Journal: Frontiers in molecular biosciences

Article Title: Circular RNA TLK1 Promotes Sepsis-Associated Acute Kidney Injury by Regulating Inflammation and Oxidative Stress Through miR-106a-5p/HMGB1 Axis.

doi: 10.3389/fmolb.2021.660269

Figure Lengend Snippet: FIGURE 2 | Depletion of circTLK1 decreases oxidation stress, inflammation, and apoptosis in the sepsis-related AKI rat model. (A–G) The CLP-induced sepsis- related AKI rat model was established in Wistar rats (N 4), followed by the treatment of circTLK1 shRNA. The cecum was exposed without perforation in the sham groups. (A) MDA levels in the rats. (B) ROS production in the rats. (C) GSH levels in the rats. (D) CAT activities in the rats. (E) SOD activities in the rats. (F) IL-1β and IL-6 release in the rats. (G) TUNEL assays of apoptosis analysis in the rats, bar 50 μm. The experiments were replicated at least three times, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cell apoptosis was assessed by employing the Annexin V-FITC Apoptosis Detection Kit (CST, United States) following the manufacturer’s instruction.

Techniques: shRNA, TUNEL Assay

Journal: Frontiers in molecular biosciences

Article Title: Circular RNA TLK1 Promotes Sepsis-Associated Acute Kidney Injury by Regulating Inflammation and Oxidative Stress Through miR-106a-5p/HMGB1 Axis.

doi: 10.3389/fmolb.2021.660269

Figure Lengend Snippet: FIGURE 3 | CircTLK1 knockdown relieves LPS-induced inflammation and apoptosis in the HK-2 cells. (A) Expression of circTLK1 was detected by qPCR in HK-2 cells treated with circTLK1 shRNA. (B–E) For the induction of the cell injury model, the HK-2 cells were treated with LPS (1 mg/L) for 24 h after the cell concentration reached 80%, followed by the treatment of circTLK1 shRNA. (B) qPCR analysis of circTLK1 expression. (C) Western blot analysis of TNF-α, IL-1β, and IL-6. (D) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3. (E) Flow cytometry analysis of apoptosis. The experiments were replicated at least three times, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cell apoptosis was assessed by employing the Annexin V-FITC Apoptosis Detection Kit (CST, United States) following the manufacturer’s instruction.

Techniques: Knockdown, Expressing, shRNA, Concentration Assay, Western Blot, Flow Cytometry

Journal: Frontiers in molecular biosciences

Article Title: Circular RNA TLK1 Promotes Sepsis-Associated Acute Kidney Injury by Regulating Inflammation and Oxidative Stress Through miR-106a-5p/HMGB1 Axis.

doi: 10.3389/fmolb.2021.660269

Figure Lengend Snippet: FIGURE 6 | CircTLK1 induces sepsis-related AKI by the miR-106a-5p/HMGB1 axis. (A–C) The CLP-induced sepsis-related AKI rat model was established in Wistar rats (N 6), followed by the indicated treatment. The cecum was exposed without perforation in the sham groups. (A) qPCR analysis of HMGB1 expression. (B) H&E staining of kidney tissues, bar 50 μm. (C) TUNEL assays of apoptosis analysis in the rats, bar 50 μm. (D–G) For the induction of the cell injury model, the HK-2 cells were treated with LPS (1 mg/L) for 24 h after the cell concentration reached 80%, followed by the indicated treatment. (D) qPCR analysis of HMGB1 expression. (E) Western blot analysis of TNF-α, IL-1β, and IL-6. (F) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3. (G) Flow cytometry analysis of apoptosis, mean ± SD, **P < 0.01, ***P < 0.001.

Article Snippet: Cell apoptosis was assessed by employing the Annexin V-FITC Apoptosis Detection Kit (CST, United States) following the manufacturer’s instruction.

Techniques: Expressing, Staining, TUNEL Assay, Concentration Assay, Western Blot, Flow Cytometry

Journal: Oncotarget

Article Title: Oncogenic features of neuromedin U in breast cancer are associated with NMUR2 expression involving crosstalk with members of the WNT signaling pathway.

doi: 10.18632/oncotarget.16121

Figure Lengend Snippet: Figure 8: NMU signaling modulates the WNT receptor pathway in NMUR2-positive SKBR3 breast cancer cells. (A) Real-time PCR-based validation of candidate NMU downstream genes in independent stably transfected SKBR3 NMU (n=5) and mock clones (n=5). * P < 0.05; ns: not significant (Mann-Whitney-U test). (B) Representative western blots showing differential protein expression of candidate NMU downstream genes in independent stably transfected SKBR3 NMU and mock clones. Loading controls: β-actin, β-tubulin, total RAC1. All experiments were performed in triplicate. (C) Densitometrical evaluation of the western blot results shown in B depicted as box plots. Box plot showing RAC1-GTP in relation to total RAC1 amounts in SKBR3 NMU (n=4) and mock clones (n=4), combines data of three independent experiments. * P < 0.05; ns: not significant (Mann-Whitney-U test). (D) Hypothetical model of NMU’s oncogenic role in dependency of NMUR2 in breast cancer: crosstalk of NMU signaling with WNT, TGFβ and ERK cascade results in decreased expression of the canonical WNT target MYC and enhanced activation of the non-canonical WNT/planar cell polarity (PCP) pathway effector RAC1 among others, contributing to growth inhibition and promotion of cell migration.

Article Snippet: Measurement of RAC1 activation in stable SKBR3 NMU and mock clones was achieved by using the Active RAC1 Detection Kit (#8815, Cell Signaling, Danvers, MA, USA) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Stable Transfection, Transfection, Clone Assay, MANN-WHITNEY, Western Blot, Expressing, Activation Assay, Inhibition, Migration

Journal: Clinical and Translational Medicine

Article Title: Uterus globulin associated protein 1 (UGRP1) binds podoplanin (PDPN) to promote a novel inflammation pathway during Streptococcus pneumoniae infection

doi: 10.1002/ctm2.850

Figure Lengend Snippet:

Article Snippet: Active Rho Detection Kit , Cell Signaling Technology , Cat# 8820S.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Cell Counting, Extraction, Software, Real-time Polymerase Chain Reaction

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: Regulation of CDC42 activity by NDRG1 in CRC cells. A) Immunoblotting for total protein level or activated form of indicated Rho GTPase in NDRG1-modified HCT116 and RKO cells. Results are representative of at least three biological repeats, and the values in histograms are represented by mean ± S.D.; *P value <0.05, **P value <0.01, relative to the respective control cells. B) Confocal images were taken to show immunofluorescence staining of active-CDC42 (red) accompanied by the cell nucleus (blue) stained by DAPI in NDRG1 overexpression and NDRG1 knockdown HCT116 and RKO cells relative to the control cells, respectively. Fluorescence quantification was performed by comparing the integrated optical density (IOD)/area value of active-CDC42 to the IOD/area value of the nucleus (DAPI) in the same image. Results are representative of three to five images from different visual fields, and the histogram values are mean ±S.D. *P value <0.05, ***P<0.001, relative to the respective control cells. Scale bars: 25 µm.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Activity Assay, Western Blot, Modification, Control, Immunofluorescence, Staining, Over Expression, Knockdown, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: Inhibition of CDC42 prevents NDRG1 loss induced CRC cell filopodial protrusion formation through suppression of PAK1/Cofilin signaling. A) Immunoblotting analysis of the expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. B) Knockdown of CDC42 in HCT116 (left) and RKO (right) cells confirmed with immunoblotting analysis. Pool, combined siCDC42 sequences. C) Expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. D) Confocal images were taken to show immunofluorescence staining of MYO10 (green) and rhodamine-phalloidin (red) accompanied by the cell nucleus (blue) in colorectal cancer cells. Quantification of the MYO10-associated filopodial protrusions density and length is represented as mean ± S.D.; results are representative of 3-5 images from different visual fields, n>50 cells. *P value <0.05, **P value <0.01, ***P < 0.001, relative to the sh-Con/si-Con groups. # P value <0.05, ## P value <0.01, ### P < 0.001, relative to the sh-NDRG1/si-Con groups.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Inhibition, Western Blot, Expressing, Phospho-proteomics, Knockdown, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: NDRG1 suppresses CDC42 activity by stabilizing the RhoGDIα-CDC42 binding. A) The STRING network view of interactive proteins of CDC42 in humans. Gray lines between the nodes indicate various types of interaction evidence. B) Co-immunoprecipitation to examine the interaction of RhoGDIα and CDC42 in both HCT116 and RKO cell lines. C) Immunoblotting assay to evaluate the influence of NDRG1 modification on RhoGDIα expression in indicated cells. GAPDH was used as loading control. D) Double stained confocal immunofluorescence assay and co-localization analysis to confirm the interaction of RhoGDIα and CDC42 in indicated cells (red: CDC42, green: RhoGDIα, blue: DAPI, scale bar: 20 µm). Co-localization analysis on wide-field merged images was performed via Leica Application Suite X. Results are representative of five images from different visual fields.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Activity Assay, Binding Assay, Immunoprecipitation, Western Blot, Modification, Expressing, Control, Staining, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: Silence of NDRG1 promotes the peritoneal metastasis and correlates with upregulated CDC42 GTP expression. A) Peritoneal metastasis of CRC cells in BALB/c nude mice. Tumors in two groups were measured in situ and assessed by bioluminescence imaging in the fourth week. B) Statistical analysis of the bioluminescence in peritoneal foci of both groups. Results are shown as mean ± S.D. C) Tumors in two groups are demonstrated after laparotomy with hematoxylin-eosin staining of peritoneal foci on the lower panel. Scale bars are as indicated. D) Immunofluorescence staining of NDRG1 (left) or CDC42 GTP (right) accompanied by the cell nucleus stained by DAPI in peritoneal foci derived from sh-NDRG1 and control groups. Results are representative of 3-5 images from different visual fields and the histogram values are mean ± S.D.; *P value <0.05, ***P< 0.001, relative to the respective control groups. Scale bar: 50 µm.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Expressing, In Situ, Imaging, Staining, Immunofluorescence, Derivative Assay, Control

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: CDC42 GTP is frequently upregulated in CRC tissues and correlated with NDRG1 expression and clinicopathological parameters. A) IHC staining of NDRG1 and active CDC42 expression in tumor and adjacent tissues in microarray. Magnification on the right with a scale bar of 100 µm. B) Heatmap illustrating different clinicopathological parameters between CDC42 GTP -high and -low-expression tumors of the 86 cases. Statistical significance was analyzed by the χ 2 test. P values are as indicated.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: International Journal of Biological Sciences

Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

doi: 10.7150/ijbs.56694

Figure Lengend Snippet: Schematic diagram for the mechanism of NDRG1's regulation of CDC42/PAK1/Cofilin axis as a switch that modulates actin cytoskeleton rearrangement in human colorectal cancer invasion by stabilizing the RhoGDIα-CDC42 binding.

Article Snippet: GST-pull down assay to detect active CDC42 and RAC1 was carried out as the protocol of Active CDC42 Detection Kit (Cat.8819, Cell Signaling Technology) and Active RAC1 Detection Kit (Cat.8815, Cell Signaling Technology).

Techniques: Binding Assay