Journal: Immunity

Article Title: Human Antibodies Fix Complement to Inhibit Plasmodium falciparum Invasion of Erythrocytes and Are Associated with Protection against Malaria

doi: 10.1016/j.immuni.2015.02.012

Figure Lengend Snippet: Invasion Inhibition by IgG and Complement and Complement Deposition on the Merozoite Surface (A) Invasion-inhibitory activity of purified IgG from Kenya and PNG was tested in invasion assays performed with 50% normal serum (NS; complement active) or heat-inactivated serum (HIS; complement inactivated). Data represent the mean ± range from two independent assays performed in duplicate. (B) C1q and C3 deposition on merozoites incubated with purified PNG IgG, purified malaria-naive IgG (Australian donors), or PBS together with 25% NS for 1, 5, 15, and 30 min. MSP1-19, a merozoite surface protein, was used as a loading control. (C) C3b deposition on merozoites incubated with purified PNG IgG and 25% NS or HIS via immuno-electron microscopy. Gold labeling is indicated with arrows. Scale bars represent 0.1 μm. (D) Formation of the membrane attack complex (MAC; complement components C5–C9) on merozoites incubated with purified PNG or Australian (Melbourne) IgG and 25% NS or HIS via IF microscopy. (E) MAC deposition as quantified by ELISA on merozoites incubated with NS and PNG or Australian IgG. Immunoblots and microscopy images are representative of two independent experiments. See also Figure S1 .

Article Snippet: C1q and C3 were detected with anti-C1q (Goat polyclonal, Calbiochem, Merck) and anti-C3 (HRP-conjugated goat polyclonal, MP Biomedicals), respectively.

Techniques: Inhibition, Activity Assay, Purification, Incubation, Immuno-Electron Microscopy, Labeling, Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Pathogens

Article Title: Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin

doi: 10.3390/pathogens14040354

Figure Lengend Snippet: Inhibition of C3b and C4b deposition by r Em CRT and its functional binding regions measured by ELISA. ( A ) Pre-incubation of 100 μL C1qD (1:50) as source of natural MBL with different amounts of r Em CRT, r Em CRT-S, or r Em CRT-NP (0, 2, 4 μM) before adding into mannan-coated plates (50 μg/mL). After being washed, C1qD (1:150) was added as source of complement components to initiate lectin pathway of complement activation. Deposition of C3b ( a ) and C4b ( b ) was detected with anti-C3 or C4 monoclonal antibodies. ( B ) The similar ELISA procedure was performed using NHS as source of physiological MBL instead of C1qD serum to detect the generation of C3b (a) and C4b (b). Data are expressed as mean of OD 450 ± SDs of three independent experiments (* p < 0.005, ** p < 0.001, *** p < 0.0005, ns, no significant difference compared to plates without added r Em CRT).

Article Snippet: After washing with 1 × TBST containing 5 mM CaCl 2 , the C1qD diluted at 1:150 in 1 × Veronal Buffer (VB, Lonza, Basel, Switzerland) containing 0.1% gelatin, 0.05% Tween-20 was added (100 μL) as supplement of other complement components (without C1q) into each well of the plates and incubated at 37 °C for 1 h. The lectin pathway-activated C3b/C4b deposition was detected with rabbit anti-C3b monoclonal antibody (1:1000, BOSTER Biological Technology, Wuhan, China) and goat anti-C4b polyclonal antibody (1:3000, Abcam, Cambridge, UK).

Techniques: Inhibition, Functional Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay, Bioprocessing

Journal: Nature Communications

Article Title: Anoctamin 1 controls bone resorption by coupling Cl − channel activation with RANKL-RANK signaling transduction

doi: 10.1038/s41467-022-30625-9

Figure Lengend Snippet: a Representative chloride currents recorded from voltage ramps from −80 to +160 mV in whole-cell patch-clamp during osteoclast differentiation. b QRT-PCR analysis of Clcn1 , Clcn2 , Clcn3 , Clcn4 , Clcn5 , Clcn6 , Clcn7 , Ano1 , Ano2 , and Cftr mRNA levels after bone marrow monocytes (BMMs) were induced by RANKL for 5 days. c , d QRT-PCR analysis of Ano1 mRNA level ( c ) and western blot analysis of Ano1 protein level ( d ) during osteoclast differentiation. e Representative images of TRAP staining in osteoclasts after treatment with 20 μM CaCC inh -A01 (A01) or its control (DMSO) for 5 days (left). Scale bar, 200 μm. Quantification of the number of multinucleated cells per well (right) ( n = 6). f Representative images of TRAP staining in osteoclasts after treatment with 10 μM Benzbromarone or its control (DMSO) for 5 days (left). Scale bar, 200 μm. Quantification of the number of multinucleated cells per well (right) ( n = 6). g Schematic representation of the topology of Ano1 mutant (E702/705Q). h Representative images of TRAP staining in osteoclasts knockdown with Ano1 siRNA, rescued with Ano1 or its mutant Ano1 (E702/705Q) (left). Scale bar, 200 μm. Quantification of the number of multinucleated cells per well (right). i QRT-PCR analysis of NFATc1 , Acp5 , Ctsk , and Mmp9 mRNA levels in osteoclasts knockdown with Ano1 siRNA, rescued with Ano1 or its mutant Ano1 (E702/705Q). All data are the mean ± s.e.m. from three independent experiments. Two-tailed unpaired Student’s t -test was used for statistical evaluations of two group comparisons. Statistical analysis with more than two groups was performed with one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test to determine group differences. Source data are provided as a Source Data file.

Article Snippet: The antibody of used were listed: rabbit anti-Ano1 (1:1000, abclonal, Cat. No. A10498, polyclonal), rabbit anti-p-Syk antibody (1:1000, abclonal, Cat. No. AP0501, polyclonal), rabbit anti-Syk antibody (1:1000, abclonal, Cat. No. A2123, polyclonal), rabbit anti-Clcn4 antibody (1:1000, abclonal, CatNo.A13790, polyclonal), rabbit anti-Clcn7 antibody (1:1000, abclonal, Cat. No. A6886, polyclonal), rabbit anti-CFTR antibody (1:1000, abclonal, Cat. No. A8386, polyclonal), rabbit anti-p-Akt antibody (1:1000, Cell Signaling Technology, Cat. No. 4060, polyclonal), rabbit anti-Akt antibody (1:1000, Cell Signaling Technology, Cat. No. 2920, polyclonal), rabbit anti-p-CaMKIV antibody (1:1000, ImmunoWay, Cat. No. YP0043, polyclonal), rabbit anti-CaMKIV antibody (1:1000, Cell Signaling Technology, Cat. No. 4032, polyclonal), rabbit anti-Creb antibody (1:1000, Cell Signaling Technology, Cat. No. 9197, Monoclonal), rabbit anti-p-Creb antibody (1:1000, Cell Signaling Technology, Cat. No. 9198, Monoclonal), rabbit anti-Plcγ2 antibody (1:1000, Cell Signaling Technology, Cat. No. 55512, Monoclonal), rabbit anti-p-Plcγ2 antibody (1:1000, Cell Signaling Technology, Cat. No. 3871, polyclonal), rabbit anti-Btk antibody (1:1000, Cell Signaling Technology, Cat. No. 5082, polyclonal), rabbit anti-Btk antibody (1:1000, Proteintech, Cat. No. 21581-1-AP, polyclonal), rabbit anti-TRAF6 antibody (1:200, Abcam, CatNo.ab137452, polyclonal), mouse anti-RANK antibody (1:200, Abcam, Cat. No. ab13918, polyclonal), rabbit anti-Gapdh antibody (1:5000, Abways, Cat. No. AB0036, Monoclonal).

Techniques: Patch Clamp, Quantitative RT-PCR, Western Blot, Staining, Mutagenesis, Two Tailed Test

Journal: Cell reports

Article Title: Autocrine Effects of Tumor-Derived Complement

doi: 10.1016/j.celrep.2014.02.014

Figure Lengend Snippet: (A) We measured total tumor weight in an orthotopic murine model of ovarian cancer induced by ID8-VEGF murine ovarian cancer cells in C3 −/− and WT control mice, both in C57BL/6 background. n.s, not significant. (B) Immunostaining of tumors induced by ID8-VEGF in WT and C3 −/− mice, using anti-C3 antibody compared to negative control stain (secondary antibody alone). Scale bar length is 100 μm. (C) Quantitative real-time PCR for C3 mRNA on RNA isolated from murine and human ovarian cancer cell lines. Expression of C3 mRNA in cancer cell lines was compared to that in MOECs and normal human ovarian surface epithelial cell lines (HIO 180) (n = 3; **p ≤ 0.01, t test). (D) C3 gene knockdown in SKOV3ip1 human ovarian cancer cells using C3 siRNA, reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (**p ≤ 0.01, t test). (E) C3 gene knockdown in SKOV3ip1-induced tumors by intraperitoneal injection of hC3 siRNA into tumor-bearing NU/NU mice reduced total weight (*p = 0.017) and number of tumor nodules (*p = 0.05). (F) Representative immunostaining for C3, Ki67, and CD31 in tumors resected from hC3 siRNA-injected and scrambled siRNA-injected mice. Scale bar, 100 μm. (G) The proliferation index in resected tumors was quantified as the percentage of Ki67 positivity shown in dot plots (39% in C3 siRNA versus 74% in scrambled siRNA, n = 5 mice in each group; *p = 0.05, t test). The number of blood vessels in resected tumors was quantified by counting the number of CD31+ lumen structures in five high-power fields (HPFs) per section and in five sections per tumor nodule and in five mice per group. Average number of CD31+ lumens per HPF is shown as dot plots (22/HPF in C3 siRNA versus 42/HPF in scrambled siRNA; *p = 0.05, t test). (H) We investigated the effect of complement on proliferation of endothelial cells by measuring the proliferation rate of RF24 endothelial cells after transfection with C3 siRNA. C3 knockdown did not reduce the proliferation rate in RF24 endothelial cells (n = 3; p = 0.07, t test).

Article Snippet: Rabbit anti-mouse C3 antibody (Abgent), mouse anti-hC3 antibody (Acris Antibodies), rabbit anti-hC5 antibody (Abcam), rabbit polyclonal anti-Ki67 antibody (Thermo Scientific/Lab Vision), rat anti-mouse CD31 antibody (BD Biosciences, BD PharMingen), mouse anti-C5b-9 antibody (Dako), mouse anti-C9 antibody (Hycult Biotech), anti-CD8 and anti-CD11b antibodies (AbD Serotec), rabbit-anti-h AKT, p-AKT, P85, and p-P85 antibodies (Cell Signaling Technology), C5aR antagonist (W-54011) ( ):N-((4-dimethylami-nophenyl)methyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1-carboxamide, HCl, and C3aR antagonist (SB290157) ( ):N2-((2,2-dipheny-lethoxy)acetyl)-L-arginine (EMD Chemicals), 3,3′-diaminobenzidine (DAB; Open Biosystems), and Gill’s #3 hematoxylin (Sigma-Aldrich) were purchased from the perspective commercial sources.

Techniques: Immunostaining, Negative Control, Staining, Real-time Polymerase Chain Reaction, Isolation, Expressing, Migration, In Vitro, Injection, Transfection

Journal: Cell reports

Article Title: Autocrine Effects of Tumor-Derived Complement

doi: 10.1016/j.celrep.2014.02.014

Figure Lengend Snippet: (A) Quantification of C3 mRNA in h breast, ovarian, lung, and endometrium cancer cell lines using quantitative real-time PCR (n = 3). (B and C) Reducing C3 gene expression in (B) H226 human squamous cell lung cancer and in (C) Hec265 human endometrium cancer cell lines using C3 siRNA reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (*p ≤ 0.01 and **p ≤ 0.001, t test). (D) The relative abundance of AKT mRNA in total mRNA isolated from SKOV3ip1 cells after 24 hr exposure to C3aR and C5aR agonists was quantified by quantitative real-time PCR, and the result of three experiments are summarized as bar graphs (*p ≤ 0.001). (E) A representative immunoblot on total cell lysate prepared from SKOV3ip1 cells after 48 hr exposure to C3aR and C5aR agonists and inhibitors using antibodies to p-85, AKT, and their phosphorylated forms (n = 3). (F) Immunostaining of tumors induced by ID8-VEGF cells expressing scrambled shRNA or mC3 shRNA in C57BL/6 mice using anti-pAKT antibody. (G) To investigate the effect of AKT or PI3K silencing on C3aR and C5aR agonist-induced enhancement of SKOV3ip1 proliferation, expression of AKT or PI3K in SKOV3ip1 was reduced using AKT or PI3K siRNA (data not shown), and cell proliferation was quantified 48 hr after exposure to C3aR-AG and C5aR-AG and was compared to SKOV3ip1 cells transfected with scrambled siRNA (n = 3; *p ≤ 0.001). (H) C3 mRNA level in the tumor specimens of 75 patients diagnosed with ovarian cancer in MDACC was determined using quantitative real-time PCR and correlated with their OS (p = 0.004) and presented as Kaplan-Meier survival curve. (I) Correlation between expression of C5aR in tumor and OS in 562 patients with ovarian cancer in TCGA database (p = 0.019). (J) The amount of C5aR mRNA in tumors resected from 167 patients with a diagnosis of lung squamous cell carcinoma was quantified using quantitative real-time PCR and was correlated to the OS of these patients as documented in TCGA database. The results are shown as Kaplan-Meier survival curve (p = 0.026).

Article Snippet: Rabbit anti-mouse C3 antibody (Abgent), mouse anti-hC3 antibody (Acris Antibodies), rabbit anti-hC5 antibody (Abcam), rabbit polyclonal anti-Ki67 antibody (Thermo Scientific/Lab Vision), rat anti-mouse CD31 antibody (BD Biosciences, BD PharMingen), mouse anti-C5b-9 antibody (Dako), mouse anti-C9 antibody (Hycult Biotech), anti-CD8 and anti-CD11b antibodies (AbD Serotec), rabbit-anti-h AKT, p-AKT, P85, and p-P85 antibodies (Cell Signaling Technology), C5aR antagonist (W-54011) ( ):N-((4-dimethylami-nophenyl)methyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1-carboxamide, HCl, and C3aR antagonist (SB290157) ( ):N2-((2,2-dipheny-lethoxy)acetyl)-L-arginine (EMD Chemicals), 3,3′-diaminobenzidine (DAB; Open Biosystems), and Gill’s #3 hematoxylin (Sigma-Aldrich) were purchased from the perspective commercial sources.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Migration, In Vitro, Isolation, Western Blot, Immunostaining, shRNA, Transfection