EMBO/soda-nel huggingface dataset:
PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS (2020)
Figure 1

Figure 1. p110α and δ are required for rat DRG axon regeneration; p110δ functions in the axonal compartment

(A) Laser injured DRG axons showing growth cone regeneration (upper panels) and regenerative failure (lower panels). Arrows as indicated.

(B) Upper graph shows percentage of regenerating axons of DIV 1-2 adult DRG neurons in the presence of p110 inhibitors, 2 h after injury. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: LY294002, 20 μM. A66 5 μM. XL-147 5 μM. TGX221 500 nM. IC-87114 10μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance analysed by Fishers exact (upper graph) or Kruskal-Wallis test (lower graph).

(C) Examples of static vs. growing uninjured axons treated with p110α or δ inhibitors as indicated. Both arrows in bottom right image indicate growing axons.

(D) Percentage of uninjured, growing axons in the presence of p110 inhibitors. Inhibitors were added as for (B). Lower graph shows length grown in 2 h. Data are shown as the mean +/- SEM. P values indicate significance measured by Fishers exact test (upper graph) or ANOVA with Tukey's post-hoc analysis (lower graph).

(E) Adult DRG neurons in microfluidic compartmental chambers. Cell bodies on the left side, axons extending through microchannels on the right. Lower panels are schematics.

(F) Upper graph shows percentage of regenerating axons in microfluidic chambers. p110α or δ inhibitors were applied to either soma or axons. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: A66 5 μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance as measured by Fishers exact test (upper graph) or by Kruskal-Wallis test (lower graph).

Data information: Numbers on bars are the total numbers of axons cut, from n=4 experiments for B, D and F.
PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS (2020)
Figure 3

Figure 3. p110δ or p110αH1047R expression elevates PIP3 in the soma and axon, whilst native p110α does not

(A) PIP3 immunofluorescence in the soma of DIV 16 cortical neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity.

(B) PIP3 immunofluorescence at the axonal growth cone of DIV 16 neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity.

(C) Quantification of PIP3 immunofluorescence in the soma. Data are shown as the mean +/- SEM. P values indicate significance as measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons.

(D) Graph showing PIP3 quantification of immunofluorescence at the axon growth cone. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.
PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS (2020)
Figure 4

Figure 4. Expression of p110δ or p110αH1047R increases axon and dendrite growth of rat cortical neurons developing in vitro, whilst native p110α does not

(A) DIV 4 cortical neurons expressing p110 isoforms and GFP. Arrow marks the axon tip.

(B) Quantification of axon length. n = 3 experiments, 45 neurons per group.

(C) Quantification of dendrite length. n = 3 experiments, 45 neurons per group.

(D) Quantification of the axon:dendrite length ratio. n = 3 experiments, 45 neurons per group.

(E) DIV 14 cortical neurons expressing p110 isoforms and GFP, immunolabelled for pS6.

(F) Sholl analysis of branches. n = 60 neurons per group. Data are shown as the mean +/- SEM. P values indicate significance measured by repeated measure ANOVA with Bonferonni's post-hoc test.

(G) Quantification of the total neurite length. n = 3 experiments, 60 neurons per group.

(H) Quantification of soma area. n = 3 experiments, 60 neurons per group.

(I) Quantification of the pS6 immunofluorescence. n = 3 experiments, 60 neurons per group.

Data information: B-D and G-I, Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis.
PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS (2020)
Figure 5

Figure 5. p110δ and p110αH1047R promote axon regeneration of CNS neurons in vitro

(A) Axotomised DIV 14-16 rat cortical neurons expressing p110δ and GFP or GFP alone. Axons were cut >1000 μm from the soma imaged for 14 h. Black arrows mark the cut site, white arrows marks the axon tip. See also Movies EV7 and EV8.

(B) Percentage of regenerating axons 14 h after laser axotomy. Numbers on bars are injured axons per group, from 4 experiments.

(C) Quantification of axon regeneration length 14 h post-axotomy. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments.

(D) Quantification of time to start of regeneration. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments.

(E) Axotomised human embryonic stem cell neurons p110δ and GFP or GFP alone. Black arrow marks the cut site, white arrows mark the axon tip.

(F) Percentage of regenerating axons of human hESC neurons, Numbers on bars are injured axons per group, from 5 experiments.

(G) Quantification of axon regeneration length. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test.

(H) Quantification of time to start of regeneration. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test.

Data information: (B)-(D), and (F), Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis.
PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS (2020)
Figure 6

Figure 6. p110δ functions through multiple downstream pathways

(A) Percentage of regenerating axons after laser axotomy of DIV 14-17 rat cortical neurons expressing either GFP (control) or GFP and p110δ in the presence of the indicated inhibitors, 14h after laser injury. Numbers on bars are injured axons per group.

(B) Single confocal section through a DIV 16 neuron expressing p110δ and GFP (upper panels). Green colour is GFP, red is overexpressed p110δ, detected with anti-p110δ. Lower panels show p110δ and GFP in a distal axon section, and at the growth cone. Arrow indicates the axon.

(C) Active ARF and total ARF6 (red) in axons of DIV 16 rat cortical neurons expressing GFP or GFP and p110δ, as indicated. Arrows indicate axons.

(D) Quantification of mean axonal ARF activity and total (mean) ARF6 in DIV 16 neurons expressing GFP or GFP and p110δ n=60 axons for each condition.

(E) Kymographs showing dynamics of α9 integrin-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ.

(F) Quantification of α9 integrin-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition.

(G) Quantification of total α9 integrin-GFP number in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 47 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.

(H) Kymographs showing dynamics of Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ.

(I) Quantification of Rab11-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition.

(J) Quantification of total Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 53 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.

Data information: Data are shown as the mean +/- SEM. (A) P values indicate statistical significance analysed by Fishers exact test. (F and I) P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. (D, G and J) data were analysed by two tailed Student's T-test.
PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS (2020)
Figure 7

Figure 7. p110δ and p110αH1047R support RGC survival and axon regeneration in the mouse optic nerve.

(A) Schematic representation of Rosa26 transgene expression via AAV.Cre recombinase.

(B) Time course of optic nerve regeneration experiments using Rosa26 p110αH1047R and Rosa26 p110δ mice.

C) Retinal sections from AAV-injected mice, immunolabelled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image.

(D) Percentage of phospho-S6 positive cells in the RGC layer 2 weeks after delivery of AAV-Cre-GFP or AAV-GFP.

(E) Retinal whole mounts of AAV-injected mice immunolabelled for the RGC marker Brn3A to indicate cell survival.

(F) Analysis of RGC survival 28 days after optic nerve crush (counts of Brn3A positive RGC neurons).

G) Representative images of CTB-labelled RGC axons 4 weeks after optic nerve crush in wild type (c57bl/6), Rosa26-p110αH1047R, and Rosa26-p110δ transgenic mice injected with AAV2.Cre.GFP. Dashed line indicates crush site.

(H) Regenerating axons at different distances distal to lesion sites.

Data information: (D), (F) and (H) Data are shown as the mean +/- SEM. P values indicate significance as measured by ANOVA with Tukey's post-hoc analysis. n=7 mice (wt), 10 mice (p110αH1047R), and 6 mice (p110δ).
PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS (2020)
Figure 8

Figure 8. AAV2-p110δ facilitates axon regeneration in the mouse optic nerve.

(A) Retinal sections from mice injected with AAV2 viruses as indicated, immunolabeled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image.

(B) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2.shScramble.GFP or AAV2.shPTEN.GFP.

(C) Number of pS6-postive RGCs of mice injected with AAV2.GFP or AAV2.p110δ.

(D) Representative images of retinal whole mounts from AAV-injected mice stained for the RGC marker Brn3A to indicate RGC survival.

(E) RGC survival 28 days after optic nerve crush.

(F) CTB-labelled RGC axons 4 weeks after optic nerve crush. Boxes i. to iv. show regenerating axons. Dashed line indicates crush site. White arrows mark the end of axons. Graph shows quantification of regenerating axons at different distances distal to the lesion sites.

Data information: (B) and (C) Data are shown as the mean +/- SEM and P values indicate significance measured by two tailed Students T-test. (E) and (H) Data are shown as the mean +/- SEM and P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. n= 8 mice (scrambled shRNA control) 6 mice (shPTEN) 4 mice (GFP) and 7 mice (p110δ).

Expanded View Figure Legends
Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy. (2012)
Figure 1

1,25D3 inhibits HIV and <italic>M. tuberculosis</italic> replication.

MDM were incubated with 1,25D3 for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h, washed then incubated with or without 1,25D3 for 7 days. (A) Top, extracellular release of HIV p24 antigen into the cell supernatant at days 0, 4 and 7 was detected by ELISA. Bottom, MDM were harvested and stained for HIV p17. Histograms are shown for a representative donor. (B) Top, cells were lysed after 3 h exposure to infectious agents or at the completion of the infection phase. Intracellular mycobacteria were harvested and assayed for mycobacterial growth at day 0 and day 7. Bottom, MDM were harvested and stained for mycobacterium. Histograms are shown for a representative donor. (C) Cell lysates from panel B were diluted and run in a MGIT 960. Bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.
Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy. (2012)
Figure 2

1,25D3 induces autophagy in human macrophages co-infected with HIV and <italic>M. tuberculosis</italic>.

HIV and/or M. tuberculosis (TB) infected MDM were treated for 7 days with 100 pmol/L 1,25D3. (A) Cells were lysed and immunoblots of LC3B isoforms using antibody to LC3B or β-actin performed. (B) Cells were incubated with 10 µg/mL pepstatin A for 4 h on day 7 prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin. (C) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown. (D) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d followed by 10 µg/mL pepstatin A for a further 4 h. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown. (E) Immunoblots of p62 using antibody to p62 or β-actin 7 d after macrophages treated with 1,25D3. (F) At 7 d post-infection, aliquots of supernatant taken before the addition of WST-1 were tested for lactate dehydrogenase (LDH) spectrophotometrically using the LDHPLUS assay. For the last hour cells were incubated with WST-1, and the reduction of the WST-1 reagent to its formazan product was monitored spectrophotometrically.
Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy. (2012)
Figure 3

1,25D3 induces autophagy in human macrophages co-infected with HIV and <italic>M. tuberculosis</italic>.

HIV (A), M. tuberculosis (TB) (B), and HIV/TB dual infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIV p17, mycobacteria, and saponin-resistant LC3B-II. Representative density plots from three donors are shown. (D) HIV/M. tuberculosis infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIV p17, mycobacteria, and saponin-resistant LC3B-II. Left, representative density plots from three donors from HIV/M. tuberculosis infected MDM are shown for HIV/M. tuberculosis co-infection. Right, histograms of saponin-resistant LC3B-II in macrophages that were HIV+
M. tuberculosis− or HIV+
M. tuberculosis+ at day 7 post-infection.
Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy. (2012)
Figure 4

1,25D3 inhibition of HIV and <italic>M. tuberculosis</italic> is Beclin-1 dependent.

MDM were transduced with non-specific scrambled shRNA (shNS) or Beclin-1 shRNA (shBCLN1) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to Beclin-1 or β-actin after initial pathogen exposure (Day 0) or after 7 days. (B) ELISA performed for HIV p24 antigen release over 7 d. (C) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. (D) Intracellular mycobacteria harvested and assayed for viability based on time to positivity (75 growth units) at day 0 and day 7 using the MGIT 960. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.
Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy. (2012)
Figure 5

1,25D3 inhibition of HIV and <italic>M. tuberculosis</italic> is ATG5 dependent.

MDM were transduced with non-specific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to ATG5 or β-actin after initial pathogen exposure (Day 0) or after 7 days. (B) ELISA performed for HIV p24 antigen release over 7 d. (C) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. (D) Intracellular mycobacteria harvested and assayed for viability based on time to positivity (75 growth units) at day 0 and day 7 using the MGIT 960. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.
Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy. (2012)
Figure 6

Bafilomycin A<sub>1</sub> inhibits the 1,25D3 mediated inhibition of HIV and <italic>M. tuberculosis</italic> replication.

MDM were pretreated with bafilomycin A1 (Baf A1) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7. (B) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7. (C) Intracellular mycobacteria harvested and assayed for viability using the MGIT 960 based on time to positivity at day 0 and 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.01.
Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy. (2012)
Figure 7

SID 26681509 inhibits the 1,25D3 mediated inhibition of HIV and <italic>M. tuberculosis</italic> replication.

MDM were pretreated with SID 26681509 (SID) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7. (B) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7. (C) Intracellular mycobacteria harvested and assayed for viability using the MGIT 960 based on time to positivity at day 0 and 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.01.
Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy. (2012)
Figure 8

Inhibition of HIV and <italic>M. tuberculosis</italic> by 1,25D3 is CAMP and autophagy dependent.

MDM were transduced with non-specific scrambled shRNA (shNS) or CAMP shRNA (shCAMP) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to CAMP or β-actin after initial pathogen exposure (Day 0) or after 7 days. (B) Day 7 HIV-1Ba-L and TB co-infected cells were harvested and stained for saponin-resistant LC3B-II at day 7. A representative histogram from three donors are shown. (C) ELISA performed for HIV p24 antigen release over 7 d. (D) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. * P<0.001.
MicroRNA-483 amelioration of experimental pulmonary hypertension (2020)
Figure 1

Figure 1. Lower serum miR-483 level in IPAH patients.

(A) Serum levels of miR-483-3p/-5p in IPAH patients (n = 139) and HC (n = 95) measured by qPCR. The data are fold change normalized to the averaged level of HC.

(B) ROC curve with sensitivity and specificity of serum levels of miR-483-3p/-5p for differentiating IPAH patients from HCs at diagnosis (IPAH, n = 139; HC, n = 95).

(C) Levels of miR-483-3p/-5p associated with PAH risk in 3 groups. IPAH patients were divided into a low risk group (Low) and an intermediate plus high risk group (Inter&high) according to the World Symposium on Pulmonary Hypertension 2018 [14].

(D) Levels of miR-483-3p were inversely correlated with serum levels of ET-1 (IPAH, n = 118; HC, n = 95) and IL-6 (IPAH, n = 112; HC, n = 93).

Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test (A), Kruskal-Wallis U test (C).
MicroRNA-483 amelioration of experimental pulmonary hypertension (2020)
Figure 2

Figure 2. MiR-483 targets PAH-related genes and pathways in PAECs.

(A) CD144-enriched EVs were isolated from serum of IPAH patients (n = 37) and HCs (n = 34). The levels of miR-483-3p/-5p were measured by qPCR.

(B, C) PAECs were transfected with miR-483-3p/-5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA-seq. Data are results from two biological repeats. (B) PAH-related GO enrichment delineated by DAVID for the top 300 up-regulated or down-regulated genes with the cutoff of p<0.05. (C) Heat map comparison of log2 fold changes of the indicated genes.

Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test.
MicroRNA-483 amelioration of experimental pulmonary hypertension (2020)
Figure 3

Figure 3. MiR-483-targeted genes.

(A) Predicted binding sites for miR-483-3p/-5p on the 3'UTR of mRNAs as indicated.

(B-D) PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of miR-483-3p/-5p, TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA and protein were measured by qPCR and Western blot, respectively.

(E) Bovine aortic ECs were transfected with a luciferase reporter fused with the 3'UTR of TGF-β (Luc-TGF-β WT), TGFBR2 (Luc-TGFBR2 WT), IL-1β (Luc-IL-1β WT) or ET-1 (Luc-ET-1 WT) or a binding site mutation (Luc-TGF-β Mut, Luc-TGFBR2 Mut, Luc-IL-1β Mut, ET-1-Mut), then infected with Lenti-pre-miR-483 for additional 36 hr. Luciferase activity was measured.

(F, G) PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 36 hr. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by immunoprecipitation with anti-Ago1 or anti-Ago2. Levels of miR-483-3p/-5p and TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein.

Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups, exact P values were shown in Appendix Table S3).