e2 ubiquitin conjugation enzyme  (OriGene)

Journal: Acta Pharmaceutica Sinica. B

Article Title: TRIB3 promotes pulmonary fibrosis through inhibiting SLUG degradation by physically interacting with MDM2

doi: 10.1016/j.apsb.2023.01.008

Figure Lengend Snippet: Dysfunction of MDM2 suppresses SLUG degradation. (A) Quantitative analyses of Slug mRNA content in AEC2s from the indicated mice. N.S.: not significant. (B) Quantitative analyses of SLUG degradation in AEC2s in the presence of protein synthesis inhibitor cycloheximide (CHX) (10 μg/mL) and 3 MA (5 mmol/L) or MG132 (10 μmol/L). The half-lives ( t 1/2 ) of Slug degradation were calculated by one phase decay analysis ( n = 3). GAPDH was used as the loading control for IB. (C, D) IP assay analyses of ubiquitinated Slug in lung tissues from mBLM mice (C) and SiO 2- challenged mice (D) ( n = 5). (E) Protein levels of Slug-related E3 ligases in AEC2s sorted from the indicated mice ( n = 3). GAPDH was used as the loading control for IB. (F–H) Confocal images showing the co-localization of MDM2 and pro-SPC in lung tissues from PF patients (F) and from mice after mBLM (G) or SiO 2 (H) exposure ( n = 3). Scale bars, 50 μm. (I) E-cad mRNA levels in sorted AEC2s after transfection with the indicated siRNA ( n = 3). (J) Sample immunoblots showing the E3 activity of MDM2 in AECs from mBLM mice in the presence of E1, E2 (UbcH5c), Ub, ATP, and subtract (SLUG) ( n = 3). Statistical significance between the two groups was determined by unpaired two-tailed Student's t -test; Statistical significance among groups was determined by one-way ANOVA ∗ P < 0.05 ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Purified SLUG protein (200 ng, OriGene) was pre-incubated with MDM2 protein (200 ng, Millipore) on ice for 30 min.

Techniques: Control, Transfection, Western Blot, Activity Assay, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: TRIB3 promotes pulmonary fibrosis through inhibiting SLUG degradation by physically interacting with MDM2

doi: 10.1016/j.apsb.2023.01.008

Figure Lengend Snippet: MDM2 inhibits SLUG degradation by physically interacting with TRIB3. (A) AEC2s were sorted form lung tissues of the vehicle- or mBLM-exposed mice. AEC2s lysate was IP with anti-Mdm2 antibody, and silver-staining shows the MDM2 binding proteins in AEC2s. (B) Quantitation of TRIB3 expression in lung samples from healthy controls or patients with pulmonary fibrosis (PF) ( n = 6). GAPDH was used as the loading control for immunoblotting. (C) Sample confocal images showing co-localization of TRIB3 and pro-SPC in lung tissues from PF patients ( n = 3). Scale bars, 50 μm. (D) The protein level of TRIB3 in AEC2s was detected with IB ( n = 3). GAPDH was used as the loading control for IB. (E) IP assay analyses of the TRIB3/MDM2 interaction in lung epithelial cells. Cells extracts were IP with normal IgG or anti-TRIB3 antibody (Ab) and blotted with anti-MDM2 Ab ( n = 3). (F) GST pull-down assay showing in vitro interaction of TRIB3/MDM2 ( n = 3). GST-only protein was used as the negative control. GST fusion proteins were stained with Coomassie Blue. (G–I) Confocal images showing the co-localization of MDM2 and TRIB3 in lung tissues from PF patients (G) and in lung tissues from the mice after mBLM (H) or SiO 2 injury (I) ( n = 3). Scale bars, 50 μm. (J) Competitive ELISA analyses of binding of SLUG to MDM2 in the presence of TRIB3-GST or GST-tagged protein ( n = 3). (K) Sample immunoblots showing the E3 activity of MDM2 in the presence of E1, E2 (UbcH5c), Ub, ATP, and subtract (SLUG), plus or minus TRIB3, in vitro ( n = 3). (L) Sample immunoblots showing UbcH5c–Ub conjugation in the presence of E1, Ub, and ATP plus or minus TRIB3 in vitro ( n = 3). (M) Sample immunoblots showing the binding ability of MDM2 to UbcH5c–Ub ( n = 3). (N) Sample immunoblots showing UbcH5c–Ub conjugation in the presence of TRIB3 ( n = 3). Statistical significance between the two groups was determined by unpaired two-tailed Student's t -test. ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Purified SLUG protein (200 ng, OriGene) was pre-incubated with MDM2 protein (200 ng, Millipore) on ice for 30 min.

Techniques: Silver Staining, Binding Assay, Quantitation Assay, Expressing, Control, Western Blot, Pull Down Assay, In Vitro, Negative Control, Staining, Competitive ELISA, Activity Assay, Conjugation Assay, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: TRIB3 promotes pulmonary fibrosis through inhibiting SLUG degradation by physically interacting with MDM2

doi: 10.1016/j.apsb.2023.01.008

Figure Lengend Snippet: Disturbing the TRIB3/MDM2 interaction promotes SLUG degradation. (A) I-TASSER server analyses of the secondary structure of the TRIB3 binding region in MDM2 and the peptide sequences containing the indicated α -helical sequence and protective AA. Highlighted sequence is the α -helix structure in the peptides. (B) Surface plasmon resonance (SPR) analyses of the kinetic interaction between MR2 and TRIB3 ( n = 3). (C, D) Sample immunofluorescence image (C) and flow cytometry plot (D) showing the cellular penetration of MR2, PMR2 and PMR3 conjugated with FAM ( n = 3). Scale bars, 20 μm. (E) Sample immunoblot shows PMR2 but not PMR3 disturbing the interaction of TRIB3/MDM2 in primary human alveolar epithelial cells (PHAECs) ( n = 3). (F) Sample immunoblot showing PMR2 restoring the interaction of MDM2/SLUG in PHAECs ( n = 3). (G) Sample immunoblots showing PMR2 but not PMR3 restoring the TRIB3-suppressed ubiquitination of SLUG in cells ( n = 3). (H) Quantitative analyses of SLUG and MDM2 expression in TRIB3-overexpressing PHAECs treated with 5 μmol/L PMR3 or PMR2 or 10 μmol/L MG132 ( n = 3). GAPDH was used as the loading control for IB. Statistical significance among groups was determined by one-way ANOVA. ∗∗ P < 0.01.

Article Snippet: Purified SLUG protein (200 ng, OriGene) was pre-incubated with MDM2 protein (200 ng, Millipore) on ice for 30 min.

Techniques: Binding Assay, Sequencing, SPR Assay, Immunofluorescence, Flow Cytometry, Western Blot, Ubiquitin Proteomics, Expressing, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: TRIB3 promotes pulmonary fibrosis through inhibiting SLUG degradation by physically interacting with MDM2

doi: 10.1016/j.apsb.2023.01.008

Figure Lengend Snippet: Targeting SLUG degradation restores LAR and reduces PF. (A) Structural analysis of the stapled MR2 peptide helices. (B) The indicated concentration of stapled MR2 peptides was passed over immobilized TRIB3 on CM5 sensor chips, and the kinetic interaction of the peptides with TRIB3 was determined by SPR analysis ( n = 3). (C) The helicity of the peptides was detected via circular dichroism analysis. (D–F) Masson staining (D), hydroxyproline content in the right lung (E), and Crs (F) were assessed to evaluate PF and lung function ( n = 8). Scale bars, 200 μm. (G) Sample blots show the expression of Slug and Slc34a2 in AEC2s from the indicated mice ( n = 5). (H) Co-staining and quantification of pro-SPC and Ki67 in lung tissues from fibrotic mice ( n = 3). Scale bars, 50 μm. (I) Co-localization of the AEC2 marker NKX2.1 and AEC1 marker HOPX in lung tissues from mBLM-challenged mice ( n = 3). Scale bars, 50 μm. (J) Images of the indicated AEC2 colonies growing in a 3D co-culture system and the colony-forming efficiency of AEC2s from vehicle or mBLM-challenged mice ( n = 3). (K) The colony-forming efficiency of AEC2s from patients with or without PF treated with SMR3 (5 mmol/L) ( n = 5). (L) The Aqp5 mRNA level in colonies formed by AEC2s from the indicated mice ( n = 3). (M) Schematic diagram illustrating that MDM2 dysfunction-induced Slug accumulation promotes PF development by inhibiting LAR and that the anti-fibrotic role of SMR3 results from disturbance of the TRIB3/MDM2 interaction. Statistical significance between the two groups was determined by unpaired two-tailed Student's t -test; Statistical significance among groups was determined by one-way ANOVA. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Purified SLUG protein (200 ng, OriGene) was pre-incubated with MDM2 protein (200 ng, Millipore) on ice for 30 min.

Techniques: Concentration Assay, Circular Dichroism, Staining, Expressing, Marker, Co-Culture Assay, Two Tailed Test

Journal: Oncology reports

Article Title: Fangchinoline exerts antitumour activity by suppressing the EGFR‑PI3K/AKT signalling pathway in colon adenocarcinoma.

doi: 10.3892/or.2020.7857

Figure Lengend Snippet: Figure 3. FAN represses EMT and EMT regulatory factors in colon adenocarcinoma cells. (A and B) E‑Cadherin, N‑cadherin, vimentin, MMP2 and MMP9 expression following treatment with FAN (0‑7 µM, 48 h) was determined by western blotting. (C and D) The expression of related transcription factors after FAN treatment (0‑7 µM, 48 h) was measured by western blotting. *P<0.05 and **P<0.01 vs. 0 µM group. FAN, fangchinoline; EMT, epithelial‑mesenchymal transition; SNAIL, zinc finger protein SNAI1; SLUG, zinc finger protein SNAI2; ZEB1, zinc finger E‑box‑binding homeobox 1.

Article Snippet: After the membranes were blocked with 5% non-fat milk in TBS with 0.1% Tween-20 for 1 h at room temperature, they were incubated overnight at 4 ̊C with primary antibodies targeting the following proteins (as appropriate): Phosphorylated (p)‐AKT (Ser473; polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 4060S; Cell Signaling Technology, Inc.), EGFR (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 18986‐1‐AP; ProteinTech Group, Inc.), E‐cadherin (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 20874‐1‐AP; ProteinTech Group, Inc.), N‐cadherin (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 22018‐1‐AP; ProteinTech Group, Inc.), vimentin (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 10366‐1‐AP; ProteinTech Group, Inc.), MMP2 (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 10373‐2‐AP; ProteinTech Group, Inc.), MMP9 (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 10375‐2‐AP; ProteinTech Group, Inc.), CD133 (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 66666‐1‐Ig; ProteinTech Group, Inc.), cyclin D1 (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 60186‐1‐Ig; ProteinTech Group, Inc.), Bax (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 50599‐2‐Ig; ProteinTech Group, Inc.), Bcl‐2 (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 12789‐1‐AP; ProteinTech Group, Inc.), zinc finger E‐box‐binding homeobox 1 (ZEB1; polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 21544‐1‐AP; ProteinTech Group, Inc.), ZEB2 (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 14026‐1‐AP; ProteinTech Group, Inc.), zinc finger protein SNAI1 (SNAIL; polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 13099‐1‐AP; ProteinTech Group, Inc.), zinc finger protein SNAI2 (SLUG; polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 12129‐1‐AP; ProteinTech Group, Inc.), AKT (polyclonal, rabbit anti‐human IgG; 1:1,000; cat. no. 10176‐2‐AP; ProteinTech Group, Inc.) and β-actin (polyclonal, mouse anti‐human IgG; 1:1,000; cat. no. TA‐09; OriGene Technologies, Inc.).

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: Myristoylated alanine rich protein kinase C substrate is a potential cancer prognostic factor that regulates cell migration and invasion in glioblastoma.

doi: 10.3892/or.2019.7009

Figure Lengend Snippet: Figure 3. MARCKS regulates the expression of EMT‑associated genes in GBM cells. (A) Knockdown of the expression of MARCKS by si‑MARCKS in U87 and LN229 cells enhanced E‑cadherin expression, but decreased the expression of EMT‑marker genes including β‑catenin, N‑cadherin and vimentin. *P<0.05, **P<0.01 vs. si‑NC. (B) In si‑MARCKS‑transfected U87 and LN229 cells, the protein expression of Snail was downregulated and E‑cadherin was upregulated. Slug expression was unchanged. *P<0.05, **P<0.01 vs. si‑NC. MARCKS, myristoylated alanine‑rich C kinase substrate; si, small interfering RNA; NC, negative control; Snail, zinc finger protein SNAI1; Slug, zinc finger protein SNAI2.

Article Snippet: Western blot analysis was performed as previously described (15,16) with primary rabbit polyclonal antibodies including those against: MARCKS (cat. no. ab52616; 1:5,000; Abcam, Cambridge, UK), PI3K, phosphorylated (p)PI3K (Tyr458; cat. no. 9655; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), Akt (cat. no. 4691; 1:1,000, Cell Signaling Technology, Inc.), pAkt (Ser473; cat. no. 4060; 1:2,000; Cell Signaling Technology, Inc.), β‐catenin (1:1,000; cat. no. 8480; Cell Signaling Technology, Inc.), N‐cadherin (1:1,000; cat. no. 13116; Cell Signaling Technology, Inc.), vimentin (1:1,000; cat. no. 5741; Cell Signaling Technology, Inc.), E-cadherin (1:1,000; cat. no. 3195; Cell Signaling Technology, Inc.), Zinc finger protein SNAI1 (Snail; 1:1,000; cat. no. 3879; Cell Signaling Technology, Inc.), Zinc finger protein SNAI2 (Slug; 1:1,000; cat. no. 9585; Cell Signaling Technology, Inc.) and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.).

Techniques: Expressing, Knockdown, Small Interfering RNA, Negative Control