Journal: Journal of Cancer Research and Clinical Oncology

Article Title: zDHHC3-mediated S-palmitoylation of SLC9A2 regulates apoptosis in kidney clear cell carcinoma

doi: 10.1007/s00432-024-05737-y

Figure Lengend Snippet: PAK7 and SLC9A2 expression in the CEPIA and GSE213324 datasets. A , B , E , F ) PAK7 and SLC9A2 expression in tumor and healthy tissues. CEPIA ( A , B ) and GSE213324 ( E , F ) data, Wilcoxon test. C , D Correlations of PAK7 and SLC9A2 expression with zDHHC3 expression

Article Snippet: The following primary antibodies were used: zDHHC3 (sc-377378, 1:500; Santa), P21-activated kinase 7 (PAK7; 35,292–1, 1:500; SAB), solute carrier family 9 member A2 (SLC9A2, also sodium–hydrogen exchanger 2; 46,256–1, 1:500; SAB), caspase 3 (19,677–1-AP, 1:500; Proteintech), and β-actin (66,009–1-LG, 1:10,000; Proteintech).

Techniques: Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: Discovery and verification of mmu_Circ_26986/hsa_Circ_0072463 as a potential biomarker and intervention target for sepsis-associated acute kidney injury

doi: 10.1007/s00018-023-05079-x

Figure Lengend Snippet: PAK7 is a direct target of miRNA-29b-1-5p. BUMPT cell line was transfected with miRNA-29b-1-5p mimic or PAK7 siRNA or SC, and then treated with/without (LPS 300 μg/mL) for 24 h. A miRDB database predicts that miRNA-29b-1-5p has a complementary binding site in the 3'-UTR of PAK7 mRNA. B miRNA-29b-1-5p was co-transfected with the 3’-UTR DLR vector of PAK7-1-MUT, PAK7-2-MUT or PAK7-WT, and the luciferase activities were then examined. C qRT-PCR detection of the mRNA expression of PAK7. D Immunoblot evaluation of PAK7 and β-tubulin levels. E Densitometric assessment of PAK7 and β-tubulin. F , G FCM analysis of BUMPT cell apoptosis. H Immunoblot analysis of C3, CC3 and PAK7. I Densitometric assessment of immunoblot bands. Mean ± SD (n = 6). #p < .05, vs. SC + Saline group; *p < .05, PAK7 siRNA + LPS group, vs. SC + LPS group. △p < .05, PAK7 WT/miRNA-29b-1-5p, vs. other groups

Article Snippet: Anti-PAK7 and anti-β-tubulin antibodies were procured from Proteintech (USA).

Techniques: Transfection, Binding Assay, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Expressing, Western Blot, Saline

Journal: Cellular and Molecular Life Sciences

Article Title: Discovery and verification of mmu_Circ_26986/hsa_Circ_0072463 as a potential biomarker and intervention target for sepsis-associated acute kidney injury

doi: 10.1007/s00018-023-05079-x

Figure Lengend Snippet: Downregulation of Circ_26986 enhances LPS-stimulated BUMPT cell apoptosis, while miRNA-29b-1-5p inhibitor reverses this process. BUMPT cell line was co-transfected with Circ_26986 (100 nM) and anti-miRNA-29b-1-5p or SC and then exposed to LPS for 24 h. A , B qRT-PCR assessment of Circ_26986 and miRNA-29b-1-5p levels. C , D Flow cytometry analysis of BUMPT cell apoptosis. E Immunoblot analysis of C3, CC3, PAK7 and β-tubulin. (F) Grey evaluation of immunoblot bands. Mean ± SD (n = 6). #p < .05, vs. SC + saline group; ▲p < .05, Circ_26986 siRNA + anti-miRNA-29b-1-5p + LPS group, vs. Circ_26986 siRNA + LPS group

Article Snippet: Anti-PAK7 and anti-β-tubulin antibodies were procured from Proteintech (USA).

Techniques: Transfection, Quantitative RT-PCR, Flow Cytometry, Western Blot, Saline

Journal: Cellular and Molecular Life Sciences

Article Title: Discovery and verification of mmu_Circ_26986/hsa_Circ_0072463 as a potential biomarker and intervention target for sepsis-associated acute kidney injury

doi: 10.1007/s00018-023-05079-x

Figure Lengend Snippet: LPS-stimulated AKI in male C57BL/6 mice can be suppressed by Circ_26986 overexpression. C57BL/6 mice were given Circ_26986 plasmid via tail vein for 12 h, and subsequently received CLP for 18 h or a sham-operated group as a control. Determination of serum BUN ( A ) and creatinine ( B ) concentrations. C Staining of renal cortex with hematoxylin and eosin. D H&E staining of the renal medulla. E TUNEL staining was also performed on the kidneys. Score bar: 50 µm. Renal cortical ( F ) and OSOM ( G ) tubular injury scores. H TUNEL-positive cell count. G , H qRT-PCR assessment of Circ_26986 and miRNA-29b-1-5p. I Immunoblot evaluation of C3, CC3, and PAK7. J Densitometric evaluation of immunoblot bands. Mean ± SD (n = 6). #p < .05, control + CLP or Circ_26986 plasmid group, vs. saline group; *p < .05, Circ_26986 plasmid + CLP group, vs. control + CLP group

Article Snippet: Anti-PAK7 and anti-β-tubulin antibodies were procured from Proteintech (USA).

Techniques: Over Expression, Plasmid Preparation, Staining, TUNEL Assay, Cell Counting, Quantitative RT-PCR, Western Blot, Saline

Journal: Cellular and Molecular Life Sciences

Article Title: Discovery and verification of mmu_Circ_26986/hsa_Circ_0072463 as a potential biomarker and intervention target for sepsis-associated acute kidney injury

doi: 10.1007/s00018-023-05079-x

Figure Lengend Snippet: Overexpression of hsa_Circ_0072463 attenuates LPS-stimulated HK-2 cell apoptosis. A Sequence comparison of mmu_Circ_26986 and hsa_Circ_0072463. Sequence conservativeness was examined using the blast function of CircBase webtool ( http://circrna.org/cgi-bin/webBlat ). According to the blast result, when the homology > 80%, the higher the score, the higher the homology of hsa_Circ_RNA is considered. HK-2 cells were exposed to LPS (50 μg/mL) for 6, 12 or 24 h. B qRT-PCR assessment of hsa_Circ_0072463 levels in cells. HK-2 cell line was transfected with hsa_Circ_0072463 plasmid and then were subjected to LPS 24 h. qRT-PCR was utilized for examining hsa_Circ_0072463 ( C ) and hsa-miRNA-29b-1-5p ( D ) levels. E Flow cytometry evaluation of HK-2 cell apoptosis. F Calculation of apoptosis rate. G Immunoblot assessment of the expression of C3, CC3, and PAK7 in HK-2 cells. H Grayscale evaluation of the immunoblot bands of C3, CC3, and PAK7 in HK-2 cells. Mean ± SD (n = 6). #p < .05 vs. SC + saline group. *p < .05 vs. SC + I/R group

Article Snippet: Anti-PAK7 and anti-β-tubulin antibodies were procured from Proteintech (USA).

Techniques: Over Expression, Sequencing, Comparison, Quantitative RT-PCR, Transfection, Plasmid Preparation, Flow Cytometry, Western Blot, Expressing, Saline

Journal: Cellular and Molecular Life Sciences

Article Title: Discovery and verification of mmu_Circ_26986/hsa_Circ_0072463 as a potential biomarker and intervention target for sepsis-associated acute kidney injury

doi: 10.1007/s00018-023-05079-x

Figure Lengend Snippet: Modelling and mechanism of mmu_Circ_26986/hsa_Circ_0072463 in septic AKI. In sepsis AKI, the expression of mmu_Circ_26986 is increased. mmu_Circ_26986 competitively acts on miRNA-29b-1-5p, increases PAK7 expression, and exhibits anti-apoptotic effects on cell and animal models. Homologous hsa_Circ_0072463 is the most likely target of mmu_Circ_26986. Hsa_Circ_0072463 may serve as an early diagnostic marker and a new therapeutic target for septic AKI

Article Snippet: Anti-PAK7 and anti-β-tubulin antibodies were procured from Proteintech (USA).

Techniques: Expressing, Diagnostic Assay, Marker

Journal: Clinical and Translational Medicine

Article Title: Alternative splicing of HSPA12A pre‐RNA by SRSF11 contributes to metastasis potential of colorectal cancer

doi: 10.1002/ctm2.1113

Figure Lengend Snippet: PAK5 phosphorylates SRSF11 at serine 287 site to protect it from ubiquitination degradation. (A) Immunoblotting analysis of endogenous PAK5 protein in LoVo cells using anti‐SRSF11. (B) Immunoblotting analysis of MYC‐tag PAK5 protein in LoVo cells co‐transfected with MYC‐PAK5 and Flag‐SRSF11 plasmids using anti‐Flag. (C) Immunoblotting analysis of Flag‐tag SRSF11 protein in LoVo cells co‐transfected with MYC‐PAK5 and Flag‐SRSF11 plasmids using anti‐MYC. (D) Phos‐tag SDS‐PAGE was performed in LoVo cells after transfection with Vector, PAK5‐WT, PAK5‐K478M or PAK5‐WT+AP as illustrated in Materials and Methods. p‐SRSF11 and unp‐SRSF11 represent the phosphorylated and unphosphorylated Slug, respectively. The total protein of SRSF11 was detected by WB analysis and shown below, with GAPDH serving as an internal control. (E) Schematic diagram of SRSF11 protein, wildtype (WT) or indicated mutants (serine replaced by alanine) fused with Flag. (F) Phos‐tag SDS‐PAGE was performed in LoVo and SW480 cells after co‐transfection with MYC‐PAK5 and WT or five mutants of Flag‐SRSF11 plasmids. The asterisks represent the significant reduction of p‐SRSF11. (G) in vitro phosphorylation assay was employed to explore the effects of PAK5 on different SRSF11 and control polypeptides. WT represents the wildtype SRSF11 polypeptides with 280–294 AA sequence; Mut represents the 284 serine replaced by alanine compared with WT; random represents that the 15 amino acids are randomly arranged in disorder; positive group represents the SRSF11 polypeptides replaced by PAK5 protein ( N = 5, SNK test; * p < .05, ** p < .01). (H) WB analysis of SRSF11 after gradient increasing overexpression of MYC‐PAK5 or Vector plasmid in LoVo and SW480 cell lines. The Phos‐tag SDS‐PAGE was performed to detect the p‐ and unp‐SRSF11 levels as shown below. (I) The effect of PAK5 on the stability of SRSF11. MG‐132 eliminates the effect of si‐PAK5 on the stability of SRSF11. (J) LoVo and SW480 cells were treated with CHX (50 nM) for 8 h after treatment with Vector or PAK5 plasmid. SRSF11 protein levels were determined at the indicated time points (the marked number below the strip represents the relative gray value to β‐actin in the same group). (K) Immunoblotting analysis of SRSF11 protein in LoVo cells after transfection with indicated plasmids using anti‐Ub. (L) Immunoblotting analysis of Flag‐tag protein in LoVo cells after transfection with indicated plasmids using anti‐Ub. (M) Immunoblotting analysis of Flag‐tag protein in LoVo cells after transfection with indicated plasmids using anti‐Ub

Article Snippet: The antibodies used are listed as follows: SRSF11 (Abcam, catalog: ab254733), PAK5 (Bioworld, catalog: BS9193), MYC (Proteintech, catalog: 16286‐1‐AP) and Flag (Proteintech, catalog: 20543‐1‐AP).

Techniques: Western Blot, Transfection, FLAG-tag, SDS Page, Plasmid Preparation, Cotransfection, In Vitro, Phosphorylation Assay, Sequencing, Over Expression, Stripping Membranes

Journal: Clinical and Translational Medicine

Article Title: Alternative splicing of HSPA12A pre‐RNA by SRSF11 contributes to metastasis potential of colorectal cancer

doi: 10.1002/ctm2.1113

Figure Lengend Snippet: PAK5‐regulated phosphorylation facilitates the nuclear translocation of SRSF11 and inhibits the inclusion of HSPA12A exon 2. (A) Representative immunofluorescence images by confocal microscope indicate SRSF11 protein localization in LoVo cells. (B) WB analysis of SRSF11 protein levels in cytoplasm and nucleus, respectively. β‐actin and Lamin‐B1 were used as cytoplasmic and nuclear internal controls, respectively. (C) RT‐PCR results and quantification of HSPA12A RNA products measured as PSI after co‐transfection with indicated plasmid and siRNA in LoVo and SW480 cell lines ( N = 3, SNK test; * p < .05, ** p < .01, *** p < .001)

Article Snippet: The antibodies used are listed as follows: SRSF11 (Abcam, catalog: ab254733), PAK5 (Bioworld, catalog: BS9193), MYC (Proteintech, catalog: 16286‐1‐AP) and Flag (Proteintech, catalog: 20543‐1‐AP).

Techniques: Translocation Assay, Immunofluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Cotransfection, Plasmid Preparation