Journal: Clinical and Translational Medicine

Article Title: METTL14‐mediated m6A modification of ZFP14 inhibits clear cell renal cell carcinoma progression via promoting STAT3 ubiquitination

doi: 10.1002/ctm2.70232

Figure Lengend Snippet: ZFP14 destabilises STAT3 via the ubiquitin–proteasome pathway. (A and B) Alterations of STAT3 and phosphorylated‐STAT3 protein levels in ccRCC cells after ZFP14 over‐expression (A) and ZFP14 knockdown (B) detected by western blot assay. (C and D) Alteration of STAT3 mRNA level in ccRCC cells after ZFP14 over‐expression (C) and ZFP14 knockdown (D) detected by qRT‐PCR. (E and F) Alterations of STAT3 protein stabilities after ZFP14 over‐expression (E) and knockdown (F) detected by protein stability assay. (G) STAT3 protein levels in Caki‐1 cells of the indicated disposals detected by western blot assay. (H and I) Endogenous interaction between ZFP14 and STAT3 in Caki‐1 (H) and ACHN (I) cells detected by Co‐IP assay. (J and K) Exogenous interaction between Flag‐ZFP14 and His‐STAT3 in HEK293T cells detected by Co‐IP assay respectively using Flag‐tag (J) and His‐tag (K) antibodies for immunoprecipitations. (L and M) Alterations of ubiquitination levels of endogenous STAT3 in ccRCC cells after ZFP14 over‐expression (L) and ZFP14 knockdown (M) detected by ubiquitination assay. (N and O) Alterations of His‐STAT3 exogenous ubiquitination levels in HEK293T cells after Flag‐ZFP14 transfection (N) and ZFP14 knockdown (O) detected by ubiquitination assay. (P and Q) Alterations of His‐STAT3 exogenous ubiquitination of the indicated linkages in HEK293T cells after Flag‐ZFP14 transfection (P) and ZFP14 knockdown (Q) detected by ubiquitination assay. Data are presented as means ± SD from three independent experiments. Student's t ‐test was used for analyses. * p < .05, ** p < .01 and *** p < .001. ns, non‐significant.

Article Snippet: Cells, either stably transfected or 24 h post‐siRNA transfection, were cultured in media containing 100 μg/mL cycloheximide (Selleck, Inc., Houston, USA) for designated durations.

Techniques: Ubiquitin Proteomics, Over Expression, Knockdown, Western Blot, Quantitative RT-PCR, Stability Assay, Co-Immunoprecipitation Assay, FLAG-tag, Transfection

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 4: FAIM deficiency suppressed the effects of PACAP on alleviating hepatic lipid accumulation in HFD-fed mice. (A) FAIM protein level of mice liver was subjected to western blot. Statistical analysis of FAIM/b-actin was shown in a bar chart (right) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. CD group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFD group). (B) Schematic diagram of HFD mouse model with PACAP treatment (0.4 mg/kg) or control saline solution and lentivirus mediated Faim knockdown. (C) Hepatic Faim mRNA level was determined by qRT-PCR, n ¼ 3. (DeH) Modulation of Faim level in the liver influenced HFD-induced hepatic steatosis, (D) bodyweight, liver weight, and WAT weight; (E) liver TG and TC contents; (F) plasma TG, TC, HDL-c, and LDL-c levels; (G) fasting blood glucose level; blood glucose concentrations were measured on day 12 after lentivirus injection; (H) representative photomicrographs of Oil Red O and H&E staining of liver tissues in different groups (scale bars: 50 or 200 mM). (I and J) Ablation of Faim impaired glucose tolerance and insulin sensitivity in mice. IPGTT (I) and IPITT (J), blood glucose was measured at different time points after glucose or insulin injection (left), and quantification of the AUC (right). Data are presented as mean SEM, n ¼ 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Western Blot, Control, Saline, Knockdown, Quantitative RT-PCR, Clinical Proteomics, Injection, Staining

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 5: PACAP activates the FAIM-AMPK-IRb axis to govern the SREBP and lipid synthetic gene program. (A and B) PACAP activated AMPK-IRb signaling pathway and down- regulated the expression of lipogenesis genes in HFD-fed mice, such effects were suppressed following knockdown of Faim. Western blot analysis were used to detect the protein level of indicated antibodies. (C and D) The overexpression of FAIM activated AMPK-IRb signaling pathway, the protein levels of FAIM, p-AMPK, AMPK, p-IRb, IRb, and lipid synthesis genes SREBP1, SREBP2, FAS, SCD1, HMGCR were determined by western blot with quantifications. Data are presented as mean SEM, n ¼ 3 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Knockdown, Western Blot, Over Expression

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 6: Knockdown of Faim inhibited the effects of PACAP on decreasing lipid accumulation and activating AMPK-IRb signaling pathway. AML12 cells were transiently transfected with Faim siRNA or scramble siRNA in cell culture medium containing 200 mM PA or 10% BSA (as control of PA solution) for 24 h, then the cells were treated with PACAP (1 mM) and Max.D.4 (10 mM) for another 24 h as indicated. (A) Representative for Oil Red O staining of AML12 (Scale bars, 50 mM). (B and C) Impacts of Faim knockdown on cellular TG and TC level (B), glucose uptake and glycogen content (C) of AML12. (D and E) Faim knockdown impaired the activation of AMPK-IRb pathway in AML12 treated with PACAP, the protein levels were analyzed by western blot and quantitative measurements (right). (F and G) AML12 were exposed to PA for 24 h, then treated with PACAP (1 mM), Max.D.4 (10 mM) and Compound C (10 mM) (F) or AICAR (0.5 mM) (G) for another 24 h. The protein level of FAIM was detected by western blot analysis and quantitative measurements (down). Data are presented as mean SEM. n ¼ 3 (&P < 0.05, &&P < 0.01, &&&P < 0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PA þ PACAP group, OP < 0.05, OOP < 0.01, OOOP < 0.001 vs. PA þ PACAP þ Max.D.4 group).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Knockdown, Transfection, Cell Culture, Control, Staining, Activation Assay, Western Blot

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 7: PACAP activates the PAC1-PKA-CREB signaling pathway to stimulate FAIM expression. (A) mRNA level of Faim in PA-induced AML12 treated with PACAP, determined by qRT-PCR (n ¼ 3). (B) The effect of PACAP on the activation of PKA and CREB in PA induced AML12 cells, the protein levels of PKA, CREB and FAIM, and phosphorylation of PKA and CREB were determined by western blot. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PACAP (1 mM) group). (C) Schematic of the sequence of two CRE binding sites that CREB targets the WT or mutated promoter region of Faim mRNA. (DeF) Luciferase activities in AML12 transiently transfected with either wild type (WT), truncated, or mutant Faim promoter-luciferase reporter constructs. Then the cells were treated with PACAP (1 mM), or co- transfected with CREB cDNA or empty vectors. (G) Luciferase activities of FAIM WT reporter plasmids in AML12 cells with the treatment of KG-501 (a specific CREB inhibitor) and PACAP (1 mM) as indicated. Data are shown as mean SEM, n ¼ 3 or 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Phospho-proteomics, Western Blot, Sequencing, Binding Assay, Luciferase, Transfection, Mutagenesis, Construct

Journal: International Journal of Molecular Sciences

Article Title: miR-132-3p Modulates DUSP9-Dependent p38/JNK Signaling Pathways to Enhance Inflammation in the Amnion Leading to Labor

doi: 10.3390/ijms23031864

Figure Lengend Snippet: The role of p38 and JNK in DUSP9 siRNA-induced expression of proinflammatory cytokines and COX2 as well as PGE2 in WISH cells. WISH cells were transfected with si- DUSP9 or NC for 24 h, followed by 24 h of treatment with DMSO, p38 inhibitor SB203580 (10 μM), or JNK inhibitor SP600125 (20 μM). RT-qPCR analysis of the expression of IL-1β , TNF-α , COX2 ( A , G ), IL-6 , and IL-8 ( B , H ). Magnetic Luminex Assays of the secretion of IL-1β, TNF-α ( C , I ), IL-6, and IL-8 ( D , J ). Western blot analysis of COX2 expression ( E , K ). ELISA analysis of PGE2 level ( F , L ). Data were shown as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01 vs. control (0), # p < 0.05, ## p < 0.01 vs. si- DUSP9 .

Article Snippet: To verify the involvement of p38 and JNK signaling pathways in the induction of IL-1β, IL-6, IL-8, TNF-α, and COX2 as well as PGE2 secretion by DUSP9 siRNA, the cells were transfected with NC or si- DUSP9 for 24 h, followed by 24 h of treatment with dimethyl sulfoxide (DMSO), p38 inhibitor SB203580 (10 μM, Selleck, Houston, TX, USA), or JNK inhibitor SP600125 (20 μM, Selleck, Houston, TX, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Luminex, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Medical Sciences

Article Title: The impact of PRDX4 and the EGFR mutation status on cellular proliferation in lung adenocarcinoma

doi: 10.7150/ijms.36071

Figure Lengend Snippet: The receiver operating characteristic (ROC) curve analysis and Kaplan-Meier curves of the disease-free survival (DFS) in patients with Stage I LUAD after surgery according to the PRDX4 expression and the EGFR mutation status. (A) We selected 40% as the cut-off point for PRDX4, since the sum of sensitivity and specificity was the highest at this point. (B) Patients with EGFR mutations and high-PRDX4 showed significantly longer postsurgical DFS. (C) Patients with EGFR wild-type and low-PRDX4 showed significantly shorter postsurgical DFS.

Article Snippet: The cells were plated in 6-well plates and cultured in growth media at approximately 60% confluence, incubated for 24 h, and transfected for 72 h with 5 μg PRDX4 plasmid DNA duplexes in Lipofectamine ® 2000 and Opti-MEM medium (Life Technologies, Carlsbad, CA, USA).

Techniques: Expressing, Mutagenesis

Journal: International Journal of Medical Sciences

Article Title: The impact of PRDX4 and the EGFR mutation status on cellular proliferation in lung adenocarcinoma

doi: 10.7150/ijms.36071

Figure Lengend Snippet: The results of the real-time PCR and Western blotting. The PRDX4 mRNA (A, B) and protein (C) expression was remarkably increased after transfection of PRDX4 plasmid in A549 and PC-9. N, negative control vector; P, PRDX4 plasmid.

Article Snippet: The cells were plated in 6-well plates and cultured in growth media at approximately 60% confluence, incubated for 24 h, and transfected for 72 h with 5 μg PRDX4 plasmid DNA duplexes in Lipofectamine ® 2000 and Opti-MEM medium (Life Technologies, Carlsbad, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control

Journal: International Journal of Medical Sciences

Article Title: The impact of PRDX4 and the EGFR mutation status on cellular proliferation in lung adenocarcinoma

doi: 10.7150/ijms.36071

Figure Lengend Snippet: The overexpression of PRDX4 inhibited cell proliferation. The proliferation of A549 (A) and PC-9 (B) cells was analyzed using a cck-8 kit, 3 days after the transfection of PRDX4 plasmid DNAs or negative vectors. N, negative control vector; P, PRDX4 plasmid DNA. *p<0.05

Article Snippet: The cells were plated in 6-well plates and cultured in growth media at approximately 60% confluence, incubated for 24 h, and transfected for 72 h with 5 μg PRDX4 plasmid DNA duplexes in Lipofectamine ® 2000 and Opti-MEM medium (Life Technologies, Carlsbad, CA, USA).

Techniques: Over Expression, CCK-8 Assay, Transfection, Plasmid Preparation, Negative Control

Journal: International Journal of Medical Sciences

Article Title: The impact of PRDX4 and the EGFR mutation status on cellular proliferation in lung adenocarcinoma

doi: 10.7150/ijms.36071

Figure Lengend Snippet: The relationship between the EGFR mutation status and the expression of PRDX4

Article Snippet: The cells were plated in 6-well plates and cultured in growth media at approximately 60% confluence, incubated for 24 h, and transfected for 72 h with 5 μg PRDX4 plasmid DNA duplexes in Lipofectamine ® 2000 and Opti-MEM medium (Life Technologies, Carlsbad, CA, USA).

Techniques: Mutagenesis, Expressing