Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

doi: 10.1186/s10020-023-00604-x

Figure Lengend Snippet: Fig. 5 Attenuated TG-induced ER stress, oxidative stress and apoptosis in HTR-8/SVneo cells by overexpressing TMBIM6. A–H Representative WB images and densitometry quantification of TMBIM6, GRP78, CHOP, NRF2, HO-1, Bax and Bcl-2 in different groups. The data were normalized to β-actin. I, J ROS production detected by DCFH-DA. Photographs were obtained at 100 × (Scale bar 100 μm). K, L Apoptosis rate in different groups detected by flow cytometry. M Representative transwell photos of HTR-8/SVneo cells in different groups (100 × magnification, Scale bar 100 μm). N Qualified data shown in M. Oe-Con: Overexpression of control plasmid; Oe-TMBIM6: Overexpression of TMBIM6 plasmid. n = 3. *p < 0.05, **p < 0.01

Article Snippet: Primary antibodies against METTL3 (15073-1-AP, 1:300; Proteintech, China) and TMBIM6 (26782-1-AP, 1:300; Proteintech, China) were

Techniques: Flow Cytometry, Over Expression, Control, Plasmid Preparation

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

doi: 10.1186/s10020-023-00604-x

Figure Lengend Snippet: Fig. 6 Overexpression of TMBIM6 partially neutralized the effects of METTL3 on TG-induced ER stress, oxidative stress and apoptosis in HTR-8/SVneo cells. A–I Representative WB images and densitometry quantification of METTL3, TMBIM6, GRP78, CHOP, NRF2, HO-1, Bax and Bcl-2 in different groups. The data were normalized to β-actin. J, K ROS production detected by DCFH-DA. Photographs were obtained at 100 × (Scale bar 100 μm). L, M Apoptosis rate in different groups detected by flow cytometry. N Representative transwell photos of HTR-8/SVneo cells in different groups (100 × magnification, Scale bar 100 μm). O Qualified data shown in L. Oe-Con: Overexpression of control plasmid; Oe-METTL3: Overexpression of METTL3 plasmid; Oe-TMBIM6: Overexpression of TMBIM6 plasmid. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Primary antibodies against METTL3 (15073-1-AP, 1:300; Proteintech, China) and TMBIM6 (26782-1-AP, 1:300; Proteintech, China) were

Techniques: Over Expression, Flow Cytometry, Control, Plasmid Preparation

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

doi: 10.1186/s10020-023-00604-x

Figure Lengend Snippet: Fig. 7 METTL3 affected the expression of TMBIM6 by regulating TMBIM6 mRNA stability via YTHDF2 involvement. A–C The knockdown efficiency of different sh-YTHDF2s determined by qRT-PCR and WB. D The relative expression levels of the TMBIM6 mRNA in sh-Con and sh-YTHDF2 group determined by qRT-PCR. E–H Representative WB images and densitometry quantification of TMBIM6 in different groups. The data were normalized to β-actin. I, J Transcript stability assay of TMBIM6 in different groups. n = 3. ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Primary antibodies against METTL3 (15073-1-AP, 1:300; Proteintech, China) and TMBIM6 (26782-1-AP, 1:300; Proteintech, China) were

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Stability Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

doi: 10.1186/s10020-023-00604-x

Figure Lengend Snippet: Fig. 9 The schematic of molecular mechanism about how METTL3 affected TMBIM6 expression resulting in trophoblastic dysfunction and PE. ① When placentas got injured by many detrimental factors, such as I/R, the trophoblasts were under ER stress and METTL3 expression was upregulated increasing the m6A modification of TMBIM6 mRNA in trophoblasts. ② The methylated TMBIM6 mRNA was recognized by YTHDF2. ③ YTHDF2 decreased the stability of TMBIM6 mRNA and increased its degradation. ④ TMBIM6 expression was downregulated due to the decreased TMBIM6 mRNA and its translation. ⑤ Downregulation of TMBIM6 could not maintain the ER homeostasis, which aggravated ER stress. Moreover, the apoptosis and ROS production were increased when TMBIM6 was decreased. ⑥ The trophoblasts functions were impaired with the placental functions abnormal, which consequently contributed to PE

Article Snippet: Primary antibodies against METTL3 (15073-1-AP, 1:300; Proteintech, China) and TMBIM6 (26782-1-AP, 1:300; Proteintech, China) were

Techniques: Expressing, Modification, Methylation

Journal: The Journal of Clinical Investigation

Article Title: MicroRNA-26a-3p rescues depression-like behaviors in male rats via preventing hippocampal neuronal anomalies

doi: 10.1172/JCI148853

Figure Lengend Snippet: (A) Representative confocal microscopic images showing expression of cleaved caspase-3 and DCX within the DG. Scale bars: 50 μm. n = 6 rats per group and at least 4–6 images from 1 animal. (B) Overexpression of miR-26a-3p decreased protein levels of proapoptotic factors in CUMS rats. n = 6 rats per group. Western blotting results of Bcl-2, Bax, and caspase-9 were from the same samples and run in parallel in different gels. Independent biological replicate experiments were repeated 3 times. (C) Representative electron micrographs showing nuclear chromatin abnormalities in DG neurons. Scale bars: 1 μm. n = 4 rats per group and at least 20 micrographs from 1 animal. Immunofluorescence and electron microscopy experiments were repeated at least 3 times and quantitation was done for representative samples from each group. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001 vs. WT; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. eGFP control (CUMS + AAV-eGFP) by 2-way ANOVA with Tukey’s post hoc correction.

Article Snippet: Proteins (30 μg) from each sample were electrophoretically resolved in 8%–15% SDS-PAGE gels, transferred to PVDF membranes, and probed with the following primary antibodies: anti-BDNF (1:300, catalog sc-546, Santa Cruz Biotechnology Inc.); anti-CREB (1:500, catalog 9197), anti–PSD-95 (1:1000, catalog 3450), anti-Syn (1:1000, catalog 5461), anti–LC3-I/LC3-II (1:1000, catalog 12741), anti–beclin-1 (1:1000, catalog 3495), anti-PARP (1:1000, catalog 9532), anti–cleaved caspase-3 (1:500, catalog 9661), anti-PTEN (1:500, catalog 9552), anti-PI3K (1:500, catalog 4292), anti–p-Akt (1:500, catalog 9271), anti-p53 (1:500, catalog 9284), anti-p62 (1:1000, catalog 23214), anti–β-actin (1:1000, catalog 4970) (all Cell Signaling Technologies); anti-caspase-9 (1:500, catalog AP0359, Bioworld); and anti-BAX (1:1000, catalog 50599-2-lg) and anti-GAPDH (1:4000, catalog 10494-1-AP) (both Proteintech Group).

Techniques: Expressing, Over Expression, Western Blot, Immunofluorescence, Electron Microscopy, Quantitation Assay

Journal: The Journal of Clinical Investigation

Article Title: MicroRNA-26a-3p rescues depression-like behaviors in male rats via preventing hippocampal neuronal anomalies

doi: 10.1172/JCI148853

Figure Lengend Snippet: (A) bpV(pic) treatment increased expression of PI3K and phosphorylated Akt and decreased p53 expression levels in miR-26a-3p–knockdown rats. Western blotting results of PI3K and p-Akt were from the same samples and run in parallel in different gels. n = 6 rats per group with 3 independent biological replicate experiments. (B) bpV(pic) treatment increased LC3-II/LC3-I and beclin-1 expression, and decreased expression of p62 in miR-26a-3p–knockdown rats. Western blotting results for p62 and beclin-1 were from the same samples and run in parallel in different gels. n = 6 rats per group with 3 independent biological replicate experiments. (C) bpV(pic) treatment increased protein levels of BDNF, PSD-95, and Syn within the DG of miR-26a-3p–knockdown rats. Western blotting results for Syn and BDNF were from the same samples and run in parallel in different gels. n = 6 per rats group with 3 independent biological replicate experiments. (D) bpV(pic) treatment decreased mRNA levels of Bax, caspase-3, and caspase-9, and increased Bcl-2 mRNA levels in miR-26a-3p–knockdown rats. n = 6 rats per group with 3 independent biological replicate experiments. (E) bpV(pic) treatment in miR-26a-3p–knockdown rats increased sucrose consumption in the sucrose preference test and (F) decreased immobility times and increased swimming times in the forced-swim test. (G) bpV(pic) treatment in DG neurons produced changes in spontaneous burst activity. n = 16 rats per group. Each data point represents 1 animal. Electrophysiological recordings were repeated at least 3 times. Data are presented as mean ± SEM. n = 18 rats per group in behavioral tests. **P < 0.01, ***P < 0.001 vs. WT; ##P < 0.01, ###P < 0.001 vs. AAV-26a-sponge (WT + AAV-miR-26a-sponge) by 2-way ANOVA with Tukey’s post hoc correction.

Article Snippet: Proteins (30 μg) from each sample were electrophoretically resolved in 8%–15% SDS-PAGE gels, transferred to PVDF membranes, and probed with the following primary antibodies: anti-BDNF (1:300, catalog sc-546, Santa Cruz Biotechnology Inc.); anti-CREB (1:500, catalog 9197), anti–PSD-95 (1:1000, catalog 3450), anti-Syn (1:1000, catalog 5461), anti–LC3-I/LC3-II (1:1000, catalog 12741), anti–beclin-1 (1:1000, catalog 3495), anti-PARP (1:1000, catalog 9532), anti–cleaved caspase-3 (1:500, catalog 9661), anti-PTEN (1:500, catalog 9552), anti-PI3K (1:500, catalog 4292), anti–p-Akt (1:500, catalog 9271), anti-p53 (1:500, catalog 9284), anti-p62 (1:1000, catalog 23214), anti–β-actin (1:1000, catalog 4970) (all Cell Signaling Technologies); anti-caspase-9 (1:500, catalog AP0359, Bioworld); and anti-BAX (1:1000, catalog 50599-2-lg) and anti-GAPDH (1:4000, catalog 10494-1-AP) (both Proteintech Group).

Techniques: Expressing, Western Blot, Produced, Activity Assay