Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Inhibition of miR-29b-1-5p ameliorates lipopolysaccharide (LPS)-induced septic myocardial injury in mice (A) Mice received a single intraperitoneal injection of 25 mg/kg LPS (lethal dose) or a tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving LPS injection), after which the survival conditions were assessed for 96 h, and survival rate curves were generated. An equal amount of saline was injected into the control group. n=20. Mice received a single intraperitoneal injection of 10 mg/kg LPS or a tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving LPS injection). (B) Twenty-four hours after LPS injection, echocardiography tests were performed. (C,D) Mice were euthanized, and histological analysis of myocardial tissue injury was performed via hematoxylin and eosin (HE) staining and Masson’s trichrome staining. Scale bar: 50 μm. (F,G) The cardiac injury score and fibrotic area were assessed via HE and Masson’s trichrome staining, respectively. (E,H) α-Smooth muscle actin (α-SMA) expression in mouse myocardial tissues was determined via immunohistochemistry (IHC) staining. Scale bar: 20 μm. (I‒L) Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and cardiac troponin I (cTnI) levels in mouse serum were determined by enzyme-linked immunosorbent assay (ELISA). (M) The miR-29b-1-5p expression level in mouse myocardial tissue was determined by real-time quantitative reverse transcription PCR (qRT-PCR). n=6. (N) The protein levels of TLR2, TLR4, TFEB, LAMP1 and LC3II in mouse myocardial tissues were determined by western blot analysis. n=3. **P<0.01 vs the control group; #P<0.05 and ##P<0.01 vs. the LPS+NC antagomir group.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Injection, Generated, Saline, Staining, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Inhibition of miR-29b-1-5p ameliorates CLP-induced septic myocardial injury in mice (A) Mice were subjected to CLP surgery or tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving CLP surgery), after which the survival conditions were assessed for 96 h, and survival rate curves were generated. n=20. A sham operation was performed on the control group. n=20. (B) Twenty-four hours after CLP surgery, echocardiography was performed. (C,D) Mice were euthanized, and histological analysis of myocardial tissue injury was performed via HE staining and Masson’s trichrome staining. Scale bar: 50 μm. (F,G) The cardiac injury score and fibrotic area were assessed via HE and Masson’s trichrome staining, respectively. (E,H) The protein levels and distribution of α-SMA in mouse myocardial tissues were examined using IHC staining. Scale bar: 20 μm. (I‒L) TNF-α, IL-1β, IL-6 and cTnI levels in mouse serum were determined by ELISA. (M) The miR-29b-1-5p expression level in mouse myocardial tissue was determined by qRT-PCR. n=6. (N) The protein levels of TLR2, TLR4, TFEB, LAMP1 and LC3II in mouse myocardial tissues were determined by western blot analysis. n=3. **P<0.01 vs the control group; #P<0.05 and ##P<0.01 vs the CLP+NC antagomir group.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Injection, Generated, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Inhibition of miR-29b-1-5p ameliorates LPS-induced cardiomyocyte dysfunction in mice in vitro (A) Twenty-four hours after transfecting HL-1 cells with the NC or miR-29b-1-5p antagomir, LPS (1 μg/mL) stimulation was applied for another 24 h, and miR-29b-1-5p expression was validated in each group by qRT-PCR. (B) After LPS treatment for 0, 24, 48, or 72 h, MTT assay was performed to examine the viability of the HL-1 cells. (C) Flow cytometry was used to evaluate apoptosis in HL-1 cells. (D‒F) TNF-α, IL-1β, and IL-6 levels in HL-1 cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA). (G‒I) Western blot analysis was used to measure Bax, Bcl-2, cleaved caspase-3, pro-caspase-3, TLR2, TLR4, TFEB, LAMP1 and LC3II protein levels in HL-1 cells. n=3. **P<0.01 vs the control group; ##P<0.01 vs the LPS+NC antagomir group.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, In Vitro, Expressing, Quantitative RT-PCR, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Silencing of TERF2 partially reverses the ameliorative effect of miR-29b-1-5p inhibition on LPS-induced cardiomyocyte apoptosis and inflammation (A) HL-1 cells were transfected with sh-TERF2 or sh-NC vector. Forty-eight hours later, HL-1 cells were collected to detect the protein expression of TERF2. (B‒I) HL-1 cells were transfected with sh-TERF2 or the miR-29b-1-5p antagomir for 24 h, followed by stimulation with LPS. MTT assay was performed to examine HL-1 cell viability (B). Flow cytometry was performed to evaluate HL-1 cell apoptosis (C). ELISA was also conducted to evaluate TNF-α, IL-1β, and IL-6 levels in HL-1 cells (D‒F). Western blot analysis was conducted to determine the protein levels of TERF2, cleaved PARP, cleaved caspase-3, pro-caspase-3, TLR2, TLR4, TFEB, LAMP1 and LC3II in HL-1 cells (G–I). **P<0.01 vs LPS+NC antagomir+sh-NC; #P<0.05, ##P<0.01 vs LPS+miR-29b-1-5p antagomir+sh-TERF2.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: PLAT-E cells were transfected with retroviral pMX-Flag-NEMO- WT -RFP (NM-WT) and pMX-flag-NEMO- K270 -RFP (NM-KA) expression vector. ( A ) Fluorescence images showing distribution of NM-WT-RFP in cytoplasm compared to puncta (yellow arrows) (juxtaposed to nuclei- DAPI stained) formation in case of NM-KA-RFP in PLAT-E cells. ( B ) Western blot for LC3 using WES (protein simple). BMMs were cultured for 2 days with RANKL (preOC) followed by 6 hr of serum starvation and western blotting. Fold change of LC3 relative to actin is indicated on top. ( C ) Quantification of LC3+ cells per high magnification field. ( D ) For flow cytometry, BMMs were transduced with pMX-GFP-LC3-RFP retrovirus generated in PLAT-E packing cells, and flow analysis was done to detect GFP signal or LC3 flux. Contour plots showing LC3-GFP+ expressing cells in NM-WT and NM-KA preOC (Blue: NM-WT without serum starvation, Red: NM-KA without serum starvation, and Black: after 6 hr of serum starvation), ( E ) Histograms representing shift in LC3-GFP+ cells following induction of autophagy (Red histogram: background signal in uninfected cells, Blue histogram: No serum starvation or 10% FBS control, yellow: 6 hr serum starvation, and pink: chloroquine), ( F ) Change in Mean fluorescent intensity (MFI) showing LC3-GFP signal in NM-WT and NM-KA preOC cells post autophagy induction. LysM-cre-NEMO-WT-f/f (NM-WT), LysM-cre-NEMO-K270A-f/f (NM-KA) mice. (*p<0.05). (*p<0.05, **p<0.01 and ***p<0.001). Figure 4—source data 1. Western blot for LC3 using WES (protein simple). Figure 4—source data 2. Quantification of LC3+ cells. Figure 4—source data 3. LC3-GFP FACS analysis.

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Transfection, Retroviral, Expressing, Plasmid Preparation, Fluorescence, Staining, Western Blot, Cell Culture, Flow Cytometry, Transduction, Generated, Control

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: ( A ) Pre-OC (RANKL-treated BMMs) from NM-WT and NM-KA mice were pelleted and processed for electron microscopic and Immunofluorescence (IF) analysis after 6 hr of serum starvation. Representative Electron microscopic images (x7500) showing nucleus, Cell membrane (CM) lysosome ( L ) in NM-WT preOC and cytoplasmic aggregates (yellow arrow) in NM-KA preOC. ( B ) Representative IF images for NEMO (red), LC3 (green) and NEMO-LC3 colocalization (yellow). Arrows indicate accumulation of NEMO in LC3 positive vacuole-like structures. ( C ) Representative western blot showing expression of LC3 from BMMs starved and stimulated with RANKL as shown. ( D ) Representative Western blot for mTOR expression in BMMs from NM-WT and NM-KA mice treated as shown. ( E ) Pre-osteoclasts were treated with chloroquine as indicated and number of TRAP+ multi nucleated osteoclasts (MNC) per well were counted in triplicate wells from three independent experiments (*p<0.05).

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Immunofluorescence, Membrane, Western Blot, Expressing

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: BMMs from NM-WT and NM-KA mice were transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing ISG15 and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for 4 days. ( A ) Representative TRAP staining for osteoclast (n = 6) and ( B ) quantification of TRAP positive OCs. ( C ) BMMs from NM-WT and NM-KA mice were transduced with ISG15 and pMRX-GFP-LC3-RFP retrovirus generated in PLAT-E packing cells. The cells were cultured for 2 days (preOC) followed by 6 hr of serum starvation and flow analysis to detect GFP signal or LC3 flux. ( C ) Histograms representing shift in LC3-GFP+ cells following induction of autophagy. Blue histogram: serum starvation, yellow histogram: serum starvation + ISG15 expression ( D ) Change in Mean Fluorescent Intensity (MFI) showing LC3-GFP signal. ( E ) Wild type BMMs transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing NEMO+/-ISG15, NEMO-K270A+/-ISG15 NEMO-WT::ISG15 (fused) and NEMO-K270A::ISG15 (fused) protein and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for 4 days.( E ) Representative TRAP staining for osteoclast (n = 3) and ( F ) quantification of TRAP positive OCs. ( G ) NEMO puncta regulation by ISG15: Live images of preOC expressing RFP-NEMO WT +/-ISG15, RFP-NEMO K270A +/-ISG15, GFP-NEMO WT ::ISG15 and GFP-NEMO K270A ::ISG15 fusion protein. Yellow arrows indicate NEMO K270A puncta. ISG15 panel which is not tagged serves as background control. ( H ) Quantification of LC3 puncta+ preOC cells shown in . ( I ) WB for LC3 in preOC expressing NEMO WT +/-ISG15, NEMO K270A +/-ISG15, NEMO WT ::ISG15 and NEMO K270A ::ISG15 fusion protein. (*p<0.05, **p<0.01 and ***p<0.001). (::) denotes fusion. ( J ) Representative IF images (NEMO (Red); LAMP1(green)). NEMO localization in preOC expressing NEMO WT +/-ISG15, NEMO K270A +/-ISG15, NEMO WT ::ISG15 and NEMO K270A ::ISG15 fusion protein. Green arrow- Lysosome and Yellow arrow-localization of NEMO in Lysosome. Figure 7—source data 1. quantification of TRAP positive OCs. Figure 7—source data 2. LC3-GFP FACS analysis. Figure 7—source data 3. Quantification of TRAP positive OCs. Figure 7—source data 4. Quantification of LC3 positive puncta in pre-OC cells shown in . Figure 7—source data 5. Western blot for LC3 expression in preOC expressing different NEMO and ISG15 constructs.

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Transduction, Generated, Retroviral, Expressing, Cell Culture, Staining, Control, Western Blot, Construct

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: ( A ) Representative IF images for LC3 puncta+ cells (arrow) in preOC expressing NEMO WT +/-ISG15, NEMO K270A +/-ISG15, NEMO WT ::ISG15 and NEMO K270A ::ISG15 fusion protein (quantified in ).

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Expressing

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Luciferase, Recombinant, Retroviral, Plasmid Preparation, FLAG-tag, Mutagenesis, Construct, Transfection, Activity Assay, BIA-KA, Quantitation Assay, Lysis, Western Blot, Electron Microscopy, Multiplex Assay, Marker, Isolation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Sequencing, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Amyloid β1-42 (Aβ1-42) Induces the CDK2-Mediated Phosphorylation of Tau through the Activation of the mTORC1 Signaling Pathway While Promoting Neuronal Cell Death

doi: 10.3389/fnmol.2017.00229

Figure Lengend Snippet: Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of p70S6K1 (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.

Article Snippet: The antibodies of hypoxia inducible factor (HIF1α; NB100-105), p70S6K1 (NB600-1049), LC3 (NB100-2220) and p62 (NBP1-48320) were obtained from Novus Biologicals (Littleton, CO, USA) and the HRP-conjugated goat anti-rabbit IgG was purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Incubation, Phospho-proteomics, Immunoprecipitation, Confocal Microscopy, MTT Assay, Trypan Blue Exclusion Assay, Control