Journal: Cell Stem Cell

Article Title: Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

doi: 10.1016/j.stem.2021.03.022

Figure Lengend Snippet: Temporal changes in marker expression during duct-like and acinus-like lineage specification (A–D) RNA expression of pancreatic ductal markers SOX9 (A), HNF1B (B), CAII (C), and CFTR (D) in DUs and ACs during 16-day culture. Floating column charts represent RNA measurements from quantitative PCR, hinges represent maximal and minimal values, central lines indicate mean values, and dots represent individual measurements. (E and F) Expression of SOX9 (E) and CAII (F) in organoids, detected by immunofluorescence. Numbers embedded in the images indicate the average percentage and standard deviation of marker-positive organoids. Scale bars, 50 μm. (G–I) RNA expression of the pancreatic acinar markers PTF1A (G), RBPJL (H), and CPA1 (I) in DUs and ACs during 16-day culture. Floating column charts: representation as in (A). (J and K) Expression of PTF1A (J) and CPA1 (K) in organoids, detected by immunofluorescence. Numbers embedded in images indicate the average percentage and standard deviation of marker-positive organoids. Scale bars, 50 μm. All results represent the sum of three independent cultures.

Article Snippet: Antibodies used: SOX9, 1:200, #AB5535, Millipore ; CAII, 1:100, #sc-133111, Santa Cruz; PTF1A, 1:100, #s25-763, BD ; CTRC, 1:100, #11456-T24, Sino Biological; CPA1,1:100, #10504-RP02, Sino Biological; Muc2, 1:100, #sc7314, Santa Cruz; Muc5AC, 1:100, #sc33667, Santa Cruz; phosphor-ERK, 1:200, #4370, Cell Signaling; KRT19, 1:200, #ab9221, Abcam;

Techniques: Marker, Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Immunofluorescence, Standard Deviation

Journal: Cell Stem Cell

Article Title: Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

doi: 10.1016/j.stem.2021.03.022

Figure Lengend Snippet:

Article Snippet: Antibodies used: SOX9, 1:200, #AB5535, Millipore ; CAII, 1:100, #sc-133111, Santa Cruz; PTF1A, 1:100, #s25-763, BD ; CTRC, 1:100, #11456-T24, Sino Biological; CPA1,1:100, #10504-RP02, Sino Biological; Muc2, 1:100, #sc7314, Santa Cruz; Muc5AC, 1:100, #sc33667, Santa Cruz; phosphor-ERK, 1:200, #4370, Cell Signaling; KRT19, 1:200, #ab9221, Abcam;

Techniques: Activity Assay, Colorimetric Assay, Staining, Plasmid Preparation, Recombinant, Software

Journal: Cell

Article Title: Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells

doi: 10.1016/j.cell.2023.08.041

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant Human IL-15 , Miltenyi Biotec , Cat#130-095-760.

Techniques: Recombinant, Modification, Activation Assay, Staining, DNA Extraction, Reverse Transcription, SYBR Green Assay, Cell Isolation, RNA Sequencing, Cell Culture, Software

Journal: bioRxiv

Article Title: LIR-dependent LMX1A/LMX1B autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience

doi: 10.1101/636712

Figure Lengend Snippet: (A) Immunofluorescence images and quantitation of LMX1A/LMX1B and dopaminergic marker expression in human iPSC-derived mDANs (imaged at: [i] D40; [ii, iii] D20). Cells were stained for anti-TH (red) and either anti-TUJ1 (green), anti-LMX1A (green) or anti-LMX1B (green). Cells were counterstained with DAPI (blue). Bar = 100µm. Data show counts of cells positive for the marker combinations shown (mean ± SD; n=3). (B-E) qRT-PCR analysis of (B) TUJ1, (C) LMX1A, (D) LMX1B, and (E) NURR1 differentiated at days 5, 10, 15, 20, 40 and 50 (or iPSC control). Levels were normalized to GAPDH. Mean ± SEM (n = 3) of mDAN cultures plated from a single neuralization; one-way ANOVA followed by a Dunnett’s multiple comparisons test. *p<0.05, ** p<0.01 and *** p<0.001 vs. iPSCs. (F, G) ChIP qRT-PCR analysis of (F) LMX1A and (G) LMX1B promoter occupancy in human mDANs. (H) shRNA suppression of LMX1A/LMX1B expression in iPSC-derived mDANs. A schematic representation of the pRRL plasmid construct expressing GFP under human synapsin promoter (hsyn) and shRNA for LMX1A or LMX1B (or a non-target shRNA control) under the U6 promoter is shown to the top. Example fields of mDANs stained for anti-LMX1A (top, red) or anti-LMX1B (bottom, red) and anti-TH (cyan) after transduction with hsyn-GFP-U6-shControl, hsyn-GFP-U6-shLMX1A or hsyn-GFP-U6-shLMX1B (green). To the right, LMX1A and LMX1B qRT-PCR quantitation after viral transduction at D30-45 (normalized to GAPDH) of two different shRNAs for LMX1A and LMX1B. Mean ± SEM (n=3); student’s t test: *p<0.05, ** p<0.01 and *** p<0.001 vs. shRNA control. Bars = 20µm. (I) qRT-PCR analysis of candidate gene expression in iPSC-derived mDANs (D30-45) following hsyn-GFP-U6-shControl/shLMX1A/shLMX1B lentiviral transduction (normalized to GAPDH). Means ± SEM (n=3); student’s t test: *p<0.05, ** p<0.01 and *** p<0.001 vs. shRNA control.

Article Snippet: The following primary antibodies were used (key: IB, immunoblot concentration; IF, immunofluorescence concentration; IP, used for immunoprecipitation): anti-ATG16L1 (MBL, PM040: IB, 1:1000; IF, 1:400); anti-ATG3 (MBL, M1333: IB, 1:1000); anti-ATG7 (Cell Signalling, 2631: IB, 1:1000); anti-FLAG (Genscript, A00187: IB, 1:1500; IF, 1:300); anti-GAPDH (Sigma, G8796: IB, 1:2000); anti-GFP (Covance, MMS-118R: IB, 1:2000); anti-Lamin B (Santa Cruz, sc-6216: IB, 1:500); anti-LC3A/B (Sigma, L8918: IB, 1:1000); anti-LC3B (Sigma, L7543: IF, 1:400; IP); anti-LMX1A (Millipore, AB10533: IB, 1:1000; IF, 1:1000; IP); anti-LMX1A (ProSci,PSI-7087: IP); anti-LMX1B (Proteintech, 18278: IB, 1:1000; IF, 1:250; IP); anti-LMX1B (Witzgall lab: IB, 1:2000; IF, 1:350); anti-p62 (Abnova, H0008878: IB, 1:1000; IF, 1:400); anti-mCherry (Novus, 25157; IB, 1:1000); anti-TH (Millipore, AB152: IB, 1:1000; IF, 1:300); anti-TH (Santa Cruz, sc-25269: IF, 1:100); anti-TUJ1 (Bio-Legend, 801201: IB, 1:1000; IF, 1:1000); anti-WIPI2 (BioRad, MCA5780GA: IF, 1:400).

Techniques: Immunofluorescence, Quantitation Assay, Marker, Expressing, Derivative Assay, Staining, Quantitative RT-PCR, shRNA, Plasmid Preparation, Construct, Transduction

Journal: bioRxiv

Article Title: LIR-dependent LMX1A/LMX1B autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience

doi: 10.1101/636712

Figure Lengend Snippet: (A) Representative immunofluorescence images and quantitation of basal WIPI2 puncta in TH/GFP positive iPSC-derived mDAN cell bodies (D30-45) following hsyn-GFP-U6-shControl/shLMX1A/shLMX1B lentiviral transduction. Mean ± SD (>30 neurons/condition; three independent experiments); student’s t test: *** p<0.001 vs. shRNA control. Bar = 20µm. (B) MitoSOX intensity in TH-positive iPSC-derived mDANs transduced with hsyn-GFP-U6-shControl/shLMX1A/shLMX1B (D30-50). Cells were treated with CCCP (10µM, 4h) as a positive control. Mean ± SEM of 3 experiments, >30 neurons per condition; student’s t test: *p<0.05, ** p<0.01 and *** p<0.001 vs. shRNA control. (C, D) Mitochondrial respiration profiles in iPSC-derived mDAN cultures (D30-50) transduced with (C) hsyn-GFP-U6-shRNA LMX1A or (D) hsyn-GFP-U6-shRNA LMX1B lentiviruses. Pairwise comparisons were performed against non-targeting shRNA control. Oxygen consumption rate (OCR) was normalized to protein levels after sequential exposure to oligomycin (1µM), FCCP (1µM) and Rotenone/Antimycin (0.5µM) as indicated. Mean ± SEM (n = 3); student’s t test: *p<0.05 and **p<0.01 vs. shRNA control. (E) Basal mDAN cell death analysis following hsyn-GFP-U6-shControl/shLMX1A/shLMX1B lentiviral transduction (D25-50 mDANs). Fluorometric caspase assay normalized to total protein. Mean ± SEM (n=3) (not significant). (F) Rotenone-induced cell death in iPSC-derived mDANs (D25-50) transduced with TRE-empty (control), TRE-LMX1A, and/or TRE-LMX1B; expression induced by doxycycline (500ng/mL, 3 days). mDANs were treated with rotenone (15µM) for 24h. Caspase activity measured relative normalized to total protein. Mean ± SEM (n=3); one-way ANOVA followed by a Tukey’s multiple comparison post-hoc test: *p<0.05 and **p<0.01 vs rotenone-treated and ## p<0.01 untreated vs rotenone-treated.

Article Snippet: The following primary antibodies were used (key: IB, immunoblot concentration; IF, immunofluorescence concentration; IP, used for immunoprecipitation): anti-ATG16L1 (MBL, PM040: IB, 1:1000; IF, 1:400); anti-ATG3 (MBL, M1333: IB, 1:1000); anti-ATG7 (Cell Signalling, 2631: IB, 1:1000); anti-FLAG (Genscript, A00187: IB, 1:1500; IF, 1:300); anti-GAPDH (Sigma, G8796: IB, 1:2000); anti-GFP (Covance, MMS-118R: IB, 1:2000); anti-Lamin B (Santa Cruz, sc-6216: IB, 1:500); anti-LC3A/B (Sigma, L8918: IB, 1:1000); anti-LC3B (Sigma, L7543: IF, 1:400; IP); anti-LMX1A (Millipore, AB10533: IB, 1:1000; IF, 1:1000; IP); anti-LMX1A (ProSci,PSI-7087: IP); anti-LMX1B (Proteintech, 18278: IB, 1:1000; IF, 1:250; IP); anti-LMX1B (Witzgall lab: IB, 1:2000; IF, 1:350); anti-p62 (Abnova, H0008878: IB, 1:1000; IF, 1:400); anti-mCherry (Novus, 25157; IB, 1:1000); anti-TH (Millipore, AB152: IB, 1:1000; IF, 1:300); anti-TH (Santa Cruz, sc-25269: IF, 1:100); anti-TUJ1 (Bio-Legend, 801201: IB, 1:1000; IF, 1:1000); anti-WIPI2 (BioRad, MCA5780GA: IF, 1:400).

Techniques: Immunofluorescence, Quantitation Assay, Derivative Assay, Transduction, shRNA, Positive Control, Caspase Assay, Expressing, Activity Assay

Journal: bioRxiv

Article Title: LIR-dependent LMX1A/LMX1B autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience

doi: 10.1101/636712

Figure Lengend Snippet: (A) LMX1B-FLAG turnover in HEK293T cells treated with cycloheximide (CHX, 50µg/mL) for 16h in the absence or presence of MG132 (10µM) or BafA1 (20nM). Mean ± SEM (n = 3); one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: *p<0.05 and ** p<0.01. (B) LMX1B-FLAG degradation during starvation (6h) in the presence of CHX ± BafA1. Mean ± SEM (n = 3); one-way ANOVA followed by Dunnett’s multiple comparisons test (*p<0.05 and *** p<0.001 vs. control), and student’s t test: ### p<0.001 starvation vs. starvation + BafA1. (C) Immunoblot and densitometry of cytosolic and nuclear fractions from HEK293T cells expressing LMX1B-FLAG with or without 2h nutrient starvation. Lamin B was used to normalize the nuclear fractions and GAPDH for the total and cytosolic fractions. Mean ± SEM (n = 3); student’s t test: *** p<0.001 vs. untreated controls. (D) LMX1B relocates to cytosolic puncta that co-label with LC3B during prolonged nutrient starvation. Representative immunofluorescence images of HEK293T cells co-transfected with GFP-LC3B (green) and LMX1B-FLAG, fixed and stained with anti-FLAG antiserum (red) after 0h, 2h or 6h starvation. Bar = 20µm. (E) Domain schematic of human LMX1A and LMX1B showing predicted nuclear localisation sequences (NLS) and a possible LIR motif in LMX1B (left) (the same schematic is shown in Appendix Fig S3A depicting the location of a possible LIR in LMX1A). Alignment of the LMX1B LIR in different species (right). (F) GFP-TRAP co-precipitation of LMX1B-FLAG with GFP-ATG8 family members in HEK293T cells. 5% protein lysate from equivalent GFP-expressing cells is shown as “input”. (G) Anti-FLAG co-precipitation of GFP-LC3B from lysates of HEK293T cells stably expressing FLAG-LMX1B. A non-specific IgG control is included. Arrow indicates position of GFP-LC3B; * indicates the position of the antibody heavy chain. (H) In vitro pull-down of LMX1B-FLAG from lysates of HEK293T stably expressing FLAG-LMX1B using sepharose beads covalently attached to recombinant His-ATG8 family members. (I) GFP-TRAP co-precipitation of LMX1B-FLAG with wild-type of LIR docking mutant (F52A/L53A) GFP-LC3B in HEK293T cells 5% of protein lysate was used as control for protein expression (input). (J) Sequence alignments of LIR motifs in various human (Hs) proteins designated as tryptophan-, phenylalanine- and tyrosine-type LIRs (refers to the residue at P 0 of the LIR). * C-terminal ATG4B LIR; # N-terminal ATG4B LIR. (K) LMX1B LIR overlayed upon a space-filling model of LC3B (5d94.pdb) in final simulation pose, showing docking of the key LIR residues at P 0 and P 3 within hydophobic pockets (HP) 1 and 2 of LC3B respectively. (L) Ribbon structure overlay of human LC3B (grey) in complex with the FYCO1 LIR (Magenta) (5d94.pdb) with the final model simulation pose of LC3B (green) and LMX1B LIR (cyan). Close alignment between FYCO1 and LMX1B is seen within the core LIR binding region. (M) Side-by-side comparison of the LMX1B (left) and FYCO1 (right) LIRs docked at HP2 of LC3B, (5d94.pdb) to show how LMX1B Q316 and FYCO1 E1287 fold back towards HP2 in both structures to stabilise LIR binding.

Article Snippet: The following primary antibodies were used (key: IB, immunoblot concentration; IF, immunofluorescence concentration; IP, used for immunoprecipitation): anti-ATG16L1 (MBL, PM040: IB, 1:1000; IF, 1:400); anti-ATG3 (MBL, M1333: IB, 1:1000); anti-ATG7 (Cell Signalling, 2631: IB, 1:1000); anti-FLAG (Genscript, A00187: IB, 1:1500; IF, 1:300); anti-GAPDH (Sigma, G8796: IB, 1:2000); anti-GFP (Covance, MMS-118R: IB, 1:2000); anti-Lamin B (Santa Cruz, sc-6216: IB, 1:500); anti-LC3A/B (Sigma, L8918: IB, 1:1000); anti-LC3B (Sigma, L7543: IF, 1:400; IP); anti-LMX1A (Millipore, AB10533: IB, 1:1000; IF, 1:1000; IP); anti-LMX1A (ProSci,PSI-7087: IP); anti-LMX1B (Proteintech, 18278: IB, 1:1000; IF, 1:250; IP); anti-LMX1B (Witzgall lab: IB, 1:2000; IF, 1:350); anti-p62 (Abnova, H0008878: IB, 1:1000; IF, 1:400); anti-mCherry (Novus, 25157; IB, 1:1000); anti-TH (Millipore, AB152: IB, 1:1000; IF, 1:300); anti-TH (Santa Cruz, sc-25269: IF, 1:100); anti-TUJ1 (Bio-Legend, 801201: IB, 1:1000; IF, 1:1000); anti-WIPI2 (BioRad, MCA5780GA: IF, 1:400).

Techniques: Western Blot, Expressing, Immunofluorescence, Transfection, Staining, Stable Transfection, In Vitro, Recombinant, Mutagenesis, Sequencing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Studying the Effect of MBNL1 and MBNL2 Loss in Skeletal Muscle Regeneration

doi: 10.3390/ijms25052687

Figure Lengend Snippet: Skeletal muscle is histologically similar between WT (FVB) and MBNL1-OE mice. ( A ) A representative image of H&E staining of TA muscles of non-injured muscle from WT and MBNL1-OE mice, and PAX7 IF (red) in TA muscle, showed increased satellite cells (white arrows) in MBNL1-OE as compared to wildtype mice. RT-PCR of Mbnl1 mRNA confirmed overexpression in MBNL1-OE mice. Muscle fiber counts showed no difference, and MBNL1-OE mice had higher counts of MuSCs in TA muscles (at least 10 independent fields from n ≥ 4 mice/group). RT-PCR showed increased Pax7 expression in MBNL1-OE mice; scale bar is 50 µm. ( B ) Immunofluorescence of recombinant MBNL1 using the anti-Flag rabbit polyclonal antibody (F7425, Sigma) in MBNL1-OE TA muscle showed MBNL1 is not expressed in the satellite cells; PAX7 (green), Flag (red), and nuclei are stained blue. Recombinant MBNL1 protein was not detected in wildtype (WT) controls. Scale bar is 10 µm. ( C ) Immunofluorescence for PAX7 (red) and MyoD (green) in non-injured TA sections from MBNL1-OE mice showed MuSCs co-expressing PAX7 and MyoD (a marker of “activated” MuSCs); this was rarely seen in WT mice; scale bar is 10 µm. ( D ) Quantitative RT-PCR of relative expression, Myog , and Myod in whole TA muscles ( n ≥ 4 mice per group) showed no significant difference. Error bars are mean ± SEM; Student’s t -test; ns = not significant; * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: Primary antibodies were anti-PAX7 (1:50, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-MBNL1 (1;1000, A2764, gift from Dr. Charles A. Thornton), anti-MYH3 (1:200, clone F1.652, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-laminin (1:1000, catalog L9393, Sigma-Aldrich, St. Louis, MO, USA)), anti-MyoD rabbit polyclonal (1:200, SC-760, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-Ki67 antibody rabbit polyclonal (#ab15580, Abnova, Taipei, Taiwan), and anti-Flag rabbit polyclonal (F7425, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Staining, Muscles, Reverse Transcription Polymerase Chain Reaction, Over Expression, Expressing, Immunofluorescence, Recombinant, Marker, Quantitative RT-PCR

Journal: mBio

Article Title: Nonmuscle Myosin Heavy Chain IIA Recognizes Sialic Acids on Sialylated RNA Viruses To Suppress Proinflammatory Responses via the DAP12-Syk Pathway

doi: 10.1128/mBio.00574-19

Figure Lengend Snippet: Sialic acids on VSV are recognized by NMHC-IIA to activate the DAP12-Syk pathway for inhibition of inflammatory responses. (A) The NMHC-IIA–DAP12-Syk pathway is involved in VSV-triggered proinflammatory cytokine transcription. RAW 264.7 cells with MYH9 , DAP12 , or Syk knockdown were infected with VSV (MOI = 1) for 1 h. qRT-PCR was performed to measure mRNA abundance of TNF-α, IL-6, IL-1β, and VSV-G. (B) Heat-inactivated VSV virions induce activation of the DAP12-Syk pathway. RAW 264.7 cells were inoculated with heat-inactivated virions (MOI = 20) for indicated time periods (1 and 2 h). DAP12 was immunoprecipitated by anti-DAP12 MAb. IB was performed to detect phosphorylated DAP12, as well as abundance of NMHC-IIA, DAP12, and Syk. (C) Heat-inactivated VSV virions increase association of NMHC-IIA and DAP12 on the membranes. RAW 264.7 cells were treated with heat-inactivated virions (MOI = 20) for 1 h. Membrane proteins were extracted to conduct an IP assay with anti-NMHC-IIA MAb. IB was then performed to detect the indicated proteins. (D) NMHC-IIA inhibitor subverted heat-inactivated VSV-mediated inhibition of proinflammatory cytokine transcription. LPS-stimulated RAW 264.7 cells were inoculated with the virions (MOI = 20) for 1 h in the presence of 5 μM blebbistatin and then subjected to qRT-PCR detection. (E and F) Heat-inactivated VSV virions inhibit LPS-induced NF-κB activation through the NMHC-IIA–DAP12-Syk pathway. RAW 264.7 cells with MYH9 , DAP12 , or Syk knockdown were stimulated with LPS (10 μg/ml) for 1 h and then treated with heat-inactivated VSV virions (MOI = 20) for 1 h. qRT-PCR analysis was applied to measure the mRNA abundance of TNF-α, IL-6, and IL-1β (E). IB was used to examine the indicated proteins in NF-κB, p38, and ERK1/2 pathways (F). (G and H) Heat-inactivated VSV virions suppress LPS-induced proinflammatory responses dependent on sialic acids. LPS (10 μg/ml)-stimulated RAW 264.7 cells were inoculated with PNGase F- or α2-3,6,8 neuraminidase-treated virions (MOI = 20) for 1 h. IB was performed to detect NF-κB pathway proteins (G). qRT-PCR was performed to detect TNFA , IL-6 , and IL1B transcription (H). Experiments in all panels were repeated at least three times, and similar results were obtained. Quantitation shown was from three independent experiments (mean ± SD from three replicates). Statistical analysis used in qRT-PCR was performed using Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Reagents used were as follows: protein A Sepharose 4 Fast Flow (catalog no. 17-5280-01; GE Healthcare, Pittsburgh, PA, USA), protein G Sepharose 4 Fast Flow (catalog no. 17-0618-01; GE Healthcare), DyLight 350 goat anti-mouse IgG (catalog no. A23010; Abbkine, Wuhan, China), ERK inhibitor PD 98059 (catalog no. 513000; Calbiochem, San Diego, CA, USA), JNK inhibitor II-129566 (catalog no. 420119; Calbiochem), p38 MAP kinase inhibitor SB253080 (catalog no. tlrl-sb20; InvivoGen, Hong Kong, China), NF-κB inhibitor BAY11-7082 (catalog no. tlrl-b82; Beyotime Biotechnology, Shanghai, China), 3′-sialyllactose (catalog no. A8681; Sigma-Aldrich), 6′-sialyllactose sodium salt (catalog no. A8556; Sigma-Aldrich), lipopolysaccharides (LPS) from Escherichia coli O111:B4 (catalog no. L3024; Sigma-Aldrich), (±)-blebbistatin (catalog no. 760; Tocris Bioscience, Bristol, United Kingdom), α2-3,6,8 neuraminidase (catalog no. P0720; New England Biolabs, Ipswich, MA), α2-3 neuraminidase S (catalog no. P0743; New England Biolabs), PNGase F (catalog no. P0704; New England Biolabs), Syk inhibitor R406 (catalog no. S2194; Selleck, Houston, TX, USA), Pierce protease and phosphatase inhibitor minitablets (catalog no. A32959; Thermo Fisher Scientific, Waltham, MA, USA), PrimeScript reverse transcription (RT) reagent kit with genomic DNA (gDNA) Eraser (catalog no. RR047B; TaKaRa, Dalian, China), FastStart Universal SYBR Green Master (Rox) (catalog no. 4913850001; Roche, Basel, Switzerland), porcine TNF-α Quantikine enzyme-linked immunosorbent assay (ELISA) kit (catalog no. PTA00; R&D Systems), CellTiter 96 AQueous One Solution cell proliferation assay (MTS) (catalog no. G3582; Promega, Madison, WI, USA), nuclear and cytoplasmic protein extraction kit (catalog no. P0028; Beyotime), ProteoExtract native membrane protein extraction kit (catalog no. 444810; Merck Millipore).

Techniques: Inhibition, Knockdown, Infection, Quantitative RT-PCR, Activation Assay, Immunoprecipitation, Membrane, Quantitation Assay

Journal: Cell Research

Article Title: Oncostatin M inhibits myoblast differentiation and regulates muscle regeneration

doi: 10.1038/cr.2010.144

Figure Lengend Snippet: OSM inhibited myoblast differentiation. (A, B) Near-confluent C2C12 cells were induced to differentiate for either 72 h (A) or 24 h (B), with or without different doses of OSM. (C, D) Primary myoblasts grown in either GM or DM were treated with or without 20 ng/ml of OSM for various time periods as indicated. In A and C, cells were fixed and subjected to immunostaining for MHC (red) or MyoD (green). Nuclei were counter-stained with DAPI (blue). Phase: phase-contrast images. PBS: phosphate-buffered saline. In B and D, 50 μg of whole cell extracts (WCE) were subjected to SDS-PAGE and western blot analysis for myogenin, MHC, and β-actin (loading control). GM: growth medium. DM: differentiation medium.

Article Snippet: Antibodies against myogenin (SC-12732), MEF2 (SC-313), MyoD (SC-760), Id1 (SC-488), Id2 (SC-489), and β-actin (SC-8432) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunostaining, Staining, Saline, SDS Page, Western Blot, Control

Journal: Cell Research

Article Title: Oncostatin M inhibits myoblast differentiation and regulates muscle regeneration

doi: 10.1038/cr.2010.144

Figure Lengend Snippet: OSM suppressed the expression of MEF2A, upregulated the expression of Id1/Id2, and inhibited the transcriptional activity of MyoD and MEF2. (A) Primary myoblasts (top three panels) and C2C12 cells (bottom three panels) grown in GM or DM were treated with or without 20 ng/ml of OSM for 12-18 h. Cells were harvested and 50 μg of WCE were subjected to SDS-PAGE and western blot analysis for MEF2, MyoD, or β-actin. (B) C2C12 cells were transfected in triplicate with 3xMEF2-luc or gal4-luc, together with constructs encoding Gal4-MyoD or Gal4-MEF2. At 24 h after transfection, cells were induced to differentiate with or without 20 ng/ml of OSM for another 24 h before harvest. WCE were subjected to luciferase assays and SDS-PAGE/western blot analysis for Gal4-MyoD and Gal4-MEF2C. Fold change was calculated as the ratio of the luciferase activity of the OSM-treated cells over that of PBS-treated cells. The results were presented as mean±s.d. (C) C2C12 cells grown in GM or DM were treated with or without 20 ng/ml of OSM for 18 or 24 h before harvest. Total RNA was prepared and subjected to RT-PCR analysis for MEF2A and MEF2D genes. GAPDH: glyceraldehyde 3-phosphate dehydrogenase (loading control). (D) C2C12 cells grown in GM or DM were treated with or without 20 ng/ml of OSM for 18 or 12 h. Cells were harvested and 50 μg of WCE were subjected to SDS-PAGE and western blot analysis for Id1, Id2, and β-actin. (E) C2C12 cells grown in GM were treated with 20 ng/ml of OSM for 0, 10, 30, and 60 min. Total RNA was prepared and subjected to RT-PCR analysis for Id1, Id2, Id3, and GAPDH.

Article Snippet: Antibodies against myogenin (SC-12732), MEF2 (SC-313), MyoD (SC-760), Id1 (SC-488), Id2 (SC-489), and β-actin (SC-8432) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Activity Assay, SDS Page, Western Blot, Transfection, Construct, Luciferase, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Cell Research

Article Title: Oncostatin M inhibits myoblast differentiation and regulates muscle regeneration

doi: 10.1038/cr.2010.144

Figure Lengend Snippet: STAT1 interacted with MEF2 and repressed its transcriptional activity. (A) C2C12 cells were cotransfected with STAT1 together with Flag-MyoD or Flag-MEF2C, with or without OSM (20 ng/ml) treatment. Flag-MyoD/or Flag-MEF2C was immunoprecipitated from WCE. (B) WCE were prepared from nontransfected C2C12 cells grown in GM or DM. The endogenous MEF2 was immunoprecipitated. HA: anti-HA antibody. (C) C2C12 cells were cotransfected with STAT1 together with Flag-MyoD, full-length Flag-MEF2C, or two truncated Flag-MEF2C constructs. Flag-tagged proteins were immunoprecipitated from WCE. The immunoprecipitates from A to C were subjected to SDS-PAGE and western blot analysis for either the transfected (A, C) or the endogenous STAT1 (B). (D) Equal amount of purified His-MEF2C (1-209 aa) was incubated with WCE prepared from 293T cells expressing either the full-length Flag-STAT1 or three-truncated Flag-STAT1 together with Talon beads. After extensive washing, the retained proteins were subjected to SDS-PAGE and western blot analysis. (E) C2C12 cells were transfected in triplicate with different reporter constructs together with either an empty vector or STAT1c in the absence or presence of Gal4-MEF2C. At 24 h after transfection, cells were further induced to differentiate in DM for another 24 h. WCE was subjected to luciferase assays and SDS-PAGE/western blots for Gal4-MEF2C. (F) Fold change was calculated as the ratio of the luciferase activity in cells transfected with STAT1c over that in cells transfected with the empty vector. The results were presented as mean±s.d.

Article Snippet: Antibodies against myogenin (SC-12732), MEF2 (SC-313), MyoD (SC-760), Id1 (SC-488), Id2 (SC-489), and β-actin (SC-8432) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Immunoprecipitation, Construct, SDS Page, Western Blot, Transfection, Purification, Incubation, Expressing, Plasmid Preparation, Luciferase

Journal: Cell Research

Article Title: Oncostatin M inhibits myoblast differentiation and regulates muscle regeneration

doi: 10.1038/cr.2010.144

Figure Lengend Snippet: Prolonged expression of OSM delayed muscle regeneration. (A) 6- to 8-week-old mice were either left untreated (D0) or injected with 20 μl of 10 μM cardiotoxin (CTX) into tibialis anterior (TA) muscles of both legs. TA muscles were surgically isolated at different time points as indicated. Total RNA was prepared and subjected to semiquantitative RT-PCR analysis for OSM, OSMR, and myogenin genes. GAPDH gene was used as a loading control. The experiment was repeated three times with similar results and one set of the representative data was shown. Dx, x day after injury; L, left leg; R, right leg. (B) Upper two panels: C2C12 cells were transfected with either an empty vector or Flag-OSM. At 24 h after transfection, cells were either left in GM for another 24 h or induced to differentiate in DM for 18 h before harvest. The conditioned media were collected for the following experiment. Bottom two panels: near-confluent C2C12 cells were treated with the conditioned media prepared above (in the same order) for 15 min or 24 h before harvest. WCE were subjected to SDS-PAGE and western blot analysis for Flag-OSM, p-STAT3, and myogenin. (C-E) TA muscles of 6-week-old mice were electroporated with either a GFP-expressing vector (C, and left panel in D) or an OSM-expressing vector (right panel in D). At 3 (C, D) or 10 (E) days after electroporation, TA muscles were dissected and embedded for cryostat sectioning. Muscle sections were then subjected to direct microscopic examination for autofluorescence (C), immunostaining for MyoD (D), or hematoxylin/eosin staining (E). The experiments in C to E were repeated three times with similar results. Representative images were shown here. Bars: 100 μm.

Article Snippet: Antibodies against myogenin (SC-12732), MEF2 (SC-313), MyoD (SC-760), Id1 (SC-488), Id2 (SC-489), and β-actin (SC-8432) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Injection, Muscles, Isolation, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, SDS Page, Western Blot, Electroporation, Immunostaining, Staining

Journal: Molecular Biology Reports

Article Title: GLP-1(7–36) protected against oxidative damage and neuronal apoptosis in the hippocampal CA region after traumatic brain injury by regulating ERK5/CREB

doi: 10.1007/s11033-024-09244-8

Figure Lengend Snippet: Real-time PCR primer sequences

Article Snippet: The sections were incubated overnight with the primary antibodies p-ERK5 (cat. no. sc-135,760; 1:1,000; AB_2250338; mouse polyclonal; Santa Cruz Biotechnology) and NeuN (cat. no. EPR12763; 1:500; RRID: AB_2532109; rabbit monoclonal; Abcam) at 4 °C overnight.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Molecular Biology Reports

Article Title: GLP-1(7–36) protected against oxidative damage and neuronal apoptosis in the hippocampal CA region after traumatic brain injury by regulating ERK5/CREB

doi: 10.1007/s11033-024-09244-8

Figure Lengend Snippet: GLP-1(7–36) promoted the activation of the ERK5/CREB signalling pathways. A Representative western blots of proteins examined. The expression of ( B and D ) p-ERK5 and ( C and E ) CREB was determined by western blot analysis and reverse transcription-quantitative PCR. β-Actin was used as an internal control. F Representative double immunofluorescence staining of p-ERK5-positive cells (green) and NeuN-positive cells (red) in brain sections is displayed. G Statistical analysis of the p-ERK5-positive cell numbers in the observed area. Values are expressed as the means ± standard deviations ( n = 3 per group). TBI, traumatic brain injury; GLP-1, glucagon-like peptide-1; ERK5, extracellular signal-regulated kinase; CREB, cAMP response element binding. (# P < 0.05, ## P < 0.01 compared with the Sham group; * P < 0.05, ** P < 0.01 compared with the TBI group; & P < 0.05, && P < 0.01 compared with the GLP-1 (7–36)-L group). (Color figure online)

Article Snippet: The sections were incubated overnight with the primary antibodies p-ERK5 (cat. no. sc-135,760; 1:1,000; AB_2250338; mouse polyclonal; Santa Cruz Biotechnology) and NeuN (cat. no. EPR12763; 1:500; RRID: AB_2532109; rabbit monoclonal; Abcam) at 4 °C overnight.

Techniques: Activation Assay, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Double Immunofluorescence Staining, Binding Assay

Journal: Molecular Biology Reports

Article Title: GLP-1(7–36) protected against oxidative damage and neuronal apoptosis in the hippocampal CA region after traumatic brain injury by regulating ERK5/CREB

doi: 10.1007/s11033-024-09244-8

Figure Lengend Snippet: GLP-1 (7–36) increased cellular antioxidant capacity and inhibited apoptosis by activating ERK5/CREB signalling. A Cellular viability was detected using a CCK-8 assay. B The results show the release of H2O2 in the different groups. C Bar graphs showing the relative DCF fluorescence intensity in hippocampal neurons. D CAT activity, E GSH levels and F SOD activity were detected to determine the antioxidant capacity. G Representative western blots of proteins examined. The expression of ( H and J ) CC-3 and (I and K) Bax/Bcl-2 was determined by western blot analysis and reverse transcription-quantitative PCR. β-Actin was used as an internal control. L Bar graph shows the relative percentage of apoptotic neuronal cells in primary hippocampal neurons based on TUNEL staining. M Representative confocal images (scale bar, 50 μm) show neuronal apoptosis in primary hippocampal neurons based on TUNEL (green) and DAPI (blue) staining. Values are expressed as the means ± standard deviations. These experiments were repeated three times with five samples for each group. TBI, traumatic brain injury; GLP-1, glucagon-like peptide-1; catalase, CAT; glutathione, GSH; superoxide dismutase, SOD; CC-3, cleaved caspase-3; Bcl-2, B-cell lymphoma-2. (# P < 0.05, ## P < 0.01 compared with the Sham group; * P < 0.05, ** P < 0.01 compared with the TBI group; & P < 0.05, && P < 0.01 compared with the TBI + GLP-1 (7–36) group). (Color figure online)

Article Snippet: The sections were incubated overnight with the primary antibodies p-ERK5 (cat. no. sc-135,760; 1:1,000; AB_2250338; mouse polyclonal; Santa Cruz Biotechnology) and NeuN (cat. no. EPR12763; 1:500; RRID: AB_2532109; rabbit monoclonal; Abcam) at 4 °C overnight.

Techniques: CCK-8 Assay, Fluorescence, Activity Assay, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, TUNEL Assay, Staining

Journal: Molecular Biology Reports

Article Title: GLP-1(7–36) protected against oxidative damage and neuronal apoptosis in the hippocampal CA region after traumatic brain injury by regulating ERK5/CREB

doi: 10.1007/s11033-024-09244-8

Figure Lengend Snippet: The mechanism’s diagram. GLP-1(7-36) mitigated neuronal damage following traumatic brain injury. The underlying mechanisms likely involve reducing oxidative stress by modulating the ERK5/CREB pathway, which includes decreasing ROS accumulation and enhancing the expression of antioxidant genes. Simultaneously, this pathway facilitates neuronal cell survival by inhibiting the activation of caspase family members and suppressing the proapoptotic Bcl-2 family members Bax or Bak

Article Snippet: The sections were incubated overnight with the primary antibodies p-ERK5 (cat. no. sc-135,760; 1:1,000; AB_2250338; mouse polyclonal; Santa Cruz Biotechnology) and NeuN (cat. no. EPR12763; 1:500; RRID: AB_2532109; rabbit monoclonal; Abcam) at 4 °C overnight.

Techniques: Expressing, Activation Assay