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: 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