Journal: Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology

Article Title: Rho and Rac, but not ROCK, are required for secretion of human and mouse eosinophil-associated RNases

doi: 10.1111/cea.13292

Figure Lengend Snippet: CCL11-mediated EAR secretion from eosinophils requires Rho activity. RNase activity was measured in supernatants of mCCL11-stimulated mouse (A) and hCCL11-stimulated human (B) eosinophils in the presence of Rho GTPase blocker, C3-transferase, as described in Material and Methods. Data are mean ± SD of five independent experiments (A) or mean ± SD from triplicate wells and are representative of four independent experiments (B). Asterisks represent P values < 0.05 (*), or < 0.01 (**).

Article Snippet: In some experiments, cells were pretreated for two hrs at 37°C with the Rho blocker, C3 transferase (5 and 10 μg/ml, Cytoskeleton Inc., Denver, CO, USA), or for 20 min at 37°C with the vesicle formation inhibitor, brefeldin A (10 μM, Enzo Life Sciences LTD., Exeter, UK), the ROCK inhibitor, Y27632 (30, 50 and 100 μM, Calbiochem-EMD), the Rac inhibitor, EHT 1684 (5 and 10 μM, Tocris Bioscience, Bristol, UK) and the microtubule polymerization blocker, nocodazole (10 and 100 μM, Calbiochem-EMD), before and during chemokine stimulation.

Techniques: Activity Assay

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 2. In vitro function of islets pre-cultured with exogenous complement component C3a. Insulin release at 2 mmol/L and 20 mmol/L glucose of 1020 replicates of three mouse islets per Eppendorf tube: (A) pre-cultured alone, with 10 nmol/L C3a alone or with 100 nmol/L C3a alone, for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a; (B) pre-cultured alone, with 5 nmol/L ANXA1 alone or with 10 nmol/L C3a alone for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a or ANXA1; (C) pre-cul- tured alone, with 5 nmol/L ANXA1 alone or with a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a for 48 h followed by subse- quent GSIS assays in the absence of exogenous C3a and/or ANXA1, P < 0.05 versus islets pre-cultured alone at the same glucose concentration. (D) Protection from cytokine-induced apoptosis following a 48 h pre-culture with 5 nmol/L ANXA1-alone, 10 nmol/L C3a alone or a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a, and the subsequent presence of specified MSC biotherapeutics during the final 20 h cytokine incubation. Eight to 12 replicates of five islets per well in each culture group assayed, *P < 0.05 versus islets pre-cul- tured alone in the presence of cytokines for the final 20 h of the 3-day culture period. The P values (AD) were calculated using two-way ANOVA Bonferroni’s post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vitro, Cell Culture, Concentration Assay, Incubation

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 3. Pre-culturing islets with a cocktail of MSC secretory factors ensures sustained improvements to islet insulin secretory function and protection from cytokine-induced apoptosis. (A, B) Insulin release at 2 and 20 mmol/L glucose of 30 replicates of three mouse islets per Eppendorf tube, pre-cultured alone, with 5 nmol/L ANXA1 alone, with 5 nmol/L ANXA1 and 10 nmol/L SDF-1, with 5 nmol/L ANXA1 and 10 nmol/L C3a, or with a cocktail of 5 nmol/L ANXA1, 10 nmol/L SDF-1 and 10 nmol/L C3a, for 48 h, before removal of the MSC- derived biotherapeutics for 1 day (A) or 3 days (B), *P < 0.05 and **P < 0.01 versus islets cultured alone at the same glucose concentration. (C, D) Protection of islets from cytokine-induced apoptosis after pre-culture with MSC-derived biotherapeutics alone, in dual combination or a cocktail of all three factors (as of legend) for 48 h, before removal of the MSC-derived biotherapeutics for 1 day (C) or 3 days (D), 8 to 12 replicates of five islets per well were assayed, *P < 0.05 and **P < 0.01 versus islets cultured alone with cytokines, +P < 0.05 vs. islets cul- tured alone without cytokines. The P values (AD) were calculated using two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: Cell Culture, Derivative Assay, Concentration Assay

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 4. In vivo function of islets pre-cultured alone, with ANXA1 alone or with a cocktail of MSC secretory factors. (A) Average blood glucose concentrations of STZ diabetic mice trans- planted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (repeated-meas- urements ANOVA with Bonferroni post hoc test, n = 79). (B) Area under the curve (AUC) of STZ diabetic mice transplanted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (one-way ANOVA with Dunn’s post hoc test, n = 79).

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vivo, Cell Culture

Journal: Toxins

Article Title: Shiga Toxin 2a Binds to Complement Components C3b and C5 and Upregulates Their Gene Expression in Human Cell Lines

doi: 10.3390/toxins13010008

Figure Lengend Snippet: C3 and C5 mRNA upregulation in HK-2 cell line. Evaluated qPCR analyses of mRNA expression of specific complement genes in HK-2 cells after stimulation for 6 h with increasing concentrations of Stx2a. qPCR data were normalized against TOP2B and GAPDH , and the fold change ± SD is shown ( n = 3). Unpaired Student’s t test was applied to compare untreated cells with the treatment groups, following Bonferroni–Holm correction with ** p < 0.01 and *** p < 0.001.

Article Snippet: Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and, after blocking, incubated with mouse anti-human C3 Ab or rabbit anti-human C5 Ab at 4 °C overnight.

Techniques: Expressing

Journal: Toxins

Article Title: Shiga Toxin 2a Binds to Complement Components C3b and C5 and Upregulates Their Gene Expression in Human Cell Lines

doi: 10.3390/toxins13010008

Figure Lengend Snippet: C3 and C5 mRNA expression in HCT-8 cell line. Evaluated qPCR analyses of C3 ( A ) and C5 ( B ) mRNA expression in HCT-8 cells after stimulation for 6 h with increasing concentrations of Stx2a. Data were normalized against GAPDH , and the fold change of a sample size of n = 4 ± SD is shown. Unpaired Student’s t test was applied to compare untreated cells with the with the treatment groups, following Bonferroni–Holm correction with * p < 0.05 and *** p < 0.001.

Article Snippet: Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and, after blocking, incubated with mouse anti-human C3 Ab or rabbit anti-human C5 Ab at 4 °C overnight.

Techniques: Expressing

Journal: Toxins

Article Title: Shiga Toxin 2a Binds to Complement Components C3b and C5 and Upregulates Their Gene Expression in Human Cell Lines

doi: 10.3390/toxins13010008

Figure Lengend Snippet: C3 and C5 protein synthesis in HK-2 cell line. OD values of sandwich ELISA assessing intracellular ( A , C ) and extracellular ( B , D ) protein expression of C3 ( A , B ) and C5 ( C , D ) in HK-2 cells after 9 h stimulation with increasing concentrations of Stx2a (0.001–10 µg/mL) are shown. Several controls have been performed: unstimulated cells (unstimulated), TNF-α- and IFN-γ-stimulated cells as positive control, lysis buffer or medium as negative controls and lysate or concentrated supernatant of unstimulated cells spiked with 325 ng of C3b and C5 (Spiked C3b/C5) as additional positive controls. Dashed line indicates the cut-off of the ELISA (mean of the negative control + 2.6 times its SD). Means ± SDs with a sample size of n = 3 are shown. One-way ANOVA was applied for the comparison of all groups excluding spiked and negative controls, following Tukey correction with * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and, after blocking, incubated with mouse anti-human C3 Ab or rabbit anti-human C5 Ab at 4 °C overnight.

Techniques: Sandwich ELISA, Expressing, Positive Control, Lysis, Enzyme-linked Immunosorbent Assay, Negative Control, Comparison

Journal: Toxins

Article Title: Shiga Toxin 2a Binds to Complement Components C3b and C5 and Upregulates Their Gene Expression in Human Cell Lines

doi: 10.3390/toxins13010008

Figure Lengend Snippet: Intracellular protein synthesis of C3 by HK-2 cell line. Western blot results assessing intracellular protein expression of C3 in HK-2 cells after stimulation for 9 h with 1 ng/mL or 10 µg/mL Stx2a. The presence of C3 was also studied in concentrated lysates of TNF-α- and IFN-γ-stimulated cells and unstimulated cells. Unstimulated cells spiked with 325 ng C3b or 200 ng of pure C3b were used as positive controls, while lysis buffer was used as a negative control. Mouse anti-human C3 antibody (Ab) was used to detect C3. Exposure time for each blot was 1 s ( A ) and 10 min ( B ).

Article Snippet: Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and, after blocking, incubated with mouse anti-human C3 Ab or rabbit anti-human C5 Ab at 4 °C overnight.

Techniques: Western Blot, Expressing, Lysis, Negative Control

Journal: Toxins

Article Title: Shiga Toxin 2a Binds to Complement Components C3b and C5 and Upregulates Their Gene Expression in Human Cell Lines

doi: 10.3390/toxins13010008

Figure Lengend Snippet: Gene name, gene symbol and assay ID from TaqMan to assess gene expression by real-time PCR.

Article Snippet: Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and, after blocking, incubated with mouse anti-human C3 Ab or rabbit anti-human C5 Ab at 4 °C overnight.

Techniques: Gene Expression

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: Excluded variables of multivariate logistic regression analyses.

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques:

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: NDRG2 reshapes the pathological structure of astrocytes by inhibiting NF-κB/C3 signaling. (a) GSEA of proteome indicated complement and coagulation cascade signaling were upregulated in diabetic mice (∗∗∗ p = 0.0002 between vehicle and STZ) and downregulated after exercise (∗∗ p = 0.0035 between STZ and STZ + Run). The cumulative enrichment scores were normalized (NES) ( n = 3/group). (b) Heatmaps showing the fold changes of significantly altered proteins in complement and coagulation cascades ( n = 3/group). (c – d) Representative immunofluorescent staining of intact C3 and its cleaved products in the hippocampus. Red: C3, green: GFAP, blue: DAPI. Scale bar, 50 μm. (∗∗∗ p = 0.0006 between vehicle and STZ, ∗∗∗ p = 0.0006 between STZ and STZ + Run) (e – f) Representative immunoblots of complement C3 in the hippocampus were increased in the STZ group compared to vehicle mice (∗∗∗∗ p < 0.0001 between vehicle and STZ), which was downregulated after exercise ( n = 6/group) (∗∗∗ p = 0.0008 between STZ and STZ + Run). (g – k) Representative immunoblots of astrocytic NDRG2 ( g, i ), complement C3 ( g, h ), NF-κB, and p–NF–κB ( j, k ) after NDRG2 loss of function ( n = 6/group) (∗ p = 0.0120 (h) , ∗∗∗∗ p < 0.0001 (i) , ∗∗ p = 0.0012 (k) between STZ + Run + acNDRG2 KO and STZ + Run + control). (l – n) Representative immunoblots of astrocytic NDRG2 ( l ), complement C3 ( m ), NF-κB, and p–NF–κB ( n ) in the hippocampus after NDRG2 gain of function ( n = 6/group) (∗∗ p = 0.0064 ( l ), ∗∗∗ p = 0.0001 ( m ), ∗∗ p = 0.0029 ( n ) between vehicle + AAV-Ctrl and STZ + AAV-Ctrl) (∗∗∗∗ p < 0.0001 ( l ), ∗ p = 0.0264 (m) , ∗∗ p = 0.0040 (n) between STZ + AAV-NDRG2 and STZ + AAV-Ctrl). Data are presented as mean ± SEM. GSEA analysis was performed in a . One-way ANOVA with Tukey's multiple comparisons test was performed in d, f, l–n . Two-tailed Student's t-test was performed in h, i, k .

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques: Coagulation, Staining, Western Blot, Control, Two Tailed Test

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: C3aR blockade rescues dendritic spine loss in diabetic mice, and C3 may be a biomarker to predict the progression of DACD in humans. (a) Schematic representing chronological order of STZ injection, C3aR antagonist, and behavioral testing. We used C3aR antagonists to clarify whether C3aR blockade could mimic the protective effect of NDRG2 overexpression on DACD. (b) Y-maze alternation triplet (%) was improved in the STZ + C3aRA group compared with STZ + PBS mice ( n = 7/group, n = 8 vehicle + PBS) (∗∗∗ p = 0.0006 between vehicle + PBS and STZ + PBS) (∗∗∗∗ p < 0.0001 between STZ + C3aRA and STZ + PBS). (c – d) Y-maze total distance ( c ) and total arm entries ( d ) ( n = 7/group, n = 8 vehicle + PBS). (e) Escape latency of MWM test was shorten at the STZ + C3aRA group compared with STZ + PBS mice ( n = 7/group, n = 8 vehicle + PBS) (∗ p = 0.0211 (day 1) , ∗ p = 0.0336 (day 2) , ∗∗ p = 0.0099 (day 3) , ∗∗ p = 0.0033 (day 4) between vehicle + PBS and STZ + PBS) ( ## p = 0.0021 (day 1) , # p = 0.0392 (day 3) between STZ + C3aRA and STZ + PBS). (f) Platform crossover of MWM test was upregulated at the STZ + C3aRA mice compared to STZ + PBS group ( n = 7/group, n = 8 vehicle + PBS) (∗∗ p = 0.0025 between vehicle + PBS and STZ + PBS) (∗∗ p = 0.0066 between STZ + C3aRA and STZ + PBS). (g) Representative images of 3D reconstruction of dendritic spines. Scale bar, 5 μm. (h – l) The densities of total spines ( h ), stubby spines ( i ), mushroom spines ( j ), long thin spines ( k ), and filopodia spines ( l ) ( n = 20/group) (∗∗∗∗ p < 0.0001 ( h ), ∗∗∗ p = 0.0008 ( i ), ∗ p = 0.0191 ( j ), ∗∗∗ p = 0.0006 ( k ) between vehicle + PBS and STZ + PBS) (∗∗∗∗ p < 0.0001 ( h ), ∗∗∗ p = 0.0005 ( i ), ∗ p = 0.0133 ( j ), ∗ p = 0.0153 ( l ) between STZ + C3aRA and STZ + PBS). (m) The DSST scores of diabetic patients ( n = 27) and non-diabetic peers ( n = 13) (∗∗∗∗ p < 0.0001 between Normal and Diabetes). (n) Fasting plasma glucose levels for diabetic patients ( n = 27) compared to non-diabetic patients ( n = 13) (∗∗∗∗ p < 0.0001 between Normal and Diabetes). (o) The serum levels of complement C3 in diabetic patients ( n = 27) and normal peers ( n = 13) (∗∗ p = 0.0072 between Normal and Diabetes). (p) The correlations between DSST score and increased C3 levels in diabetic patients ( n = 27) and normal peers ( n = 13). Correlations were found using linear regression, with r = −0.3315 and p = 0.0366. Values presented as mean ± SEM. Two-way ANOVA with Tukey's multiple comparisons test was performed in e . One-way ANOVA with Tukey's multiple comparisons test was performed in b, f–l . Two-tailed Student's t-test was performed in m–o .

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques: Biomarker Discovery, Injection, Over Expression, Clinical Proteomics, Two Tailed Test

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: Characteristics of the study population.

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques: