Journal: FEBS Open Bio

Article Title: Reduced MAGI3 level by HPV18E6 contributes to Wnt/β‐catenin signaling activation and cervical cancer progression

doi: 10.1002/2211-5463.13298

Figure Lengend Snippet: MAGI3 downregulates β‐catenin protein level and inhibits Wnt/β‐catenin‐mediated signaling in CC cells. (A) MAGI3 overexpression retarded Wnt signaling activation via reducing β‐catenin protein level. HeLa‐MAGI3 cells were treated in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of Wnt/β‐catenin activator CHIR‐99021 (0.2 µmol·L −1 ) for 4 h. The expression levels of β‐catenin and TCF1 were analyzed by western blotting. (B) MAGI3 knockdown promoted Wnt signaling activation via increasing β‐catenin level. C‐33A cells were transfected with MAGI3 siRNAs or scrambles for 24 h and then treated with Wnt/β‐catenin inhibitor IWR‐1‐endo (100 µmol·L −1 ; lanes 3 and 4) for 24h, and cell lysates were analyzed by western blotting. The gray value of protein bands of western blots has been quantified by imagej . All experiments were repeated three times ( n = 3).

Article Snippet: CHIR‐99021, a Wnt/β‐catenin activator, was purchased from TargetMol (Boston, MA, USA); IWR‐1‐endo, a Wnt/β‐catenin inhibitor, was purchased from Selleck (Houston, TX, USA). pcDNA3‐V5/His‐MAGI3 and GFP/GFP‐MAGI3 plasmids were kindly provided by R. Hall (Emory University, GA).

Techniques: Over Expression, Activation Assay, Expressing, Western Blot, Transfection

Journal: FEBS Open Bio

Article Title: Reduced MAGI3 level by HPV18E6 contributes to Wnt/β‐catenin signaling activation and cervical cancer progression

doi: 10.1002/2211-5463.13298

Figure Lengend Snippet: Downregulation of MAGI3 by HPV18 E6 rather than HPV16 E6 enhances cell migration and invasion via activation of β‐catenin signaling. (A, B) knockdown of HPV18 E6 inhibited the migration and invasion of HeLa cells via increasing MAGI3 and reducing β‐catenin level. HPV18 E6 was knocked down by HPV18 E6 siRNAs, or codepleted with MAGI3 siRNAs in the presence or absence of β‐catenin knockdown in HeLa cells. (C, D) Overexpression of HPV18 E6 promoted the migration and invasion of C‐33A cells via reducing MAGI3 and increasing β‐catenin level. HPV18 E6 was overexpressed alone or co‐overexpressed with MAGI3 in the presence or absence of Wnt/β‐catenin activator CHIR‐99021 in C‐33A cells. (E) HPV18 E6 rather than HPV16 E6 promoted CC cell migration and invasion through activating Wnt/β‐catenin signaling. HPV18 E6 and HPV16 E6 were overexpressed in C‐33A cells, or co‐treated with Wnt/β‐catenin inhibitor IWR‐1‐endo or vehicle. Cell lysates, migration and invasion were conducted as described in Fig. . The expression level of protein was quantitatively expressed by relative gray value, all experiments were repeated three times ( t‐ test; * P < 0.05, ** P < 0.01, and *** P < 0.001, values represent mean ± SD, n = 3). Scale bar, 100 μm.

Article Snippet: CHIR‐99021, a Wnt/β‐catenin activator, was purchased from TargetMol (Boston, MA, USA); IWR‐1‐endo, a Wnt/β‐catenin inhibitor, was purchased from Selleck (Houston, TX, USA). pcDNA3‐V5/His‐MAGI3 and GFP/GFP‐MAGI3 plasmids were kindly provided by R. Hall (Emory University, GA).

Techniques: Migration, Activation Assay, Over Expression, Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics and Phosphoproteomics Revealed Dysregulated Kinases and Potential Therapy for Liver Fibrosis

doi: 10.1016/j.mcpro.2025.100991

Figure Lengend Snippet: Biological function analysis of identified upregulated kinases. A , the overlap between the predicted and upregulated identified kinases. Of the predicted 198 kinases, two were upregulated at the protein level, 12 were upregulated at the phosphorylation level, and one kinase overlapped. B , the expression of STK4 was upregulated at the proteome level by LC–MS/MS, ∗∗ p < 0.01. C , the expression of GSK3α was upregulated at the proteome level by LC–MS/MS, ∗ p < 0.05. D , heat map and bar charts of kinases with upregulated phosphorylation levels. The bar chart shows the Log 2 (fold change) of kinases. E , biological process enrichment. F , KEGG pathway enrichment. The size of the circle represents the number of genes involved, and the color indicates the significance of the enrichment. G and H , relative mRNA level of STK4 ( G ) and GSK3α ( H ) in mouse livers after CCl 4 -induced early liver fibrosis was determined by RT–quantitative PCR, ∗∗∗ p < 0.001, ∗∗ p < 0.01. I , protein levels of STK4, GSK3α, and COL1A1 were determined by Western blotting. GAPDH serves as a loading control. J , the phosphorylated peptide, “GEPNVS(p)YICSR”, derived from GSK3α, was selected for quantification via selective reaction monitoring (SRM), ∗ p < 0.05. K , the phosphorylated peptide, “RGTS(p)PRPPEGGLGYSQLGDDDLK”, derived from CDK11B was selected for quantification via SRM, ∗ p < 0.05. L , the immunohistochemistry staining of STK4 and GSK3α in the liver tissues from two patients with liver cirrhosis. A negative control without antibody was selected. CCl 4 , carbon tetrachloride; GSK3α, glycogen synthase kinase 3α; KEGG, Kyoto Encyclopedia of Genes and Genomes; MS, mass spectrometry; STK4, serine/threonine-protein kinase 4.

Article Snippet: Following an 8-h culture period of isolated primary HSCs, the addition of 10 ng/ml of the recombinant TGF-β1 protein (Sino Biological; #10804-HNAC) occurred, either as a standalone treatment or in conjunction with 200 nM of the STK4 inhibitor SBP-3264 (MCE; #HY-132969), 100 nM GSK3α inhibitor laduviglusib (MCE; #HY-10182A), or 100 nM CDK11B inhibitor OTS964 (MCE; #HY-12467), respectively.

Techniques: Phospho-proteomics, Expressing, Liquid Chromatography with Mass Spectroscopy, Real-time Polymerase Chain Reaction, Western Blot, Control, Derivative Assay, Immunohistochemistry, Staining, Negative Control, Mass Spectrometry

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics and Phosphoproteomics Revealed Dysregulated Kinases and Potential Therapy for Liver Fibrosis

doi: 10.1016/j.mcpro.2025.100991

Figure Lengend Snippet: Inhibition of kinase activity by target inhibitors can reduce hepatic stellate cell (HSC) activation. A – D , the downstream substrate phosphorylation levels of three kinases in the liver of CCl 4 mice were monitored by SRM targeting. Detailed information about the phosphopeptides monitored by SRM can be found in . Phosphorylation levels of ARHGEF6 ( A ), a substrate of STK4, were upregulated after treatment with CCl 4 ; MPRIP ( B ) and ANK1 ( C ) are substrates of GSK3α. The phosphorylation level of MPRIP was upregulated after treatment with CCl 4 , whereas the ANK1 remained unchanged; phosphorylation levels of LIG1 ( D ), a substrate of CDK11B, were upregulated after induced by CCl 4 . ∗ p < 0.05; ns, not significant. E , basic information of three kinases (in Mus musculus and Homo sapiens ) and their reported small-molecule inhibitors. F , experimental design to validate the antihepatic fibrosis potential of small-molecule inhibitors targeting kinases. G and H , the expression of COL1A1 and αSMA was determined by Western blotting after treated with 10 ng/ml TGF-β1 alone or along with kinase inhibitors (200 nM STK4 inhibitor SBP-3264; 100 nM GSK3α inhibitor laduviglusib; 100 nM CDK11B inhibitor OTS964) in mouse primary HSCs ( G ) and LX-2 cell line ( H ). I , immunofluorescence showed that TGF-β stimulation could significantly inhibit the activation of HSCs by adding kinase inhibitors, and the production of COL1A1 was reduced. CCl 4 , carbon tetrachloride; GSK3α, glycogen synthase kinase 3α; SRM, selective reaction monitoring; TGF-β1, transforming growth factor beta-1.

Article Snippet: Following an 8-h culture period of isolated primary HSCs, the addition of 10 ng/ml of the recombinant TGF-β1 protein (Sino Biological; #10804-HNAC) occurred, either as a standalone treatment or in conjunction with 200 nM of the STK4 inhibitor SBP-3264 (MCE; #HY-132969), 100 nM GSK3α inhibitor laduviglusib (MCE; #HY-10182A), or 100 nM CDK11B inhibitor OTS964 (MCE; #HY-12467), respectively.

Techniques: Inhibition, Activity Assay, Activation Assay, Phospho-proteomics, Expressing, Western Blot, Immunofluorescence

Journal: Cell Proliferation

Article Title: Genome‐scale screening in a rat haploid system identifies Thop1 as a modulator of pluripotency exit

doi: 10.1111/cpr.13209

Figure Lengend Snippet: Thop1 ‐KO inhibits phosphorylation of ERK1/2 in the absence of PD0325901. (A) Respective global transcriptome profiles of Thop1 ‐KO GFP (#1 GFP , #2 GFP and #3 GFP ) and DA‐5‐3 GFP were hierarchically clustered. (B) The heatmap of representative pluripotent genes among DEGs of Thop1 ‐KO GFP and DA‐5‐3 GFP . (C) The heatmap of representative apoptosis genes among DEGs of Thop1 ‐KO GFP and DA‐5‐3 GFP . (D) Volcano plot depicting the differential expression analysis between Thop1 ‐KO GFP and DA‐5‐3 GFP and their relationships with the MAPK pathway, the GSK3 pathway and JAK–STAT pathway. Highlight points indicated well‐known genes of the three pathways, considering a fold change of 2.0 and p < 0.05. Only the MAPK pathway well‐known genes located to the DEGs. (E) Phase and FITC merged images of DA‐5‐3 GFP and Thop1‐ KO GFP when cultured withdrawing different factors (CHIR&LIF, PD&LIF, CHIR&PD, 2i&LIF) on Day 5. The red arrows indicated the differentiated cells. Scale bar, 100 μm. (F) FACS analysis of GFP‐positive cells in DA‐5‐3 GFP and Thop1‐ KO GFP when cultured withdrawing different factors (CHIR&LIF, PD&LIF, CHIR&PD, 2i&LIF) on Day 5. (G) Western blotting analysis of p‐ERK1/2, ERK1/2 and α‐TUBULIN in DA‐5‐3 GFP and Thop1‐ KO GFP when cultured in 2i/LIF medium or 2i/LIF medium without PD0325901

Article Snippet: The final concentrations of 2i (PD0325901 [MCE, HY‐10254], CHIR99021 [MCE, HY‐10182]) and human LIF (SinoBiological, 14890‐HNAH) were as follows: PD0325901, 1 μM; CHIR99021, 3 μM and LIF, 1000 U/ml.

Techniques: Expressing, Cell Culture, Western Blot