferon regulatory factor (IRF)3 and IRF7. (a) CHME3 cells were transfected with Cy3-labeled control anti-miR, anti-miR-32 and anti-miR-32 plus Tat C treatment. Phosphorylated (p)IRF3 level increased after anti-miR-32 treatment, while the total IRF3 level was downregulated in anti-miR-32-transfected cells, showing a positive relationship between cellular TRAF3 level and activation of IRF3. (b) pIRF7 was increased after anti-miR-32 treatment and anti-miR-32 plus Tat C treatment, again showing a positive role of TRAF3 in IRF7 activation. Total IRF7 level was decreased in anti-miR-32-transfected cells showing that recovery of TRAF3 could modulate the transcription of IRF3and IRF7. (c,d) Densitometry analysis of pIRF3, pIRF7, total IRF3 and total IRF7 normalized to β-tubulin. Experiments were performed three times and data are presented as mean ± SE. Results were significant (*P ≤0.05).

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Expressing, Transfection, Labeling, Control, Activation Assay

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 9 Modulation of tumor necrosis factor receptor-associated factor 3 (TRAF3) protein level alters the interferon regulatory factor (IRF)3 and IRF7 mRNA level. (a,b) CHME3 cells were treated with Tat C protein for 24 hours and transfected with miR-32, respectively. Relative fold changes in mRNA levels were determined for IRF3 and IRF7 using quantitative(q)PCR with SYBR green. As a consequence of Tat C treatment and miR-32 overexpression, the transcript expression levels of IRF3 and IRF7 increased. (c) After inhibiting the cellular miR-32 via application of anti-miR, the transcript level of both IRF3 and IRF7 was reduced. In cells treated with anti-miR-32 plus Tat C, the transcript levels of IRF3 and IRF7 were lower than those in control CHME3 cells. All experiments were repeated three times, and data are presented as mean ± SE. Relative change in IRF3 transcript in miR-32 transfected cells compared with empty vector were significant ***P ≤0.0005, **P ≤0.005 and *P ≤0.05 in respective graphs.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Transfection, SYBR Green Assay, Over Expression, Expressing, Control, Plasmid Preparation

Journal: Journal of neuroinflammation

Article Title: HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

doi: 10.1186/1742-2094-9-131

Figure Lengend Snippet: Figure 10 Proposed model for HIV-1 Tat C-induced, miR-32-mediated post-transcriptional regulation of tumor necrosis factor receptor-associated factor 3 (TRAF3). In response to HIV-1 Tat C exposure of human microglial cells, miR-32 was upregulated, consequently downregulating the protein level of TRAF3 post-transcriptionally by binding to its 3′ untranslated region. The miRNA inhibitor against miR-32, ant-miR-32, reduced the cellular level of miR-32 and rescued the expression level of TRAF3 protein. The cellular expression level of TRAF3 protein had an inverse relationship to the expression level of interferon regulatory factor (IRF)3/7 and this could perturb the expression of inflammatory genes in microglial cells after exposure to HIV-1 Tat C protein.

Article Snippet: The TRAF3 3′ UTR construct in pMirTarget (SC206836; Origene Technologies) and miR-32 construct as pCMV-Mir (SC400329; Origene Technologies) were used.

Techniques: Binding Assay, Expressing