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

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) Schematic of recombinant fPanx1 construct incorporating a thrombin cleavage site, (eGFP), and a Strep-Tag. ( B ) Schematic of proteoliposomes containing fPanx1-eGFP fusion proteins, in either orientation. ( C ) fPanx1-containing fractions from Nycodenz co-floatation assay (fractions 1–3, 20 μL each) were run on a polyacrylamide gel and analyzed by silver stain. ( D ) Negative stain electron microscopy image of fPanx1-eGFP proteoliposomes extruded at 100 nm shown at 29,000× magnification. ( E ) Western blot of fPanx1-eGFP from proteoliposomes after overnight incubation in the absence and presence of recombinant Caspase-3 (Casp3). The schematics illustrate the location of: caspase cleavage sites, N-terminally directed α-Panx1 antibody, and corresponding cleavage products. Note that residual thrombin from activating recombinant caspase cleaves at its cognate C-terminal site. ( F ) Upper : Schematic of fPanx1-eGFP embedded in bilayer in recording chambers in NanIon Orbit mini; only channels in the orientation shown are activated by recombinant Casp3 added to the chamber. Positions of recording and ground electrodes are depicted. Lower : Recordings of purified fPanx1 channels in planar lipid bilayers at the indicated voltages following activation by recombinant Casp3; current levels are indicated that correspond to closed ( C ) state and apparent openings of one or two channels (O1, O2). ( G ) Unitary current voltage relationships for caspase-activated fPanx1 show two different conductance states (N = 3 bilayers with each conductance level). Numerical data for conductance measurements from lipid bilayer recordings are presented in . Figure 1—source data 1. Conductance measurements of Xenopus Pannexin 1 (fPanx1) in lipid bilayers.

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Recombinant, Construct, Strep-tag, Silver Staining, Staining, Electron Microscopy, Western Blot, Incubation, Purification, Activation Assay

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) Size-exclusion chromatogram showing the purification of fPanx1- (eGFP). ( B ) Simply blue-stained SDS-PAGE gel showing samples taken during the purification process: flow through after gravity flow chromatography (FT), low salt wash (W1), high salt wash (W2), desthiobiotin elution fractions (e1, e2, e3), blank, concentrated sample before size-exclusion chromatography (SEC; Pre), and concentrated sample after SEC (Post). ( C ) Distribution of liposome diameters measured from negative stain EM images (97.3 ± 22 nm, 374 liposomes from 12 images). Diameters of liposomes are presented in . Figure 1—figure supplement 1—source data 1. Diameters of Xenopus Pannexin 1 (fPanx1) proteoliposomes.

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Purification, Staining, SDS Page, Chromatography, Size-exclusion Chromatography, Liposomes

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) Current–voltage traces for fPanx1-eGFP expressed in HEK 293T with normal internal solution ( A ) and with recombinant Caspase-3 (Casp3) in the internal solution ( B ; 2 µg/mL). The current activated by Casp3 was inhibited by bath application of 50 μM CBX. ( C ) Normalized current–voltage curves from whole-cell patch-clamp electrophysiology comparing hPANX1 and fPanx1-eGFP. ( D ) Schematic showing protocol for inside-out patch-clamp recordings of fPanx1-eGFP (upper) and channel activity evoked by Casp3 and inhibited by CBX. ( E ) Unitary current–voltage relationship with estimated slope conductance from inside-out patches of fPanx1 (N = 3). Numerical data for current–voltage traces of fPanx1-eGFP treated with or without Caspase-3 and comparison with hPANX1 are presented in . Numerical data for fPanx1 unitary conductance are presented in . Figure 1—figure supplement 2—source data 1. In vitro electrophysiology of Xenopus Pannexin 1 (fPanx1). Figure 1—figure supplement 2—source data 2. Xenopus Pannexin 1 (fPanx1) unitary conductance.

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Recombinant, Patch Clamp, Activity Assay, Comparison, In Vitro

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) ‘Flickery’ currents observed after addition of fPanx1 to lipid bilayers at +140 mV before addition of caspase. ( B ) Current recordings from DPhPC lipid bilayers with fPanx1, before (left) and after addition of Caspase-3 (Casp3; right) (N = 3). ( C ) Current recordings from DPhPC lipid bilayers without incorporation of fPanx1, before (left) and after addition of Casp3 (right) (N = 3).

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques:

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) Schematic depicting experimental design for treating fPanx1-containing proteoliposomes with recombinant Caspase-3 (Casp3) overnight at 4°C before incubation with SR-B (B, 1 mM) for 3 hr and loading on a G-25 spin column. ( B ) SR-B fluorescence (mean ± SEM) in eluted proteoliposomes under the indicated conditions (nine assays: >3 proteoliposome preparations). ANOVA (F 4,34 = 5.95, p=0.001) and Tukey’s multiple comparisons test (p-values from comparisons are shown). Numerical data for bulk dye uptake are presented in . Figure 2—figure supplement 1—source data 1. Bulk dye uptake in caspase-treated Xenopus Pannexin 1 (fPanx1)-containing proteoliposomes.

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Recombinant, Incubation, Fluorescence

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) Proteoliposomes containing fPanx1were incubated overnight with recombinant Caspase-3 (Casp3) and for 3 hr with sulforhodamine B (SR-B) dye prior to analysis by ImageStream flow cytometry. ( B ) Representative images of liposomes treated with vehicle (left) or Casp3 (right) viewed by brightfield, or on channels for (GFP) and SR-B fluorescence. ( C ) Frequency distributions of fluorescence intensity show that proteoliposomes treated with recombinant Casp3 show a reduction in mean GFP intensity ( left ) and an increase in SR-B intensity ( right ), relative to vehicle treated proteoliposomes. ( D ) Mean fluorescence intensity (MFI) of GFP fluorescence ( left ) (p=0.0096) and SR-B fluorescence ( right ) (p=0.0104) before and after caspase treatment are shown with individual experiments depicted according to the color shown (N = 5). A paired t-test was performed. Numerical data for changes in GFP and SR-B MFI are presented in . Figure 2—source data 1. Mean fluorescence intensity (MFI) of Xenopus Pannexin 1 (fPanx1) proteoliposomes before and after Caspase-3 treatment.

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Incubation, Recombinant, Flow Cytometry, Liposomes, Fluorescence

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) Changes in GFP and SR-B fluorescence in individual experiments are shown (N = 5). ( B–F ) Flow cytometry plots are shown for individual experiments from ( A ) (color-coded accordingly). Gray dots indicate proteoliposomes not treated with Caspase-3 (Casp3) and colored dots indicate proteoliposomes after treatment with Casp3. Only positive SR-B values are plotted on the logarithmic y-axis. Numerical data for change in GFP and SR-B MFI are presented in . Numerical data for flow cytometry dot-plots for individual experiments are presented in . Figure 2—figure supplement 2—source data 1. (GFP) and sulforhodamine-B mean fluorescence intensity (MFI) in caspase-treated Xenopus Pannexin 1 (fPanx1)-containing proteoliposomes. Figure 2—figure supplement 2—source data 2. Flow cytometry dot plots of ImageStream experiments.

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Fluorescence, Flow Cytometry

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) Schematic of experimental design to assay dye release kinetics from caspase-treated fPanx1-containing proteoliposomes by total internal reflection fluorescence (TIRF) microscopy. ( B ) Example fluorescence intensity traces for sulforhodamine B (SR-B, anionic dye, 559 Da) and (GFP) (caspase cleavage) over time in proteoliposomes after caspase treatment. Sample images of the proteoliposome fluorescence at the different time points are also provided. ( C ) Steady-state change in normalized fluorescence intensity for SR-B ( left ) and GFP ( right ) from fPanx1-GFP-containing proteoliposomes treated with either Caspase-3 (Casp3) or vehicle, or from empty liposomes (no fPanx1-GFP) treated with Casp3. Data from fPanx1-containing proteoliposomes were grouped according to whether they showed a reduction in GFP fluorescence (cleaved) or no change in GFP fluorescence (<10%, uncleaved) (N = 5 (+)Panx 1 (+)Casp3, N = 5 (+)Panx1 (−)Casp3, N = 4 (−)Panx1 (+)Casp3). ( D, E ) Fluorescence intensity traces ( D ) and steady-state change in normalized fluorescence intensity ( E ) for Rhodamine B (RhB, cationic dye, 479 Da) and GFP, as described for ( B, C ) (N = 5 (+)Panx1 (+)Casp3, N = 3 (+)Panx1 (−)Casp3, N = 4 (−)Panx1 (+)Casp3). ( F ) Efflux rates for SR-B and RhB were determined from fits of mono-exponential to the fluorescence intensity decay curves for individual caspase-treated proteoliposomes and plotted relative to the change in GFP fluorescence (i.e., fPanx1 cleavage); overlaid regression lines are depicted (with 95% confidence interval; slopes were significantly different, p=0.0014). Inset shows dye efflux rates for individual liposomes. Individual liposome dye efflux rates were analyzed by a Mann–Whitney test (p<0.0001) ( G ) Steady-state change in normalized fluorescence intensity for Alexa594 (anionic, 880 Da), Alexa555 (anionic, 980 Da), ATTO550 (cationic, 1363 Da), and Dextran 3000 (anionic, 3000 Da) from fPanx-GFP-containing proteoliposomes treated with Casp3 (Alexa 594 N = 6, Alexa 555 N = 5, ATTO 550 N = 3, Dextran 3000 N = 5). ( H ) Efflux rates for the indicated dyes represented by associated regression lines (with 95% confidence interval), with pairwise comparison of slopes: SR-B vs. RhB (p=0.0014), SR-B vs. 594 (p=0.16), SR-B vs. 555 (p=0.0074), 594 vs. 555 (p=0.15), 594 vs. RhB (p=0.16), 555 vs. RhB (p=0.53). Inset shows dye efflux rates for individual liposomes (ANOVA: F 3,267 = 36.18, p<0.0001; with Tukey’s multiple comparisons test: ****p<0.0001; ***p=0.0002). ( I ) Relative change (upper) and rate (lower) of GFP fluorescence in cleaved fPanx1-containing proteoliposomes filled with the indicated dyes. Numerical data for all dye release data and GFP cleavage are provided in . Figure 3—source data 1. Total internal reflection fluorescence (TIRF) imaging of dye release from proteoliposomes.

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Fluorescence, Microscopy, Liposomes, MANN-WHITNEY, Comparison, Imaging

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) Schematic depicting experimental design for treating fPanx1-containing proteoliposomes with recombinant Caspase-3 (Casp3) overnight at 4°C before incubation with 4 μCi each of α[ 32 P]-ATP ( B , 1 mM;~1:150000, hot:cold), [ 3 H]-Glutamate ( C , 0.8 mM;~1:2000), and [ 3 H]-Spermidine ( D , 8 µM;~1:24) for 3 hr and filtration using a Whatman GF/B filter. ( B–D ) Metabolites taken up by proteoliposomes under the indicated conditions for α[ 32 P]-ATP (N = 8), [ 3 H]-Glutamate (N = 6), and [ 3 H]-Spermidine (N = 5); molar quantities should not be compared between compounds due to different assay conditions. A box plot with the box depicting the quartiles/median, and lines drawn to points outside 25th/75th percentiles are shown. By repeated-measures one-way ANOVA (ATP: F 3,28 = 14.18, p<0.0001; Glutamate: F 3,20 = 18.29, p<0.0001; Spermidine: F 3,16 = 40.50, p<0.0001), with p-values provided from Tukey’s multiple comparisons tests. Numerical data for individual metabolite flux are shown in . Figure 4—source data 1. Metabolite flux in proteoliposomes.

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Recombinant, Incubation, Filtration

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet: ( A ) Schematic depicting experimental design for treating Xenopus Pannexin 1 (fPanx1)-containing proteoliposomes with recombinant Caspase-3 (Casp3) overnight at 4°C before concurrent incubation with 4 μCi each of α[ 32 P]-ATP ( B , 1 mM; ~1:150,000, hot:cold) and [ 3 H]-Spermidine ( C , 8 µM; ~1:24) for 3 hr and filtration using a Whatman GF/B filter. Orange boxes depict 95% confidence intervals of Casp3-treated fPanx1 proteoliposome metabolite uptake of compounds incubated individually (from ). By repeated-measures one-way ANOVA (ATP: F 3,19 = 15.14, p=0.0002; spermidine: F 3,16 = 12.17, p<0.0006), with p-values provided from Tukey’s multiple comparisons tests. Numerical data for ATP and spermidine concurrent flux are presented in . Figure 4—figure supplement 2—source data 1. ATP and spermidine concurrent flux in proteoliposomes.

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Recombinant, Incubation, Filtration

Journal: eLife

Article Title: ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels

doi: 10.7554/eLife.64787

Figure Lengend Snippet:

Article Snippet: Samples were transferred to 0.45 μM nitrocellulose membranes (Perkin Elmer), which were blocked for 1 hr at room temperature in 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4 and then incubated overnight at 4°C with fPanx1 antibody (Rabbit mAb #91137, 1:1000; Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Derivative Assay, Software