Journal: Bone research

Article Title: IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating ubiquitylation and SUMOylation of MFN1/2.

doi: 10.1038/s41413-024-00363-3

Figure Lengend Snippet: Fig. 5 ISG15 upregulated by PAPR12, increasing ISGylation of MFN1/2 and then attenuating the ubiquitylation and SUMOylation to inhibit PINK1/Parkin-dependent mitophagy. a Immunoblotting (IB) analysis of ubiquitylation and ISG15 in PHCs following transfection with KD-ISG15 or KD-NC. b Protein quantification of (a) using ImageJ. n = 3 per group. c IB analysis of ubiquitylation of mitochondrial proteins. Mitochondria were isolated from KD-NC and KD-ISG15 PHCs transfected with a HA-ubiquitin construct. d Protein quantification of (d) using ImageJ. n = 3 per group. e IB analysis of SUMO2/3 and ubiquitin with SUMO2/3-conjugated proteins in KD-NC and KD-ISG15 PHCs immunoprecipitated using SUMO2/3 affinity beads. f IB analysis of MFN1/2, MFN1, MFN2, ubiquitin, SUMO2/3 and ISG15 in KD-NC and KD-ISG15 PHCs treated with or without MG-132 after immunoprecipitation of MFN1/2. The molecular marker of IB plots of ubiquitin, SUMO2/3, and ISG15 conjugates with MFN1/2 is 70 kD and above when considering the molecular weight of MFN1/2 at 85 kD. g Quantification of MFN1/2, ubiquitin, SUMO2/3, and ISG15 of (f) using ImageJ after immunoprecipitation of MFN1/2. n = 3 per group. h IB analysis of MFN1/2, MFN1, MFN2, ubiquitin, SUMO2/3, and ISG15 in PHCs following either knockdown or overexpression of PARP12 after immunoprecipitation of MFN1/2. i Quantification of MFN1/2, ubiquitin, SUMO2/3, and ISG15 of (h) using ImageJ. n = 3 per group. j MitoTracker Red and LysoTracker Green staining of PHCs was observed by confocal microscopy. Scale bars: 10 µm. k Western blot analysis of PARP12, ISG15, LC3B, p62, MFN1, MFN2, PINK1, Parkin, COL2A1, aggrecan, MMP13, RUNX2, Bcl2, Bax, and NLRP3 inflammasome activity in PHCs coinfected with PARP12 KD-03 shRNA and ISG15-OE adenovirus. n = 3 per group. l ROS staining with PARP12 knockdown and ISG15 overexpression. Scale bars: 100 µm. m JC-1 staining in PHCs following transfection with PARP12 knockdown and ISG15 overexpression. Scale bars: 100 µm. Data are presented as the mean ± SD. Paired t-test (b, d, i) and one-way analysis of variance followed by Tukey’s multiple comparison test (g) were used for statistical analysis. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: According to the manufacturer’s instructions of SUMOylation 2/3 affinity beads (BK162, Cytoskeleton, Inc.), SUMO2/3-conjugated proteins in chondrocytes lysates were immunoprecipitated.

Techniques: Western Blot, Transfection, Isolation, Ubiquitin Proteomics, Construct, Immunoprecipitation, Marker, Molecular Weight, Knockdown, Over Expression, Staining, Confocal Microscopy, Activity Assay, shRNA, Comparison

Journal: Bone research

Article Title: IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating ubiquitylation and SUMOylation of MFN1/2.

doi: 10.1038/s41413-024-00363-3

Figure Lengend Snippet: Fig. 8 PARP12 modulates osteoarthritis (OA) pathogenesis in monosodium iodoacetate (MIA)-treated rats. a Experimental diagram of the MIA OA rat model treated with XAV-939 or PARP12 overexpression (OE) adenovirus. Rats were evaluated at age of 10 weeks. n = 6 per group. b 3D reconstruction images of micro-CT scanning of the knees of rats treated with XAV-939 or PARP12-OE adenovirus. n = 5 per group. c–f Analysis of BV/TV, BS/TV, trabecular thickness, and trabecular numbers. n = 5 per group. g Western blot analysis of PARP12, COL2A1, aggrecan, MMP13, RUNX2, Bcl2/Bax, LC3B, p62, MFN1, MFN2, PINK1, Parkin and NLRP3 inflammasome activity in chondrocytes of rats treated with XAV-939 or PARP12 overexpression (OE) adenovirus. n = 3 per group. h Representative images of Safranin O and IHC staining of PARP12, COL2A1 and MMP13. Scale bars: 250 µm (first row) and 50 µm (second row). i Quantification of macroscopic score based on staining results in (h). n = 3 per group. j–l Quantification of PARP12, COL2A1, and MMP13 positive chondrocytes based on staining results in (h). n = 3 per group. m–o ROS staining, ATP level and JC-1 staining in chondrocytes of rats treated with XAV-939 or PARP12 OE adenovirus. p Schematic representation of the mechanism by which IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating the ubiquitylation and SUMOylation of MFN1/2. Data are presented as the mean ± SD. Paired t-test (c–f, j–l, n) and non-parametric Mann-Whitney U test (i) were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: According to the manufacturer’s instructions of SUMOylation 2/3 affinity beads (BK162, Cytoskeleton, Inc.), SUMO2/3-conjugated proteins in chondrocytes lysates were immunoprecipitated.

Techniques: Over Expression, Micro-CT, Western Blot, Activity Assay, Immunohistochemistry, Staining, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: RvD2 mitigates TNFɑ-Induced mitochondrial reactive oxygen species through NRF2 signaling in placental trophoblasts

doi: 10.3389/fphys.2025.1547940

Figure Lengend Snippet: RvD2 upregulates the NRF2 signaling cascade in TNFɑ-induced JEG-3 cells. For cotreatment of TNFɑ + RvD2 (TR) groups, cells were pretreated with RvD2 for 16 h, followed by TNFɑ treatment for an additional 5 or 10 h (16 + 5 h or 16 + 10 h), resulting in total treatment durations of 21 and 26 h, respectively. For vehicle (V) or RvD2 (R) treatments, cells were treated for a total of 21 or 26 h. Cells treated with TNFɑ (T) were exposed for either 5 or 10 h. (A) Immunoblot analysis of NRF2 in 16 + 5 h and 16 + 10 h treatment strategies. The values below the immunoblot represent band intensity ratio of nNRF2/HDAC1. The same blot was used in . (B–G) Relative mRNA expression of 16 + 5 h treatment strategy of kelch-like ECH-associated protein 1 (KEAP1), hemoxygenase 1 (HOXO1), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), NADPH quinone oxidoreductase 1 (NQO1) in trophoblasts; n = 3 per group. (H) Reduced glutathione was measured with the pretreatment of RvD2 (100 nM) for 16 h followed by a 1 h treatment of TNFɑ (100 ng/mL); n = 5–7 per group. Data presented as mean ± SEM; *p < 0.05 and **p < 0.01 compared against each treatment.

Article Snippet: The Cellular Glutathione Detection Assay Kit (Cell Signaling Technology, Catalog #13859) was used to quantify reduced glutathione (GSH) concentrations, following the manufacturer’s instructions.

Techniques: Western Blot, Expressing

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

Journal: BMC Biology

Article Title: Identification of berberine as a novel drug for the treatment of multiple myeloma via targeting UHRF1

doi: 10.1186/s12915-020-00766-8

Figure Lengend Snippet: BBR induces UHRF1 degradation via the ubiquitin-proteasome system pathway. a BBR affected the stability of UHRF1 in MM cells. RPMI-8266 and MM.1S cells were treated with DMSO alone, BBR (25 μM) alone for 24 h, or pretreated with DMSO or BBR for 12, 16, and 20 h, followed by addition of CHX (50 μg/mL) for additional 4, 8, and 12 h. Cell lysates were harvested and subjected to western blotting with anti-UHRF1 and anti-GAPDH antibodies. b , c Densitometry was utilized to quantify UHRF1 protein levels after normalization with GAPDH control to obtain percent UHRF1 degradation in RPMI-8266 and MM.1S cells. The data were presented as the mean ± SD obtained from three independent experiments. d MG132 abolished the effect of BBR on UHRF1 degradation. RPMI-8266 and MM.1S cells were treated with DMSO alone, BBR (25 μM) alone for 24 h, or pretreated with DMSO or BBR for 20 h, followed by addition of MG132 for additional 4 h. Cell lysates were harvested and subjected to western blotting with anti-UHRF1 and anti-GAPDH antibodies. e RPMI-8266 cells were subsequently treated with BBR (25 μM for 24 h) prior to harvesting. The proteins modified by ubiquitination were purified from cell extracts using anti-UB beads and subjected to western blotting with anti-UHRF1 and anti-GAPDH antibodies. f RPMI-8266 cells were transiently transfected with HA-Ub constructs, and endogenous UHRF1 proteins were immunoprecipitated from BBR-treated or BBR-untreated RPMI-8266 cells (25 μM for 24 h). Immunoprecipitates were harvested and subjected to western blotting with anti-UHRF1, anti-FK2, and anti-GAPDH antibodies

Article Snippet: Ubiquitinated proteins were immunoprecipitated using Signal-SeekerTM Ubiquitination Detection Kit according to the protocol provided by the manufacturer (cat. #BK161, Cytoskeleton).

Techniques: Ubiquitin Proteomics, Western Blot, Control, Modification, Purification, Transfection, Construct, Immunoprecipitation