Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Schematic of the screening for candidate membrane proteins involved in SARS-CoV-2 entry. Schematic illustration of the labeling procedure according to EMARS. After EMARS reaction, the fluorescein-labeled proteins were purified and then analyzed using mass spectrometry.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Labeling, Purification, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: SARS-CoV-2 spike protein-based EMARS probes. ( A ) ACE2 expression in Caco-2 and A549 cells. Western blot analysis of Caco-2 and A549 cell lysates; 10 μg protein samples were subjected to SDS-PAGE (on 10% gels) and stained with anti-ACE2 antibody. Arrows indicate bands of the ACE2 protein. ( B ) Immunocytochemical staining of ACE2 in Caco-2 and A549 cells. Staining with the anti-ACE2 antibody (ACE2+2 nd 568) was performed as described in Experimental procedure . Negative control samples (2 nd 568) were also prepared simultaneously. White bar: 100 μm. ( C ) Immunocytochemical staining of SARS-CoV-2 spike proteins in Caco-2 and A549 cells. Staining of monovalent Alexa Fluor 488-labeled spike proteins (spike-488) and the two-step staining (spike protein followed by Alexa Fluor 488 secondary antibody; spike+2 nd 488) were performed with DIC images. Negative control samples (cAb-488 or 2 nd 488) were also prepared simultaneously. White bar: 100 μm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Expressing, Western Blot, SDS Page, Staining, Negative Control, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Proximity labeling near the cell membrane-bound SARS-CoV-2 spike protein. ( A, B ) Fluorescein-labeled proximal proteins around cell membrane-bound SARS-CoV-2 spike proteins. The EMARS reaction described in the “Experimental procedure” was performed in Caco-2 ( A ) and A549 ( B ) cells using a spike protein ( Spike (RBD) ) and HRP-conjugated anti-mouse IgG ( mouse HRP ). The EMARS products were subsequently subjected to Western blot analysis to detect fluorescein-labeled proteins as candidate proximal proteins. In Caco-2 cells, HRP-conjugated Cholera Toxin B Subunit B ( CTxB-HRP ) was used for EMARS reaction as the positive control for membrane protein labeling. For loading controls, the PVDF membrane was stained with Coomassie Brilliant Blue after western blot analysis (right column)

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Labeling, Western Blot, Positive Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Co-localization of the identified proteins with cell membrane-bound SARS-CoV-2 spike proteins. Representative images of co-localization with SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells were co-stained for SARS-CoV-2 spike protein (green) and the antibodies recognizing ACE2, CD133, Cadherin 17, DPP4, and VAPA (Red). The resulting specimens were subsequently stained with appropriate secondary antibodies and DAPI (Blue), then observed using confocal microscopy (20× objective). Co-localization is indicated in yellow in the “Merge” images. White bar: 10 μm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Candidate proteins located near SARS-CoV-2 spike proteins. ( A to D ) Morphological observation of SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells observed using electron microscopy. Cultured Caco-2 cells were fixed and co-stained with the SARS-CoV-2 spike protein (indicated as 20 nm particles), and candidate molecules identified. CD133 ( A ), DPP4 ( B ), CDH17 ( C ), and VAPA ( D ) are indicated as 10 nm particles. Red arrows indicate the locations of SARS-CoV-2 spike proteins. Yellow arrow heads indicate the location of each candidate protein. Scale bar; 200 or 500 nm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Electron Microscopy, Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: In vitro infection assay of SARS-CoV-2 pseudovirus. ( A ) Expression of ACE2 and candidate membrane proteins in transfectant HEK293 cells. Western blot analysis of transfectant cell lysates; Each cell lysates were subjected to SDS-PAGE (on 6 to 10% gels) and stained with antibodies recognizing ACE2 or candidate membrane proteins. Arrows indicate bands of the target proteins. The CBB staining image indicates load control. Asterisks indicate predicted nonspecific bands. ( B ) Schematic illustration of the assay procedure using HEK293T transfectant host cells. ( C ) Representative images of GFP-positive P-ACE2 cells after pSARS-CoV-2 infection. ACE2-expressing HEK293T cells were treated (pSARS-CoV-2 (+)) or not treated (pSARS-CoV-2 (-)) with pSARS-CoV-2, followed by fluorescein microscopic observation. Two independent experiments were carried out. White bar: 100 μm. ( D-F ) Flow cytometric analysis of pSARS-CoV-2-infected cells. P-ACE2 cells ( D ), candidate protein-single expressing cells ( E ), and candidate protein-coexpressing P-ACE2 cells ( F ) were analyzed using BD FACS Canto II. GFP-positive cells were defined as the infected cells with a GFP fluorescence intensity of 10 3 or higher (P3 area). Two ( E ) or five ( D and F ) independent replications were carried out in each experiment. ( G ) Increase in pSARS-CoV-2 infection in candidate protein-coexpressing P-ACE2 cells. The number of GFP-positive cells in each cell was quantified using flow cytometry. The number of infected cells (GFP-positive) in P-ACE2–CD133, –CDH17, and –VAPA was significantly higher than that in P-ACE2 cells ( P < 0.05 or P < 0.005; Dunnett's test), but not in P-ACE2-GPC3 (N.D.) as the negative control.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: In Vitro, Infection, Expressing, Transfection, Western Blot, SDS Page, Staining, Fluorescence, Flow Cytometry, Negative Control

Journal: Chinese medicine

Article Title: Combination of mangiferin and T0901317 targeting autophagy promotes cholesterol efflux from macrophage foam cell in atherosclerosis.

doi: 10.1186/s13020-023-00876-9

Figure Lengend Snippet: Fig. 3 Autophagy is implicated in lipid droplet degradation under combined treatment of T0 and MGF. Representative digital images of electron microscopy reveal autophagic vacuoles accumulating in the cytoplasm of peritoneal macrophages (A) and RAW264.7 cells (B). Scale bar, 50 μm, 1 μm, 500 nm. Expression of p-mTOR, mTOR, p-AMPK, and AMPK in peritoneal macrophages (C) and RAW264.7 cells (D) was determined by western blot with total proteins extracted from cell samples. Expression of p62, LC3, Beclin1 and ATG5 in peritoneal macrophages (E) and RAW264.7 cells (F) was determined by western blot with total proteins extracted from cell samples

Article Snippet: Primary rabbit polyclonal antibodies against α-SMA (14395), CD36 (18836), Beclin1 (11306), ATG5 (10181), and ATG7 (10088) were purchased from Proteintech Group, Inc. (Rosemont, IL, USA).

Techniques: Electron Microscopy, Expressing, Western Blot

Journal: Frontiers in Microbiology

Article Title: The role of SIRT1 in the process of Toxoplasma gondii infection of RAW 264.7 macrophages

doi: 10.3389/fmicb.2022.1017696

Figure Lengend Snippet: Murine ortholog of immunity-related GTPase family M (IRGM1) regulation by Sirtuin 1 (SIRT1) with or without interferon gamma (IFN-γ) or CD154 at different time points. RAW 264.7 macrophages were either infected with RH tachyzoites at a ratio of 5:1 (parasite/host cell) for 2 h or left in the culture plate without infection, followed by incubation with IFN-γ (1 μg/ml), CD154 (200 ng/ml), SRT1720 (5 μM), EX527 (10 μM), IFN-γ (1 μg/ml) and SRT1720 (5 μM), or IFN-γ (1 μg/ml) and EX527 (10 μM) for 2, 18, or 24 h before the collection of the cell lysates. We adjusted the protein concentration of each group of samples to be consistent and then carried out the experiment. Anti-IRGM1 antibody and anti-beta-Actin antibody were used at a 1:1,000 dilution. (A) The expression of IRGM1 in the Toxoplasma gondii infection group increased at 2 h but decreased at 18 and 24 h compared to that for the control group. Infected macrophages treated with IFN-γ displayed an upregulation of IRGM1 at 2, 18, and 24 h compared to that of the infection-only group. (B) Infected macrophages treated with CD154 showed no significant changes of IRGM1 compared to that for the infection-only group. (C) Infected macrophages treated with RH tachyzoites showed an upregulation of IRGM at 2 h and a downregulation at 18 or 24 h compared to that of the control group. Meanwhile, IRGM1 production was found to be decreased under SRT1720 stimulation and increased with the existence of EX527 at 2 h. However, both SRT1720 and EX527 failed to interfere with the IRGM1 level at 18 and 24 h. (D) When combining SIRT1 interference and IFN-γ stimulation, infected macrophages treated with IFN-γ and SRT720 showed a downregulation of IRGM at 2, 18, and 24 h compared to that of the IFN-γ group. Meanwhile, infected macrophages treated with IFN-γ and EX527 showed an upregulation of IRGM at 2, 18, and 24 h compared to that of the IFN-γ group. These results were obtained from three independent experiments. RH, T. gondii RH strain. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following reagents and equipments were used in the studies: SRT1720 (hydrochloride) (CAS No.: 1001645, MCE, USA), EX527 (CAS No.: 49843-98-3, MCE, USA), Rapamycin (CAS No.: 53123-88-9, MCE, USA), recombinant Interferon-γ (Cat. No.: I4777, Sigma-Aldrich, USA), recombinant mouse TRAP/CD40L protein (Active) (ab220551, Abcam, USA), anti-SIRT1 antibody [SirT1 (1F3) Mouse mAb #8469, Cell Signaling Technology, USA], anti-LC3 antibody [LC3A/B (D3U4C) XP Rabbit mAb #12741, Cell Signaling Technology, USA], anti-mTOR antibody [mTOR (7C10) Rabbit mAb #2983, Cell Signaling Technology, USA], anti-Phospho-mTOR antibody [Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb #5536, Cell Signaling Technology, USA], anti-IRGM1 antibody [IRGM (E6P7W) Rabbit mAb #71950, Cell Signaling Technology, USA], anti-beta-Actin mouse monoclonal antibody (Cat. No.: T0022, Affinity, China), LysoTrackerTM Red DND-99–Special Packaging (Cat. No.: L7528, Thermo Fisher, USA), ProLong Gold Antifade Mountant with DAPI (Cat. No.: P36935, Thermo Fisher, USA), Goat anti-Mouse Alexa Fluor 488 (Cat. No.: A-11017, Thermo Fisher, USA), microplate reader (Sunrise, TECAN, Switzerland), Confocal Microscope (LSM 710, Zeiss, Germany), and Transmission Electron Microscope (TEM) (HT7800, Hitachi, Japan).

Techniques: Infection, Incubation, Protein Concentration, Expressing, Control

Journal: Diagnostics

Article Title: Performance of Antigen Detection Tests for SARS-CoV-2: A Systematic Review and Meta-Analysis

doi: 10.3390/diagnostics12061388

Figure Lengend Snippet: Characteristics of the 235 studies included in the meta-analysis.

Article Snippet: Miyakawa et al. [ ] , Japan , N , nsp , LFIA/virus culture data , 1. SARS-CoV-2 Ag-RDT 2. Panbio COVID-19 Ag Rapid Test 3. SARS-CoV-2 Rapid Antigen Test 4. SD Biosensor Standard Q COVID-19 Ag 5. Espline SARS-CoV-2 , 1. YCU-FF 2. Abbott 3. Roche 4. SD Bio 5. Fujirebio , Up to 40 , Rapid , 108 , 45 , 63.

Techniques: Diagnostic Assay, Surround Optical-fiber Immunoassay, Fluorescence, Enzyme-linked Immunosorbent Assay, Raman Spectroscopy, Antigen Assay, Mass Spectrometry, Stripping Membranes

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a – e Schematic diagrams of the brain of mouse ( a ), and the red box in the cerebral cortex shows the location where the images were taken. Immunofluorescence double labelling ( b , c , 2 double-labelled neurons are indicated as examples in ( b , c )) and quantification ( d , e , n = 6 images from 3 mice) of GDF11 (green, b ) and NeuN (red, b ) or GDF11 (green, c ) and CaMKIIα (red, c ) in the cerebral cortices of the mice aged 3 months (3 M). f Representative images of immuno-electron microscopy (Immuno-EM) of GDF11 labelled with nanogold particles (there are many GDF11 labelled black dots and only some examples are indicated with red arrows) in the cerebral cortex of the mice aged 3 M ( n = 3 mice). Nuc, nucleus; Den, dendrite. g Immunofluorescence double labelling of GDF11 (green, arrow) and GABA (red, double arrowheads) ( n = 3 mice). h Immunofluorescence double labelling of GDF11 (green) together with Olig2 (red, left), GFAP (red, middle), Iba1 (red, middle) in the cerebral cortex (Cx) and Dcx (red, right) in the dentate gyrus (DG) of the mice aged 3 M ( n = 3 mice). The GDF11 negative cells are indicated by arrows in ( h ). i Schematic diagrams of the brain of the marmoset (one aged 62 M and another aged 70 M), and the red box in the cerebral cortex shows the location of the images ( n = 2 marmosets). j – o Immunofluorescence double labelling ( j , m , n , o ) and quantification ( k , l ) of GDF11 (green) together with CaMKIIα (red, j , k , l , 2 double-labelled neurons are indicated as examples in ( j ); n = 8 images from 2 marmosets) or GABA (red, m ), Olig2 (red, n ) or GFAP (red, o ). The GDF11 negative cells are indicated by arrows in ( m , n , q ). p Schematic diagrams of the human brain. The red box in the cerebral cortex shows the location of the images. q – s Immunofluorescence double labelling ( q , male patient aged 24 years (Y) and female patient aged 23Y diagnosed with intractable epilepsy and the focus of epileptic cortices had to be removed surgically) and quantification ( r , s , n = 4 patients, male patient aged 23Y, male patient aged 52Y, female patient aged 54Y and male patient aged 60Y suffered brain injury) of GDF11 (green) together with CaMKIIα (red) in the cerebral cortex of patients and 2 double-labelled neurons are indicated by arrows in ( q ). t Immunofluorescence double labelling of GDF11 (green) together with GABA (red, left), Olig2 (red, middle), GFAP (red, middle) and Iba1 (red, right) in the cerebral cortex of patients ( n = 4 patients). The GDF11 negative cells are indicated by arrows in ( t ). Scale bars, as shown on the images, 30 μm ( b , c ), 250 nm ( f ), 10 μm ( g ), 40 μm ( j , m , n , o ), 20 μm ( h , q , t ). Data are presented as mean ± SEM. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Immunofluorescence, Immuno-Electron Microscopy

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Quantification by qPCR of the relative mRNA of GDF11 in the brain of the WT mice aged 3 M, 9 M or 36 M ( n = 3 mice/group). b Immunofluorescence double labelling of GDF11 (green) and CaMKIIα (red) in the cerebral cortices of the mice aged 3 M, 9 M and 36 M. One GDF11 + CaMKIIα + neuron is indicated by an arrow as an example per group. c Quantification of the average gray value of GDF11 in GDF11 + CaMKIIα + neurons in the cerebral cortices of the mice aged 3 M, 9 M and 36 M (3 M, n = 140; 9 M, n = 160; 36 M, n = 232 cells). d – g Representative images ( d ) and quantification ( e – g ) of the SA-β-Gal + cells in layers 4 and 5 ( d , up, and e , the dashed lines indicate the borders of layers 4 and 5, WT, n = 6; GDF11 f/f , n = 8; GDF11 cKO , n = 6), layer 6a ( d , middle, and f layer 6a is the deep layer cortex near the corpus callosum (CC), WT, n = 8; GDF11 f/f , n = 8; GDF11 cKO , n = 8) of the insular cortex (IC), and layers 2 and 3 of the piriform cortex ( d , down, and g the dashed lines indicate the borders of layers 2 and 3, WT, n = 8; GDF11 f/f , n = 10; GDF11 cKO , n = 10) of GDF11 cKO or GDF11 f/f or WT mice aged 10 M. h–j Representative images ( h ) and quantification of the SA-β-Gal + cells in the cingulate cortex of GDF11 cKO or GDF11 f/f mice aged 10 M ( i , GDF11 f/f , n = 8; GDF11 cKO , n = 6) and 17 M ( j , GDF11 f/f , n = 3; GDF11 cKO , n = 4). Examples of the SA-β-Gal + cells are indicated by double arrowheads in ( d , h ). k A schematic summary on the distribution of the SA-β-Gal + cells in the brain of GDF11 cKO or GDF11 f/f mice aged 10 M and 17 M. l Representative images of double labelling of SA-β-Gal staining (blue) and immunofluorescence of NeuN (fluorescence shown in white) in the insular cortex of GDF11 cKO or GDF11 f/f mice aged 10 M. Examples of the SA-β-Gal + NeuN + neurons are indicated by red arrowheads. m Representative images of double labelling of SA-β-Gal staining (blue) and immunohistochemical staining of CaMKIIα (brown) in the cerebral cortices of GDF11 cKO or GDF11 f/f mice aged 10 M. Examples of the SA-β-Gal + CaMKIIα + ENs are indicated by black arrows. n Survival curves of GDF11 f/f ( n = 35 mice) and GDF11 cKO mice ( n = 15 mice) which died naturally, and log-rank test P value was shown. Median survival is 25 months in GDF11 f/f mice and 22.8 months in GDF11 cKO mice. Scale bars, as shown on the images, 20 μm ( b , d up, m ), 40 μm ( d , middle and down), 50 μm ( h ) and 10 μm ( l ). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. a ( F (2, 6) = 6.672, e 0.0298; 3 M versus 36 M, P = 0.0270), c ( F (2529) = 18.77, P < 0.0001; 3 M versus 9 M, P < 0.0001; 3 M versus 36 M, P < 0.0001; 9 M versus 36 M, P = 0.5477), e ( F (2, 17) = 20.14, P < 0.0001; WT versus GDF11 f/f , P = 0.9950; GDF11 f/f , versus GDF11 cKO , P < 0.0001), f ( F (2, 21) = 4.825, P = 0.0189; WT versus GDF11 f/f , P = 0.9963; GDF11 f/f , versus GDF11 cKO , P = 0.0322) and g ( F (2, 25) = 11.61, P = 0.0003; WT versus GDF11 f/ f, P = 0.4738; GDF11 f/f , versus GDF11 cKO , P = 0.0002). One-way ANOVA with post Tukey multiple comparisons test. i ( P = 0.3427) and j ( P = 0.0280), unpaired two-tailed t test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Immunofluorescence, Staining, Fluorescence, Immunohistochemical staining, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Immunofluorescence image of NeuN (green) in Neuro-2a cells ( n = 6 fields). Scale bar, 40 μm. b PCR of the cell genomes verified successful knockout of the targeted part of exon 2 of GDF11 in Neuro-2a cells (GDF11 KO ) ( n = 3 clones of GDF11 KO cells). c Verification of GDF11 knockout by comparing the mRNA enrichment tracks of GDF11 between GDF11 KO and WT Neuro2a cells by bulk RNA-seq. d Quantification of the relative mRNA of GDF11 in the GDF11 KO and WT Neuro-2a cells by qPCR ( n = 3 biological repeats/group). e , f Western blot ( e ) and Immunofluorescence of GDF11 ( f , scale bar, 40 μm) in GDF11 KO or WT Neuro-2a cells ( n = 3 biological repeats/ group). g , h Representative images ( g ) and quantification ( h , GDF11 KO , n = 13; WT, n = 12 fields) of the SA-β-Gal + cells (blue) in GDF11 KO and WT Neuro-2a cells. All cells are indicated by black stars, and a few representative SA-β-Gal + cells are indicated by black arrows. Scale bar, 50 μm. i Quantification of SA-β-Gal + cells in 3 independent clones of GDF11 KO and WT Neuro-2a cells (GDF11 KO , n = 3; WT, n = 3 clones). j , k Representative images ( j , DAPI, blue) and quantification ( k , GDF11 KO , n = 234 cells; WT, n = 211 cells) of the nuclei of GDF11 KO and WT Neuro-2a cells. Scale bar, 3 μm. l Volcano plot of upregulated (706) and downregulated (411) genes caused by deletion of GDF11 in Neuro-2a cells and revealed by bulk-RNA-seq ( n = 3 clones). m Bulk RNA-seq gene ontology (GO) analysis reveals the top 10 enriched biological processes downregulated by GDF11 deletion in Neuro-2a cells, and the logarithm base 2 of the fold change below −1 was included. n Heatmap of downregulated (11) or upregulated (1) genes involved in “lipid metabolic process” listed in m or “lipid droplets” caused by deletion of GDF11 in Neuro-2a cells, and the logarithm base 2 of the fold change above 1 or below −1 was included. o Representative images of transmission electron microscope (TEM) show the ultrastructure features of GDF11 KO and WT Neuro-2a cells. Cell nucleus (Nuc), lipofuscin (light blue arrows), neurosecretory granules (red double arrowheads) and mitochondrion (brown arrowheads) are indicated as examples. Scale bars, 2 μm. p – r Representative TEM images ( p , lipofuscins, light blue arrows) and quantification of the number (Q, GDF11 KO , n = 20 cells; WT, n = 20 cells) or the area ( r , GDF11 KO , n = 141; WT, n = 85 lipofuscins) of lipofuscins in the GDF11 KO and WT Neuro-2a cells. Scale bars, 500 nm. s – u Representative TEM images ( s , mitochondrion, brown arrowheads; neurosecretory granules, red double arrowheads) and quantification of the number ( t , GDF11 KO , n = 10 cells; WT, n = 10 cells) or the area ( u , GDF11 KO , n = 299; WT, n = 254 mitochondria) of the mitochondria of the GDF11 KO and WT Neuro-2a cells. Scale bars, 500 nm. v Quantification of the number of neurosecretory granules (GDF11 KO , n = 8 cells; WT, n = 10 cells) of the GDF11 KO and WT Neuro-2a cells. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and “ns” indicates not significant, d ( P < 0.0001), h ( P < 0.0001), i ( P = 0.0024), k ( P = 0.0030), q ( P = 0.0002), r ( P = 0.0274), t ( P = 0.8009), u ( P < 0.0001), v ( P = 0.0047), unpaired two-tailed t test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Immunofluorescence, Knock-Out, Clone Assay, RNA Sequencing Assay, Western Blot, Transmission Assay, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Schematic diagrams (left) and representative images (right) of the cingulate gyrus 2 (Cg2), in the prefrontal cortex of GDF11 f/f mice aged 4M-5M, where bilateral focal injection of AAV9-CaMKIIα-Cre-P2A-GFP virus (KO) or AAV9-CaMKIIα-GFP virus (Ctrl) was received at age of 2–3 M and survived for two more months. b Infrared-differential interference contrast (IR-DIC) image (top) and GFP fluorescent image (bottom) of an example of GFP + EN which is undergoing whole-cell patch clamp recording ( n = 64 cells from six mice). c Representative whole-cell recordings in brain slice of a control EN (in Cg2 of GDF11 f/f mice, Ctrl, blue) and a GDF11 deleted-EN (in Cg2 of fGDF11 cKO mice, KO, red) show the firing of action potentials (AP) in response to a series of step current injections. d Examples show typical firing patterns of GFP + EN of fGDF11 cKO mice. e Pie graphs show the percentage of GFP + EN with diverse firing patterns (RS, regular spiking; IS, irregular spiking; IB, intrinsic bursting; RB, repetitive bursting) in WT or KO mice. f Left, plots of the AP frequency as a function of injected currents. Curves are color coded (Ctrl, blue, n = 31 cells from three mice; KO, red, n = 33 cells from three mice). Inset shows the beginning of the curve. Right, plots of the rheobase (Ctrl: 113 ± 16 vs. KO: 81 ± 10 pA, P = 0.049) and slope (Ctrl: 0.18 ± 0.01 vs. KO: 0.30 ± 0.03, P = 0.000) in the two groups (Ctrl, n = 31 cells from three mice; KO, n = 30 cells from three mice). g Left, representative AP waveforms (top) and phase plots (bottom) from Ctrl (blue) or KO (red) group. Right, plots of the AP threshold (Ctrl: −37.9 ± 0.8 vs. KO: −35.0 ± 0.7 mV, P = 0.014), amplitude (AMP) (Ctrl: 85.8 ± 1.6 vs. KO: 78.6 ± 2.2 mV, P = 0.010) and half-width (Ctrl: 0.79 ± 0.03 vs. KO: 0.74 ± 0.03 ms, P = 0.30) in the two groups (Ctrl, n = 29 cells from three mice; KO, n = 24 cells from three mice). h Left-top, representative membrane potential responses to negative current pulses from Ctrl (blue) or KO (red) groups. Plots of the input resistance (Ctrl: 104 ± 10 vs. KO: 214 ± 21 MΩ, P = 0.000), membrane constant (Ctrl: 14.4 ± 1.1 vs. KO: 22.1 ± 2.0 ms, P = 0.003), Sag ratio (Ctrl: 1.18 ± 0.02 vs. KO: 1.27 ± 0.03, P = 0.033), membrane capacitance (Ctrl: 147 ± 11 vs. KO: 95 ± 5 pF, P = 0.000) and RMP (Ctrl: −67.3 ± 1.0 vs. KO: −63.1 ± 0.9 mV, P = 0.004) in the two groups (Ctrl, n = 31 cells from three mice; KO, n = 33 cells from three mice). i Representative whole-cell recordings of mIPSC from the EN in GDF11 f/f mice (Ctrl, blue) and fGDF11 cKO mice (KO, red). j Left, scaled mIPSC examples in the two groups. Right, plots of rising time (Ctrl: 0.65 ± 0.04 vs. KO: 0.85 ± 0.06 ms, P = 0.005) and decay time (Ctrl: 4.44 ± 0.21 vs. KO: 4.69 ± 0.34 ms, P = 0.53) of mIPSCs in the two groups (Ctrl, n = 18 cells from four mice; KO, n = 16 cells from four mice). k , l Cumulative frequency curve of the inter-event-interval ( k ) and amplitude ( l ) of mIPSCs. Insets show the group plots of mIPSC frequency ( k , Ctrl: 34.6 ± 5.2 vs. KO: 4.0 ± 0.9 Hz, P = 0.000) and amplitude ( l , Ctrl: 24.0 ± 1.6 vs. KO: 20.5 ± 1.8 pA, P = 0.16). m – p Recordings of mEPSCs (Ctrl, n = 24 cells from four mice; KO, n = 28 cells from 4 mice) and similar plots as the mIPSCs shown above. Rising time ( n , ctrl: 0.87 ± 0.05 vs. KO: 0.81 ± 0.06 ms, P = 0.46); Decay time ( n , ctrl: 3.54 ± 0.20 vs. KO: 2.98 ± 0.24 ms, P = 0.041); Frequency ( o , Ctrl: 3.66 ± 0.84 vs. KO: 3.13 ± 0.65 Hz, p = 0.82); Amplitude ( p , Ctrl: 14.5 ± 0.8 vs. KO: 14.3 ± 0.9 pA, P = 0.33). q , r Representative traces showing IPSC ( q , left) or EPSC ( r , left) evoked by extracellular electric stimulations for the comparison of paired-pulse ratio (PPR) in GDF11 f/f mice (Ctrl, blue) and fGDF11 cKO mice (KO, red). Group plots of PPR for IPSC ( q , right, Ctrl, n = 7 cells from 3 mice: 0.98 ± 0.07 vs. KO, n = 9 cells from three mice: 1.16 ± 0.20, P = 0.92) and EPSC ( r , right, Ctrl, n = 9 cells from 3 mice: 1.38 ± 0.07 vs. KO, n = 6 cells from three mice: 1.26 ± 0.06, P = 0.24). s Track diagrams in the 3-chamber test (3CT) between the fGDF11 cKO (KO) and GDF11 f/f (Ctrl) mice aged 4–5 M. O object, S1 stranger mouse, S2 new stranger mouse. t Quantification of the exploration time in 3CT (KO, n = 13; Ctrl, n = 13 mice) on objects between the fGDF11 cKO (KO) and GDF11 f/f (Ctrl) mice aged 4–5 M. O1, object 1; O2, object 2. u Quantification of the preference index (S1-O) between the S1 and object in the KO and Ctrl groups (KO, n = 13; Ctrl, n = 13 mice). v Quantification of the preference index (S2-S1) between the S2 and S1 in the KO and Ctrl groups (KO, n = 13; Ctrl, n = 13 mice). w Schematic diagram of the novel object recognition test (NORT) between the GDF11 cKO and GDF11 f/f mice aged 10 M. Red squares indicate the familiar toy while blue triangle indicates a novel toy. x Quantification of the percentage of exploration time (GDF11 cKO , n = 9; GDF11 f/f , n = 6 mice) on the familiar or a novel toy in the GDF11 cKO and GDF11 f/f mice aged 10 M. y Quantification of the novel object discrimination index ((novel-familiar)/(novel + familiar)) between the familiar or a novel toy in the GDF11 cKO and GDF11 f/f mice aged 10 M (GDF11 cKO , n = 9; GDF11 f/f , n = 6 mice). Data are presented as mean ± SEM. Whisker boxplots in ( f , h ) represent the median and interquartile range; whiskers represent 1.5× interquartile range. * P < 0.05, ** P < 0.01 and “ns” represents not significant. f (Rheobase/Slope), h (Input resistance/Membrane constant/Sag ratio/Capacitance), j (Rising time), k , n (Decay time), o – q Mann–Whitney U test. g , h (RMP), j (Decay time), l , n (Rising time), r , u ( P = 0.0118), v ( P = 0.0128), x (GDF11 f/f : Familiar versus Novel, P = 0.0331; GDF11 cKO : Familiar versus Novel, P = 0.0188) and y ( P = 0.0254), unpaired two-tailed t test. t (Ctrl: O1 versus O2, P = 0.3210; KO: O1 versus O2, P = 0.2200), two-way ANOVA with post Sidak’s multiple comparisons test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Injection, Virus, Patch Clamp, Slice Preparation, Control, Membrane, Comparison, Whisker Assay, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Schematic diagrams of the cingulate gyrus 2 (Cg2), in the prefrontal cortex of GDF11 f/f mice aged 4–5 M, where bilateral focal injection of AAV9-CaMKIIα-Cre-P2A-GFP virus (KO) or AAV9-CaMKIIα-GFP virus (Ctrl) was received at age of 2–3 M and survived for two more months. b UMAP of the clustered 16 cell types in snRNA-seq of the Cg2 in both 3 KO mice and 3 control mice (Ctrl) aged 4–5 M. c Violin chart of the relative mRNA of GDF11 by snRNA-seq in KO-GFP + , KO-GFP - , Ctrl-GFP + or Ctrl-GFP - EN. The KO-EN were divided into KO-GFP + and KO-GFP - groups whereas “Ctrl-EN” were divided into Ctrl-GFP + and Ctrl-GFP − groups. d and e , Heatmap shows the average transcription of downregulated and upregulated ageing-related genes ( d ) and SASP-related genes ( e ) in snRNA-seq of KO-GFP + , KO-GFP − , Ctrl-GFP + or Ctrl-GFP − EN. f Confocal images (Left) and 3D-reconstruction (Right) of representative EN from Ctrl (Top) or KO (Bottom) groups. Dendrites and soma are presented in red, and axons are in blue. Scale bar, 50 μm. g , h Plots of the number of intersections of dendrites ( g ) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice) and the group data showing the number of total dendrite intersections ( h , Ctrl: 448 ± 28 vs. KO: 346 ± 36, P = 0.028). i – k Group data show the total number of apical dendrite intersections ( i , Ctrl: 238 ± 17 vs. KO: 181 ± 18, P = 0.036), the total length of apical dendrites ( j , Ctrl: 3.77 ± 0.28 vs. KO: 2.83 ± 0.34 mm, P = 0.044), and the apical branch orders against the averaged dendrite length ( k , branch order 1, Ctrl: 445 ± 28 vs. KO: 403 ± 22 μm, P = 0.26; branch order 2, Ctrl: 115 ± 3 vs. KO: 93 ± 8 μm, P = 0.017; order 3, Ctrl: 91 ± 4 vs. KO: 70 ± 6 μm, P = 0.007; branch order 4, Ctrl: 72 ± 6 vs. KO: 56 ± 7 μm, P = 0.12) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice). l – n Group data comparing the number of total basal intersections ( l , Ctrl: 207 ± 15 vs. KO: 162 ± 22, P = 0.11), total basal dendrite length ( m , Ctrl: 2.73 ± 0.18 vs. KO: 2.16 ± 0.29 mm, P = 0.11) and the basal branch orders against the averaged dendrite length ( n , branch order 1, Ctrl: 102 ± 4 vs. KO: 102 ± 8 μm, P = 0.98; branch order 2, Ctrl: 82 ± 3 vs. KO: 82 ± 9 μm, P = 0.32; order 3, Ctrl: 69 ± 8 vs. KO: 59 ± 2 μm, P = 0.25) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice). o , p Plots of the axon distance from soma against the number of intersections ( o ) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice). Group data show the number of total axon branches intersections ( p , Ctrl: 239 ± 17 vs. KO: 190 ± 28, P = 0.15). q Confocal examples of dendritic spines (red arrows indicate the big mushroom spines while yellow arrows point to small mushroom spines) in the two groups. Scale bar, 5 μm. r , s Group data show total spine density per 10 μm ( r , Ctrl: 6.28 ± 0.23 vs. KO: 1.61 ± 0.13/10 μm, P = 0.000) and mushroom spine diameter ( s , Ctrl: 0.66 ± 0.01 vs. KO: 0.80 ± 0.02 μm, P = 0.000) in two groups (Ctrl, n = 68 dendrites from 16 cells; KO, n = 70 dendrites from 16 cells). t Plots of spine density against the mushroom spine diameter in the two groups (Ctrl, n = 16 cells from three mice; KO, n = 16 cells from three mice). u A schematic summary: GDF11 deletion results in hyperexcitability of the EN as reflected by an enhancement in their firing frequency (due to increased input resistance and elevated RMP) and a decrease in mIPSC frequency. In addition, GDF11 deletion in the EN prunes and shortens their apical dendrites, reduces their dendritic mushroom spine density while enlarges its size. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. h , i , j , k , l , m , n , p , unpaired two-tailed t test; r , s , Mann–Whitney U test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Injection, Virus, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a , b SnRNA-seq GO analysis reveals the top ten enriched biological processes of upregulated ( a ) or downregulated ( b ) in the KO-GFP + EN in comparison with the KO-GFP - EN, and the EN were obtained from the Cg2 of the “KO” mice and the “Ctrl” mice aged 4–5 M. c Volcano plot shows upregulated and downregulated DEGs in the KO-GFP + EN in comparison with the Ctrl-GFP + EN. Some of the top upregulated and downregulated genes were annotated. c , d FC fold change. P value was calculated using Wilcox test and adjusted for multiple testing using Benjamini–Hochberg correction. d Volcano plot shows upregulated and downregulated DEG in the KO-GFP + EN in comparison with the KO-GFP - EN. Some of the top upregulated and downregulated genes were indicated. e UMAP visualization highlights the distribution and the transcription of Cdkn1a/p21 in the identified cell types in snRNA-seq. f Dot plot representing the frequency and average transcription of Cdkn1a/p21 in the identified cell types in snRNA-seq. g , h Relative mRNA of Cdkn1a/p21 ( g ) or p53 ( h ) among four types of EN: Ctrl-GFP - , Ctrl-GFP + , KO-GFP - and KO-GFP + by snRNA-seq. i Heatmap of upregulated (10) and downregulated (6) genes involved in “cellular senescence” caused by deletion of GDF11 in Neuro-2a cells, and the logarithm base 2 of the fold change above 1 or below −1 was included. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and “ns” indicates not significant. a , b Hypergeometric test with Benjamini and Hochberg (BH) correction. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Comparison

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a , b Genetic strategy for generation of p21 f/f mice ( a ) and CaMKIIα-Cre; GDF11 f/f ; p21 f/f mice ( b ) to selectively delete both GDF11 and p21 in CaMKIIα + neurons through Cre/Loxp system. c – g Representative images ( c ) and quantification ( d – g ) of the SA-β-Gal + cells in the cingulate cortex ( c , up, and d , n = 4 per group), layers 4 and 5 ( c , middle, and e GDF11 f/f , n = 4; GDF11 cKO , n = 3; CaMKIIα-Cre; GDF11 f/f ;p21 f/f , n = 5), layer 6a ( c middle, and f layer 6a is the deep layer cortex near the corpus callosum (CC), GDF11 f/f , n = 5; GDF11 cKO , n = 4; CaMKIIα-Cre; GDF11 f/f ;p21 f/f , n = 4) of the insular cortex (IC), and layers 2 and 3 of the piriform cortex ( c down, and g the dashed lines indicate the borders of layers 2 and 3, GDF11 f/f , n = 8; GDF11 cKO , n = 4; CaMKIIα-Cre; GDF11 f/f ;p21 f/f , n = 8) of CaMKIIα-Cre; GDF11 f/f ;p21 f/f or GDF11 cKO or GDF11 f/f mice aged 17 M. Examples of the SA-β-Gal + cells are indicated by double arrows. Scale bars, as shown on the images, 50 μm ( c , up and middle) and 20 μm ( c , middle and down). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. d ( F (2, 9) = 72.52, P < 0.0001; GDF11 f/f versus GDF11 cKO , P = 0.0006; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P = 0.0004), e ( F (2, 9) = 78.16, P < 0.0001; GDF11 f/f versus GDF11 cKO , P = 0.0020; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001), f ( F (2, 10) = 49.87, P < 0.0001; GDF11 f/f versus GDF11 cKO , P < 0.0001; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P = 0.0347) and g ( F (2, 17) = 102.8, P < 0.0001; GDF11 f/f versus GDF11 cKO , P = 0.0227; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f; p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P = 0.0001), One-way ANOVA with post Tukey multiple comparisons test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques:

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Quantification by qPCR of the relative p21 mRNA in the GDF11 KO and WT Neuro-2a cells ( n = 3 clones). b – e Immunofluorescence representative images ( b ) and quantification of the number of p21 + cells per field ( c , n = 5 fields/group), the proportion of p21 + cells ( d , n = 6 fields/group) or the average gray value of p21 per cell ( e , GDF11 KO , n = 420 cells; WT, n = 280 cells) in the GDF11 KO and WT Neuro-2a cells. Scale bar, 25 μm. Examples of the p21 + cells are indicated by double arrowheads. f – h The same snRNA-seq data were used, as described in Fig. . f Rank for regulons in the EN based on regulon specificity score (RSS). The EN were obtained from the Cg2 of the “KO” mice and the “Ctrl” mice aged 4–5 M. g Regulons activity analysis based on area under the curve (AUC) in the identified cell types in snRNA-seq of the “KO” mice and the “Ctrl” mice aged 4–5 M. The activity of regulon Smad3 (highlighted in red) is high in the EN. h Cytoscape network visualization of genes including GDF11, Cdkn1a (p21), Smad2, Smad3 (highlighted in red) and their transcription factors (TFs, yellow). i – m Representative images ( i and l ) and quantification by densitometry of western blot analysis of Smad2 ( j ), phosphorylated Smad2 (pSmad2, k ) and Smad3 ( m ) in the total protein extracted from the GDF11 KO and WT Neuro-2a cells ( n = 3 biological repeats/group). n ChIP-qPCR assessment of the enrichment of Smad2 at the promoter of Cdkn1a/p21 in the GDF11 KO and WT Neuro-2a cells ( n = 3 biological repeats/group). o A proposed working model for loss of GDF11 on cellular senescence. Loss of GDF11 upregulates pSmad2, enhances nuclear entry of Smad2/3 tricomplex and then Smad2 binds to the promoter of p21 and promotes the pro-senescence factor p21 transcription, and eventually causes cellular senescence. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and “ns” indicates not significant. a ( P = 0.0037), c ( P = 0.0033), d ( P = 0.0157), e ( P < 0.0001), j ( P = 0.6648), k ( P = 0.0040) and m ( P = 0.0299), unpaired two-tailed t test. n (IgG: WT versus GDF11 KO , P = 0.57; Smad2: WT versus GDF11 KO , P < 0.001), two-way ANOVA with Sidak’s test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Clone Assay, Immunofluorescence, Activity Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: Evidence of both in vitro (left) and in vivo (right) indicates that growth differentiation factor 11-Smad2/3-p21 pathway acts as a brake on excitatory neuronal senescence and brain ageing.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: In Vitro, In Vivo