EMBO/soda-nel huggingface dataset:
CoQ Deficiency Causes Disruption of Mitochondrial Sulfide Oxidation, a new Pathomechanism Associated to this Syndrome (2016)
Figure 8

Figure 8. COX activity in tissues from CoQ deficient mice.

COX activity in cerebrum, kidneys and muscle (A) of Coq9+/+, Coq9R239X and Coq9Q95X mice. Images COX histochemistry (B) in gastrocnemious of Coq9+/+, Coq9R239X and Coq9Q95X mice; scale bars: 100 μm. Data are expressed as mean ± SD (one-way ANOVA with a Tukey's post hoc test; n = 3-6 for each group).
CoQ Deficiency Causes Disruption of Mitochondrial Sulfide Oxidation, a new Pathomechanism Associated to this Syndrome (2016)
Figure 9

Figure 9. Blood pressure and heart rate in Coq9R239X mice.

Systolic, diastolic and mean blood pressure in Coq9+/+ and Coq9R239X mice (A). Heart rate in Coq9+/+ and Coq9R239X mice (B). **P < 0.01; Coq9R239X mice versus Coq9+/+ mice (t test; n = 5 for each group).
A Protein Quality Control Pathway Regulated by Linear Ubiquitination (2019)
Figure 1

Fig 1. Htt-polyQ aggregates recruit LUBAC components.

(A) HOIP, HOIL-1L, and SHARPIN co-localize with Htt-Q97 aggregates. SH-SY5Y cells co-expressing Htt-Q25-GFP or Htt-Q97-GFP (green) and HOIP, HOIL-1L, SHARPIN, or HHARI (red); DAPI (blue). Scale bar, 10 µm.

(B, C) HOIP co-localizes with Htt inclusion bodies in human HD frontal cortex. Perinuclear Htt aggregates visualized by immunofluorescence (B) and DAB immunohistochemistry (C) in human HD frontal cortex. Scale bar, 10 µm (B), 15 µm (C).

Data information: See also Fig EV1.
A Protein Quality Control Pathway Regulated by Linear Ubiquitination (2019)
Figure 2

Fig 2. Linear ubiquitin chains are enriched at Htt-polyQ aggregates.

A-C M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured SH-SY5Y cells (A), R6/2 mouse cortex and striatum (B) and human HD frontal cortex (C). Autofluorescent lipofuscin appears in the green channel in human brain. Scale bar, 20 µm (A), 10 µm (B, C).

(D) Htt-Q25- or Htt-Q97-expressing HEK293T were analyzed by filter retardation assays. As indicated, the M1 ubiquitin antibody was preincubated with recombinant tetra-ubiquitin linked via M1 (4 x M1 ubiquitin) or K63 (4 x K63 ubiquitin).

Data information: See also Fig EV2.
A Protein Quality Control Pathway Regulated by Linear Ubiquitination (2019)
Figure 3

Fig 3. HOIP is recruited to Htt-polyQ via the PUB domain in a p97/VCP-dependent manner.

(A) Scheme of WT HOIP and HOIP mutants.

(B) Quantification of the co-localization of WT HOIP or HOIP mutants and Htt-Q97 aggregates. Data are displayed as mean ± SD and were analyzed by Mann-Whitney U-test, n = 3.

(C) Htt-Q97-GFP (green) was co-expressed with HA-tagged WT or HOIP mutants (red) in SH-SY5Y cells and analyzed by immunocytochemistry using an antibody against HA. DAPI (blue). Scale bar, 10 µm.

(D) Recruitment of HOIP to Htt-Q97 is decreased in p97/VCP-deficient cells. p97/VCP-deficient SH-SY5Y cells co-expressing Htt-Q97 and HA-HOIP were reconstituted with either WT VCP or VCP∆PIM and analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9.

(E) Recruitment of p97/VCP to Htt-Q97 is decreased in HOIP KO cells. WT HAP1 cells (control) or HOIP KO HAP1 cells reconstituted with either WT HOIP or HOIP∆PUB were analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Bonferroni Multiple Comparison Test, n = 5.

(F) The interaction of soluble Htt-Q60 with p97/VCP is dependent on HOIP. WT HAP1 cells or HOIP KO HAP1 cells were transfected with Htt-Q60-HA. 48 h after transfection cells were lysed with 1% Triton X-100, followed by an immunoprecipitation of Htt-Q60 via the HA tag. Immunopurified proteins were detected by Western blotting using an anti-VCP and anti-HOIP antibody.

Data information: *p ≤ 0.05, ***p ≤ 0.001.
A Protein Quality Control Pathway Regulated by Linear Ubiquitination (2019)
Figure 5

Fig 5. Linear ubiquitination of Htt-Q97 prevents sequestration of the transcription factor Sp1.

(A) The interaction between Htt-Q97 and Sp1 is decreased by linear ubiquitination. HEK293T cells were co-transfected with Htt-Q97-HA and the siRNAs indicated. Htt-Q97-GFP was transfected as a control for specific immunoprecipitation of HA-Htt-Q97. Nuclear (left panel) and cytosolic fractions (middle panel) were subjected to an anti-HA immunoprecipitation followed by Western blotting using an anti-Sp1 antibody. Right panel: Input controls (whole cell lysates).

(B) Quantification of the interaction between Htt-Q97 and Sp1 in nuclear fractions. Shown is the relative amount of Sp1 co-purifying with Htt-Q97. Signal intensities were normalized to nuclear Sp1. Data are displayed as mean ± SD and were analyzed by 2 independent Mann-Whitney U-tests, n = 4.

(C, D) Impairment of Sp1-mediated transcription by Htt-Q97 is modified by HOIP. (C) Shown is the relative luciferase activity in HEK293T cells expressing Htt-Q97-HA, a Sp1 luciferase reporter construct and HOIP cDNA. Data are displayed as mean ± SD and were analyzed by Kruskal-Wallis test followed by Dunn's Multiple Comparison Test, n = 9-11. (D) Shown is the relative luciferase activity in HEK293T cells expressing Htt-Q97-HA, a Sp1 luciferase reporter construct and HOIP siRNA. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9.

(E) Sp1 silencing decreases HOIP and SHARPIN protein expression. SH-SY5Y cells were transfected with control or Sp1-specific siRNAs and analyzed for protein expression 2 days after transfection by immunoblotting (left panel). Quantification of normalized Sp1, HOIP and SHARPIN protein expression levels (right panel). Data are displayed as mean ± SEM with n=5 from unpaired two-tailed Student's t-tests.

(F) HOIP-, HOIL-1L- and SHARPIN-specific mRNA is decreased in R6/2 striatum. Quantification of mRNA from the striatum of 9-12 weeks old R6/2 mice and non-transgenic littermates. Data are displayed as mean ± SD with n = 6-9 biological replicates from unpaired two-tailed Student's t-tests.

(G) HOIP protein levels are decreased in human HD brain. Total protein lysates from human brain (anterior cingulate cortex) analyzed by immunoblotting using antibodies against HOIP and βIII-tubulin. HOIP-positive signals were normalized to βIII-tubulin. To verify the specificity of the HOIP antibody, WT and HOIP KO HAP1 cells were analyzed in parallel on a different gel. Data are displayed as mean ± SEM with n=5 for control and n=7 for HD patients from unpaired two-tailed Student's t-tests.

Data information: See also Fig EV5. *p ≤ 0.05, ***p ≤ 0.001.
A Protein Quality Control Pathway Regulated by Linear Ubiquitination (2019)
Figure 6

Fig 6. Linear ubiquitin chains are found at multiple protein aggregates.

(A) HOIP is recruited to disease-associated protein aggregates formed by Ataxin-3-Q84, SOD1 G85R, TDP-43 Q331K or Optineurin R96L. SH-SY5Y cells co-expressing disease-associated proteins (green) and HOIP (red) were analyzed by immunocytochemistry; DAPI (blue). Scale bar, 20 µm.

(B, C) Linear ubiquitin accumulates at disease-associated protein aggregates under endogenous LUBAC expression. M1-linked ubiquitin (red) and disease-associated proteins (green) were analyzed by immunocytochemistry in SH-SY5Y cells using the 1E3 antibody (Millipore) (B) or primary striatal neurons using the 1F11/3F5/Y102L antibody (Genentech) (C). DAPI (blue). Scale bar, 20 µm.

(D) SDS-soluble SOD G85R and TDP-43 Q331K species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with disease-associated proteins and either the plasmids indicated or with control siRNA or OTULIN siRNA. 48 h after transfection cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of SOD G85R and TDP-43 Q331K via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3.

Data information: See also Fig EV5.
A Protein Quality Control Pathway Regulated by Linear Ubiquitination (2019)
Figure 7

Fig 7. Linear ubiquitination decreases the number of cells with aggregates in a proteasome-dependent manner.

(A) Linear ubiquitination reduces the fraction of cells with Htt-Q97 aggregates. SH-SY5Y cells were transfected with Htt-Q97 and either control siRNA, HOIP siRNA or OTULIN siRNA. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. Expression levels of HOIP and OTULIN were analyzed by Western blotting.

(B) Linear ubiquitination reduces the fraction of cells with Htt-Q97 aggregates. SH-SY5Y cells were transfected with Htt-Q97 and either WT HOIP or the catalytically inactive HOIP mutant C885A. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 5. Expression levels of WT and mutant HOIP and OTULIN were analyzed by Western blotting.

(C) The effect of HOIP on Htt-Q97 aggregation is independent of NF-κB signaling. SH-SY5Y cells transiently transfected with Htt-Q97 and either control vector (CO) or WT HOIP plus the NF-κB super-repressor IκB-2S/A were analyzed by immunocytochemistry. Data are mean ± SD with n=5 from an unpaired two-tailed Student's t-test. Expression levels of WT HOIP and IκB-2S/A were analyzed by Western blotting.

(D) Catalytically active HOIP decreases the number of cells with Htt-Q97 aggregates independently of autophagy. WT and ATG5 KO MEFs were transiently transfected with Htt-Q97-GFP and either control vector (CO), HOIP or HOIP C885A. Cells were treated 24 h after transfection with the proteasomal inhibitor MG132 (1 µM, 16 h). Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 7.

(E) Linear ubiquitination reduces the fraction of cells with TDP-43 Q331K aggregates. SH-SY5Y cells were transfected with TDP-43 Q331K and either control siRNA, HOIP siRNA or OTULIN siRNA. Data are displayed as mean ± SD and were analyzed by Kruskal-Wallis test, followed by Dunn's Multiple Comparison Test, n = 7. Expression levels of HOIP and OTULIN were analyzed by Western blotting.

(F) Catalytically active HOIP decreases the number of cells with TDP-43 aggregates dependent on proteasomal degradation. SH-SY5Y cells were transfected with TDP-43 Q331K and either control vector (CO), HOIP or HOIP C885A. Cells were treated 48 h after transfection with the proteasomal inhibitor MG132 (1 µM) or the p97/VCP inhibitor NSM-873 (1 µM) for 3 h. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Dunnett's Multiple Comparison Test, n = 5.

(G) Inhibition of the proetasome and p97/VCP do not have additive effects on the degradation of misfolded TDP-43 species. SH-SY5Y cells expressing TDP-43 Q331K were treated 48 h after transfection with MG132 (1 µM) and/or NSM-873 (1 µM) for 3 h. All data are mean ± SD, data were analyzed by one-way ANOVA followed by Bonferroni's Multiple Comparison Test, n = 5.

Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

EXPANDED VIEWS
Haematopoietic stem cell gene therapy with IL-1Ra rescues cognitive loss in mucopolysaccharidosis IIIA (2020)
Figure 1

Figure 1

Interleukin-1 and related cytokines are elevated in MPSIIIA mice and patients

A. Representative sections of WT and MPSIIIA brains at 9 months of age stained for astrocytes (GFAP) or microglia (isolectin B4, ILB4). Images correspond to high power views covering cortical layers II-V. Scale bar, 100 μm. (n=4 mice per group).

B. Quantitative PCR for various inflammatory cytokines in whole brains from 9 month old WT and MPSIIIA mice. Symbols above bars are versus WT. (n=6 mice per group). Data are expressed as mean ± STDEV, data for each cytokine were tested by unpaired t-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus WT. Exact P-values are indicated in Appendix Table S1.

C, D. Quantification of IL-1β and IL-1Ra levels in plasma from human healthy control and MPSIII patients (n=6 control participants and n=21 MPSIII patients). Data are expressed as mean ± STDEV and were tested by unpaired t-test. C. IL-1β, control vs. MPSIII P=0.0407; D. IL-1Ra, control vs. MPSIII P=0.0001.

E, F. Quantification of IL-1β and IL-1Ra levels in cerebrospinal fluid (CSF) from control and MPSIII patients (n=3 control participants and n=21 MPSIII patients). Data are expressed as mean ± STDEV and were tested by unpaired t-test; *P<0.05. F. IL-1Ra, control vs. MPSIII P=0.0167.
Haematopoietic stem cell gene therapy with IL-1Ra rescues cognitive loss in mucopolysaccharidosis IIIA (2020)
Figure 3

Figure 3

Both HS and secondary stimuli are required to induce secretion of IL-1β via the NLRP3 inflammasome.

A. Representative sections from 9 month old WT and MPSIIIA brains showing secondary storage of GM2 ganglioside, cholesterol, protein aggregates and amyloid beta peptide 1-40 and 1-42 within cortical layers II to V/VI, x20, scale bar: 50 μm. Inserts, 100x, scale bar: 10μm. (n=4 mice per group).

B. ATP levels in whole brain homogenate from 9 month old WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.02.

C. Brain transcription of mRNA species for various inflammasome components (n=6 mice per group). Data are expressed as mean ± STDEV, data for each cytokine were tested by unpaired t-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus WT. Exact P-values are indicated in Appendix Table S3.

D. Caspase 1 activity levels in whole brain homogenate from WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.0001.

E-J. WT (E-G) and Nlrp3-/- (H-J) mixed glia were primed with MPSIIIA GAG or LPS. ATP (5 mM), cholesterol crystals (1 mg/ml) or amyloid-β fibrils (Aβ1-40 4.3 μM and Aβ1-42 5 μM) were added 4 hours post-priming stimulus, and the secretion of IL-1β into the media measured via ELISA after a further 20 hours (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, ***P<0.001. Exact P-values are indicated in Appendix Table S3.
Haematopoietic stem cell gene therapy with IL-1Ra rescues cognitive loss in mucopolysaccharidosis IIIA (2020)
Figure 4

Figure 4

Evaluation of LV.IL1RN HSCT in MPSIIIA mice.

A. Schematic of CD11b.IL1RN.WPRE lentivirus produced to drive codon optimised human IL1RN (secreted isoform).

B. The biological activity of LV-mediated IL-1Ra was assessed in vitro. RAW264.7 macrophages were transduced with LV.IL1RN or LV.GFP vectors at a MOI of 10. After 72 hours cells were stimulated with recombinant hIL-1β and the expression of TNF-α assessed after a further 48 hours. (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus NTC within a treatment group. Exact P-values in Appendix Table S4.

C. Donor MPSIIIA bone marrow was lineage depleted and transduced with LV.IL1RN vector at a MOI of 10. 3x105 transduced HSCs were transplanted into 6-8 week old busulfan myeloablated recipient MPSIIIA mice. Chimerism was assessed 2 months post-transplant and behavioural assessment conducted 4 months post-transplant. Animals were sacrificed and biochemical or histological analysis was carried out after behavioural assessment was finished.

D-F. A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. Vector copy number (D) and protein production of secreted human IL-1Ra (E) and intracellular human IL-1Ra (F) were quantified (n=6 CFU assays). Data are expressed as mean ± STDEV and were tested by unpaired t-test. E. WT vs. MPSIIIA P=0.0001; F. WT vs. MPSIIIA P=0.0008.

G, H. Donor chimerism (G) based on peripheral blood composition (H) was determined by flow cytometry at 4 months of age (2 months post-transplant) (n=10 mice).

I. Human IL-1Ra expression was quantified in the plasma of mice at 6 months of age (4 months post-transplant) (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; WT vs. LV.IL1RN P<0.0001; MPSIIIA vs. LV.IL1RN P<0.0001. Symbols above bars are versus WT.

J. Vector copy number was evaluated in brain, spleen, liver, white blood cells (WBC) and bone marrow (BM) of mice at 6 months of age (4 months post-transplant) (n=10 mice per group).

K. Quantification of human IL-1Ra levels in the brain of transplanted MPSIIIA mice (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; WT vs. LV.IL1RN P<0.0001; MPSIIIA vs. LV.IL1RN P<0.0001. Symbols above bars are versus WT.

L. Representative sections from 6-month old MPSIIIA and LV.IL1RN transplanted MPSIIIA mice. Sections show the CA3 region of the hippocampus. Cells expressing high levels of human IL-1Ra are indicated with black arrows. 40x, scale bar, 50 μm (n=4 mice per group).
Haematopoietic stem cell gene therapy with IL-1Ra rescues cognitive loss in mucopolysaccharidosis IIIA (2020)
Figure 6

Figure 6

IL-1 signalling inhibition in MPSIIIA mice reduces glial activation.

A. Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with anti-mouse glial fibrillary acidic protein (GFAP; red)/DAPI (nuclear, blue) to identify activated astrocytes, 20x, scale bar: 50μm.

B. Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with isolectin B4 (ILB4) to identify activated microglia, 20x, scale bar: 50μm

C, D. The average number of GFAP positive astrocyte cells over three fields of view per mouse of the hippocampus (C) and the cortex (D) were counted (n=4 mice per group, average of 3 fields of view per mouse). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; **P<0.01, ***P<0.001. Symbols above bars are versus WT. Exact P-values are indicated in Appendix Table S6.

E, F. The average number of ILB4+ positive microglial cells over three fields of view per mouse of the hippocampus (C) and the cortex (D) were counted (n=4 mice per group, average of 3 fields of view per mouse). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus WT. Exact P-values are indicated in Appendix Table S6.

Data information: Non-linear adjustments were made equally in fluorescent images to reduce background.
PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity (2021)
Figure 1

Figure 1 - Genetic inhibition of PI3KC2β reduces vascular leakage, edema, cerebral infarction and improves neurological outcomes in ischemia/reperfusion stroke models.

A-D WT, heterozygous (C2βWT/D1212A) and C2βD1212A/D1212A mice were subjected to tMCAO (1h ischemia followed by 24h reperfusion). (A) Representative photographs of coronal brain sections and graph quantification of Evans blue (EB) dye leakage (dashed orange line) and edema volume. Data represent mean ± SEM (EB leakage: n=5-16 mice per group; Edema volume: n=14-30 mice per group; *p<0.05, **p<0.01 Mann-Whitney test). (B) Representative photographs of coronal brain section stained with TTC (dashed white line) and graph quantification of infarct volume measurements. Data represent mean ± SEM (n=14-27 mice per group; *p<0.05; ***p<0.001, unpaired t-test). (C) Mortality between day 0 and day 1 after tMCAO (n=30-39 mice per group; *p<0.05, Fisher's test). (D) Neurological scores evaluated 24 hours after reperfusion by Bederson test (left panel) and Grip test (right panel) based on a five point system. Data represent mean ± SEM (n=14-27 mice per group; *p<0.05, unpaired t-test).

E WT and C2βD1212A/D1212A mice were subjected to thromboembolic stroke model. Representatives T2-weighted MRI of coronal section taken 24 hours after the onset of in situ clot formation by α-thrombin were shown. Graph quantification of infarct volume measurements. Data represent mean ± SEM (n=23-25 mice per group; **p<0.01, Unpaired t-test).
PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity (2021)
Figure 2

Figure 2 - Genetic inhibition of PI3KC2β reduces inflammation 24 hours after stroke.

A-C WT and C2βD1212A/D1212A mice were subjected to tMCAO followed by 24h of reperfusion. (A) Gene expression of TNFα, IL-1β and IL-6 in the cortex and basal ganglia (B. ganglia). The mRNA levels are given as the fold change normalized to rps29 relative to the corresponding contralateral (healthy) hemisphere. Data represent mean ± SEM (n=8-10 mice per group; *p=0.05; **p<0.01, Unpaired t-test with Welch's correction). (B) Protein expression of TNFα, IL-1β and IL-6. Data represent mean ± SEM (n=4 mice per group; *p=0.05; Mann-Whitney test). (C) Representative immunohistochemistry of neutrophils (arrowheads) in ischemic hemisphere (scale bar 200 µm) and graph quantification. Data represent mean ± SEM (n=5-7 mice per group; **p<0.01; Mann-Whitney test).

D Representative T2-weighted MRI of WT and C2βD1212A/D1212A mice taken 24 hours after clot formation and graph quantification of area stained by P-Selectin using MPIOs in ipsilateral (ischemic) normalize to contralateral (healthy) cortex in percentage. Data represent mean ± SEM (n=10-11 mice per group; *p<0.05; Unpaired t-test with Welch's correction).
PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity (2021)
Figure 3

Figure 3 - In the absence of reperfusion, genetic inhibition of PI3KC2β does not affect inflammatory cytokine profile and infarct volume.

A Gene expression of TNFα (n=8-10 mice for WT and n=6 mice for C2βD1212A/D1212A), IL-1β (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A), IL-6 (n=8 mice for WT and n=10-12 mice for C2βD1212A/D1212A) and VCAM-1 (n=8-10 mice for WT and n=10 mice for C2βD1212A/D1212A) after 1 hour of ischemia without reperfusion (right panel). Gene expression of IL-6 (n=10-12 mice for WT and n=8 mice for C2βD1212A/D1212A) and VCAM-1 (n=12 mice for WT and n=10mice for C2βD1212A/D1212A) after 1 hour of ischemia followed by 4 hours of reperfusion (bottom, left panel) on WT and C2βD1212A/D1212A mice. The mRNA levels are given as the fold increase normalized to PGK1 relative to the healthy contralateral hemisphere of WT mice. Data represent mean ± SEM (***p<0.001, **p<0.01, *p<0.05, Mann-Whitney test or unpaired t-test, as accordance).

B Representative photographs of T2-weighted MRI of WT and C2βD1212A/D1212A mice and graph quantification of ischemic lesion after 24 hours permanent MCAO (electrocoagulation model). Data represent mean ± SEM (n=9-10 mice per group; ns, unpaired t-test).
PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity (2021)
Figure 4

Figure 4 - PI3KC2β inactivation in the non-hematopoietic compartment reduces cerebral infarction and edema in vivo and preserves endothelial barrier integrity in vitro.

A, B Representative photographs of coronal brain sections and graph quantification of infarct volume (dashed white line) measurements (A) and edema volume (B) in bone marrow (BM) chimeric mice 24 hours after tMCAO. WT>WT: transplantation of WT BM into WT hosts; C2βD1212A>WT: transplantation of C2βD1212A/D1212A BM into WT hosts; C2βD1212A>C2βD1212A: transplantation of C2βD1212A/D1212A BM into C2βD1212A/D1212A hosts; WT> C2βD1212A transplantation of WT BM into kinase-dead hosts. Data represent mean ± SEM; n=13-16 mice per group; *p<0.05 **p<0.01; ***p<0.001 vs WT>WT, Mann-Whitney test or unpaired t-test, as accordance.

C Sh-Control or sh-PI3KC2β hCMEC/D3 cells were cultured on Transwell and stimulated with TNFα over the time. Trans-endothelial electrical resistance (TEER) was measured with a voltohmeter Millicell ERS-2 at the indicated time. Data are shown in percentage of control at t0 ± SEM (n=4 independent experiments). ***p<0.001; **p<0.01; *p<0.05, significance differences from control, 2-way ANOVA with Bonferroni post-test.