Journal: Nucleic Acids Research

Article Title: An engineered glutamic acid tRNA for efficient suppression of pathogenic nonsense mutations

doi: 10.1093/nar/gkaf532

Figure Lengend Snippet: tRNA GluV13 rescues endogenous nonsense mutations in TP53 . ( A ) Representative western blots of cells transfected with an empty vector or a plasmid containing three copies of tRNA Pyl (control tRNA) or tRNA GluV13 CUA . Lysates were probed for p53 and CBP80 (loading control) 48 h after transfection. Bands corresponding to full-length (FL p53) and truncated (TR p53) are indicated. ( B ) p53-dependent luciferase activity in cells transfected with an empty vector or a plasmid containing three copies of tRNA Pyl (control tRNA) or tRNA GluV13 CUA . Data are displayed as the fold change in luciferase activity relative to cells transfected with an empty vector and reported as mean ± SD, n = 3. * P < 0.05, *** P < 0.0005, ns = not significant, unpaired Student’s t -test.

Article Snippet: The following day, cells were transfected with 50 ng of the indicated sup-tRNA plasmid (or an empty vector), 50 ng of p53-dependent Fluc reporter plasmid PG13-luc (a gift from Bert Vogelstein; Addgene Plasmid # 16442; http://n2t.net/addgene:16442; RRID:ADDGENE_16442 ), and 15 ng of pLX313- Renilla .

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: Alkaloids 1 – 8 and their effect on EGF-induced neoplastic transformation of JB6 P + Cl41 cells. ( A ) Structures of guanidine alkaloids 1 – 8 isolated from the marine sponge Monanchora pulchra ; ( B ) representative pictures of microscopic fields of EGF-induced colonies of JB6 P + Cl41 cells in soft agar treated with urupocidin A ( 6 ) at the indicated concentrations; ( C ) Inhibition of EGF-induced neoplastic transformation of JB6 P + Cl41 cells by compounds 1 – 4 , 7 , and 8 . INCC 50 —concentration leading to a 50% inhibition of colonies formation. Ratios of IC 50 /INCC 50 were calculated using the IC 50 values from generated by using the MTT assay. * indicates a statistically significant difference ( p < 0.05) from the control value.

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Transformation Assay, Isolation, Inhibition, Concentration Assay, Generated, MTT Assay, Control

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: IC 50 of alkaloids 1 – 8 in human cancer HeLa cells and mouse non-malignant JB6 P + Cl41 cell lines after 48 h of treatment.

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Activity Assay

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: Effect of alkaloids 1 – 8 on MAPK/AP-1 signaling. ( A ) Effect on basal AP-1-dependent transcriptional activity ( ○ ) and viability (□) of JB6 Cl41 cells stably expressing a luciferase reporter gene controlled by the AP-1 DNA binding sequence after 12 h of treatment. Cell viability was measured using MTT assay; ( B ) effect on the activation of JNK1/2 and ERK1/2. JB6 P + Cl41 cells were treated with the compounds 1 – 8 at the indicated concentrations for 48 h, and the level of protein expression was assessed by Western blotting. The intensities of p-JNK1/2 and p-ERK1/2 signals were quantified with Quantity One 4.6 software (Bio-Rad, Hercules, CA, USA) and normalized against the signals of total JNK1/2 and ERK1/2, correspondently; and ( C ) the effect of SP600125 (specific JNK1/2 inhibitor) on the survival of JB6 P + Cl41 cells treated with compounds 1 – 8 . Cells were co-treated with different concentrations of the individual drugs or their combination for 48 h. Cell viability was measured by MTT assay and the combinational index (CI) values were calculated with CompuSyn software (v.1.0., ComboSyn Inc., Paramus, NJ, USA) using the Chou-Talalay method. The ratio of the substances and the effects are presented in the . * indicates a statistically significant difference ( p < 0.05) from the control value.

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Activity Assay, Stable Transfection, Expressing, Luciferase, Binding Assay, Sequencing, MTT Assay, Activation Assay, Western Blot, Software, Control

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: Effect alkaloids 1 – 8 on basal p53-dependent transcriptional activity ( ○ ) and viability (□) of JB6 Cl41 cells stably expressing a luciferase reporter gene controlled by the p53 DNA binding sequence after 12 h of treatment. Cell viability was measured using MTT assay. * indicates a statistically significant difference ( p < 0.05) from the control value.

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Activity Assay, Stable Transfection, Expressing, Luciferase, Binding Assay, Sequencing, MTT Assay, Control

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: Summary of observed effects of compounds 1 – 8 .

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Activity Assay, Phospho-proteomics, Activation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of the lncRNA MIAT prevents podocyte injury and mitotic catastrophe in diabetic nephropathy

doi: 10.1016/j.omtn.2022.03.001

Figure Lengend Snippet: Effects of Miat on podocyte mitotic catastrophe are mediated by Sox4 in vitro (A) Flow cytometry showed the apoptosis rate of podocytes (n = 4). (B) Flow cytometry illustrated the cell-cycle progression of podocytes (n = 3). (C) Immunostaining of mitosis with antibodies against α-tubulin (green; to indicate mitotic spindles) and histone-3 phosphorylated at serine 10 (H3-Ser10) (red; to indicate metaphase) (n = 3). Scale bar, 50 μm. (D) The percentages of podocytes with normal or abnormal mitotic processes were monitored and analyzed (n = 3). Scale bar, 10 μm. (E) Western blotting depicted the expression of Sox4, p53, p21 cip1/waf1 , CyclinB, and cdc2 (n = 3). (F–J) Levels of the Sox4 (n = 3), p53 (n = 5), p21 cip1/waf1 (n = 4), CyclinB (n = 4), and cdc2 (n = 3) mRNAs. Error bars represent ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: The Mus-pmirGLO- Miat -WT ( Miat -WT) plasmid, pmirGLO- Miat -MUT ( Miat -MUT) plasmid, pmirGLO- Sox4 -WT/pmirGLO- Sox4 -MUT ( Sox4 -3′-UTR-WT/MUT) plasmids, miR-130b-3p mimic ( miR-130b-3p ), miR-130b-3p NC (miR-NC), Sox4 -NC plasmid, Sox4 -OE plasmid, pG13-Luc plasmid, and MG15-Luc plasmid were provided by RiboBio (Guangzhou, China).

Techniques: In Vitro, Flow Cytometry, Immunostaining, Western Blot, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of the lncRNA MIAT prevents podocyte injury and mitotic catastrophe in diabetic nephropathy

doi: 10.1016/j.omtn.2022.03.001

Figure Lengend Snippet: Podocyte injury and mitotic catastrophe require Sox4 in vitro (A) Western-blot analysis showed the expression of Sox4, p53, p21 cip1/waf1 , CyclinB, and cdc2 (n = 3). (B) Flow cytometry analysis illustrated the cell-cycle progression of podocytes (n = 3). (C) Immunostaining of mitosis with antibodies against α-tubulin (green) and H3-Ser10 (red) (n = 3). Scale bar, 10 μm. (D)The percentages of podocytes with normal or abnormal mitotic processes were monitored and analyzed (n = 3). (E) Morphological changes in the podocyte cytoskeleton (n = 3). Scale bar, 50 μm. (F) The expression of Desmin, podocin, synaptopodin, and ZO-1 was measured using western blotting (n = 3). (G–I) Immunofluorescence staining was performed to determine the intensities of podocin (G), Desmin (H), and p-cadherin (I) under HG conditions (n = 4). Scale bar, 50 μm. (J) Cell migration was detected using Transwell assays (n = 5). Scale bar, 100 μm. (K) Flow cytometry revealed the apoptosis rate of podocytes (n = 5). (L) Flow cytometry clarified the proportion of podocytes in G2/M phase (n = 4). (M) Immunostaining illustrated morphological changes associated with normal or abnormal mitosis using antibodies against α-tubulin (green) and H3-Ser10 (red) (n = 3). Scale bar, 10 μm. (N) The percentages of podocytes with normal or abnormal mitotic processes were monitored and analyzed (n = 3). Error bars represent ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: The Mus-pmirGLO- Miat -WT ( Miat -WT) plasmid, pmirGLO- Miat -MUT ( Miat -MUT) plasmid, pmirGLO- Sox4 -WT/pmirGLO- Sox4 -MUT ( Sox4 -3′-UTR-WT/MUT) plasmids, miR-130b-3p mimic ( miR-130b-3p ), miR-130b-3p NC (miR-NC), Sox4 -NC plasmid, Sox4 -OE plasmid, pG13-Luc plasmid, and MG15-Luc plasmid were provided by RiboBio (Guangzhou, China).

Techniques: In Vitro, Western Blot, Expressing, Flow Cytometry, Immunostaining, Immunofluorescence, Staining, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of the lncRNA MIAT prevents podocyte injury and mitotic catastrophe in diabetic nephropathy

doi: 10.1016/j.omtn.2022.03.001

Figure Lengend Snippet: Sox4 is critical for p53 stabilization and function (A–D) Western blotting (A) and real-time PCR illustrated the expression of Sox4 (B), p53 (C), and p21 cip1/waf1 (D) under HG conditions (n = 3). (E) Lysates from scramble shRNA or Sox4 shRNA-transfected podocytes treated with 20 mg/mL cycloheximide (CHX) at the indicated time points (0, 20, 40, and 60 min; left panel), and quantification of the relative p53 levels were quantified (right panel; n = 3). (F) Luciferase reporter assay revealed the interaction between Sox4 and p53 transcriptional activity in podocytes (n = 3). (G) Sox4 and p53 were endogenously expressed in podocytes, as shown by immunoprecipitation (IP) with an anti-Sox4 antibody (n = 3). (H) Podocytes were transfected with His-ubiquitin with or without Sox4 shRNA for 24 h and were incubated in the presence or absence of HG for another 48 h, followed by treatment with 20 μmmol/L MG-132 for 8 h. Cell lysates were immunoprecipitated and analyzed using immunoblotting with an anti-p53 antibody (n = 3). IB, immunoblotting. (I) Podocytes were transfected with FLAG-p53, GFP, HA-Mdm2, and Myc-Sox4 vectors (1, 2, and 4 μg) for 24 h, and the expression of p53 in podocyte lysates was analyzed using IB (n = 3). (J) Coprecipitation of p53 with Mdm2 was observed after Sox4 overexpression (2 and 4 μg) in podocytes in the presence of MG-132 for 8 h. Cell lysates were IP with an anti-HA antibody and were subjected to IB (n = 3). (K) Podocytes transfected with the control or Sox4 shRNA were treated with or without HG, and the levels of p53 acetylation at the Lys-373 and -382 residues were analyzed using IB with an anti-acetyl-p53 (Lys-373 or -382) antibody (n = 3). (L) Podocytes were transfected with FLAG-p53, Myc-Sox4, HA-CBP, and HA-p300 plasmids for 24 h. Cell lysates were IP with an anti-HA antibody and IB with an anti-FLAG antibody, and the whole-cell lysates (WCLs) were analyzed using IB (n = 3). Error bars represent ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: The Mus-pmirGLO- Miat -WT ( Miat -WT) plasmid, pmirGLO- Miat -MUT ( Miat -MUT) plasmid, pmirGLO- Sox4 -WT/pmirGLO- Sox4 -MUT ( Sox4 -3′-UTR-WT/MUT) plasmids, miR-130b-3p mimic ( miR-130b-3p ), miR-130b-3p NC (miR-NC), Sox4 -NC plasmid, Sox4 -OE plasmid, pG13-Luc plasmid, and MG15-Luc plasmid were provided by RiboBio (Guangzhou, China).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, shRNA, Transfection, Luciferase, Reporter Assay, Activity Assay, Immunoprecipitation, Ubiquitin Proteomics, Incubation, Over Expression, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of the lncRNA MIAT prevents podocyte injury and mitotic catastrophe in diabetic nephropathy

doi: 10.1016/j.omtn.2022.03.001

Figure Lengend Snippet: Miat promotes Sox4 expression by targeting miR-130b-3p in vitro (A) The levels of miR-130b-3p in the plasma from healthy controls (n = 22, −0.8066 ± 0.4477) and clinical patients with DN (n = 61, −1.124 ± 0.4351). (B) A luciferase reporter assay was performed to evaluate the interaction between miR-130b-3p and Miat in HEK293 cells (n = 3). (C) RIP revealed the interaction of Miat and miR-130b-3p in podocytes (n = 3). (D) Real-time PCR revealed the expression of miR-130b-3p in podocytes in the presence of miR-130b-3p mimic and Miat -WT plasmid or Miat -MUT plasmid (n = 3). (E) Dual-luciferase reporter assay showed the interaction between miR-130b-3p and the Sox4 3′ UTR in HEK293 cells (n = 3). (F) Western blotting manifested the expression of Sox4 in podocytes after pretreatment with the miR-130b-3p mimic (n = 3). (G) Real-time PCR revealed Sox4 mRNA expression in podocytes (n = 5). (H) Western blotting was used to assess Sox4 expression in different groups of podocytes (n = 3). EV, empty vector. (I) The expression of Sox4 in podocytes was revealed by western blotting (n = 3). (J) The expression of Sox4 was measured in podocytes using western blotting (n = 3). Error bars represent ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: The Mus-pmirGLO- Miat -WT ( Miat -WT) plasmid, pmirGLO- Miat -MUT ( Miat -MUT) plasmid, pmirGLO- Sox4 -WT/pmirGLO- Sox4 -MUT ( Sox4 -3′-UTR-WT/MUT) plasmids, miR-130b-3p mimic ( miR-130b-3p ), miR-130b-3p NC (miR-NC), Sox4 -NC plasmid, Sox4 -OE plasmid, pG13-Luc plasmid, and MG15-Luc plasmid were provided by RiboBio (Guangzhou, China).

Techniques: Expressing, In Vitro, Clinical Proteomics, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of the lncRNA MIAT prevents podocyte injury and mitotic catastrophe in diabetic nephropathy

doi: 10.1016/j.omtn.2022.03.001

Figure Lengend Snippet: Miat enhances podocyte injury and G2/M-phase arrest by binding to miR-130b-3p in vivo (A) Morphological changes in the FPs and GBM of the mice at 12 weeks were observed under TEM. The GBM thickness and podocyte effacement were qualified (n = 3). Scale bar, 1 μm. (B) PAS staining of mouse tissues at 12 weeks. The relative mesangial area and glomerular volume were qualified (n = 4). Scale bar, 50 μm. (C) Masson’s trichrome staining of mouse tissues at 12 weeks (n = 4). Scale bar, 50 μm. (D–G) Immunofluorescence staining for the podocyte-specific markers Desmin (D), podocin (E), synaptopodin (F), and ZO-1 (G) in mice at 12 weeks (n = 3). Scale bar, 50 μm. (H) Immunofluorescence staining showed the expression of WT-1 in the mouse glomeruli at 12 weeks (n = 3). Scale bar, 50 μm. (I) Levels of injury markers were confirmed in primary mouse podocytes at 12 weeks using western blotting (n = 3). (J) Western-blot analysis showed the expression of Sox4, p53, p21 cip1/waf1 , CyclinB, and cdc2 in primary mouse podocytes at 12 weeks (n = 3). (K) Cell-cycle progression of primary mouse podocytes at 12 weeks was assessed using flow cytometry (n = 4). Error bars represent ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: The Mus-pmirGLO- Miat -WT ( Miat -WT) plasmid, pmirGLO- Miat -MUT ( Miat -MUT) plasmid, pmirGLO- Sox4 -WT/pmirGLO- Sox4 -MUT ( Sox4 -3′-UTR-WT/MUT) plasmids, miR-130b-3p mimic ( miR-130b-3p ), miR-130b-3p NC (miR-NC), Sox4 -NC plasmid, Sox4 -OE plasmid, pG13-Luc plasmid, and MG15-Luc plasmid were provided by RiboBio (Guangzhou, China).

Techniques: Binding Assay, In Vivo, Staining, Immunofluorescence, Expressing, Western Blot, Flow Cytometry