Journal: bioRxiv

Article Title: ATGs ubiquitination is required for circumsporozoite protein to subvert host innate immunity against malaria liver stage

doi: 10.1101/2021.01.20.427456

Figure Lengend Snippet: ( A ) After 1.2 × 10 5 HepG2 cells were incubated with 4 × 10 4 CSP wt and CSP mut P.b ANKA sporozoites for 24 h, then cells were stained with anti-UIS4, and anti-CSP and DAPI. Translocation of CSP from the PV into HepG2 cytoplasm was observed under confocal microscopy ( left ), and quantified ( right ). ( B ) After mice (n=5) were intravenously injected with 1,000 CSP wt and CSP mut parasite sporozoites for 46 h, the parasite burden in the liver was measured as the ratio of Plasmodium 18S rRNA to mouse GAPDH using Taqman real-time PCR. ( C ) HepG2 cells were transiently transfected with the indicated amount of pcDNA3.1 vector (Vec), CSP plasmid, and then incubated with sporozoites for 46 h; the parasite burden was determined as the ratio of Plasmodium 18S rRNA to human GAPDH using Taqman real-time PCR. ( D ) After HepG2 cells were incubated with CSP wt and CSP mut sporozoites for 46 h, the parasite burden was determined as described in ( C ). ( E ) Control and CSP-stable transfected HepG2 cells were pre-treated with or without 1U/ml IFN-γ, and then incubated with sporozoites for 46 h. EEF number was determined and statistically analyzed. ( F ) HepG2 cells were transfected with or without ΔCSP plasmid and pre-treated with or without 1 U/mL IFN-γ followed by incubation with WT or CSP mut sporozoites; 46 h later, the parasite burden in hepatocytes was determined and compared as described in ( C ). ( G ) Mice (n=5) were infected with CSP wt or CSP mut sporozoites as above, and treated with anti-IFN-γ or isotype antibody. Then the parasite burden was determined as described in ( B ). ( H ) IFN-γR1 knockout and WT mice were infected with 1,000 CSP wt or CSP mut sporozoites, liver burden was determined at 46 h after infection as described as above. Med, Medium; Data are represented as mean ± SEM; ns, not significant; ** p < 0.01; *** p < 0.001. Scale bar=5μm.

Article Snippet: The cells were labeled with 1:500 goat anti-UIS4 (Sicgen, Cantanhede, Portugal) overnight at 4°C and 1:100 Green donkey anti-goat secondary antibody (Abbkine, Wuhan, China) for 1 h at room temperature.

Techniques: Incubation, Staining, Translocation Assay, Confocal Microscopy, Injection, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Infection, Knock-Out

Journal: bioRxiv

Article Title: ATGs ubiquitination is required for circumsporozoite protein to subvert host innate immunity against malaria liver stage

doi: 10.1101/2021.01.20.427456

Figure Lengend Snippet: ( A ) 1.2 × 10 5 control and CSP-stable transfected HepG2 cells were infected with Ad-mRFP-GFP-LC3 virus, and then treated with the autophagy inducer rapamycin for 24 h. Both LC3-RFP or LC3-GFP puncta in HepG2 cells were imaged ( up ) and qualified ( down ). ( B ) Control and CSP-stable transfected HepG2 cells were transiently transfected with LC3-RFP plasmid, and then incubated with 4 × 10 4 WT sporozoites for 24 h. Cells were fixed and stained with anti-UIS4, and LC3 surrounding the EEFs was observed under confocal microscopy ( up ), and the co-localization of LC3 and EEF was quantified ( down ). ( C ) HepG2 cells were transiently transfected with LC3-RFP plasmid and infected with CSP wt or CSP mut sporozoites for 24 h, The LC3 signals around WT and mutant EEFs were observed under confocal microscopy ( up ) and quantified ( down ). ( D ) Control and CSP-stable transfected HepG2 cells were treated with rapamycin for the indicated times. Protein levels of ATGs, including LC3I/LC3II, ATG3, ATG5, ATG7, Beclin-1, and p62 (SQSTM1), was determined by western blot ( left ), and the bands were quantified ( right ). ( E ) Control and CSP-stable transfected HepG2 cells transfected with or without ATG5, or ATG7 plasmid, and then treated with IFN-γ and infected with sporozoites for 46 h. The parasite load in HepG2 cells was determined and compared. ( F ) HepG2 cells were transiently transfected with Scramble or ATG5 sh RNA plasmids, and then treated with IFN-γ and incubated with CSP wt or CSP mut sporozoites. 46 h later, the parasite load in the HepaG2 cells was determined and compared. ( G ) Both control (ATG5 fl/fl ) and ATG5 fl/fl - Alb - Cre mice (n=5) were infected by intravenous injection with 1,000 WT or mutant sporozoites, 46 h later, the liver burden of the two parasites in control and ATG5 fl/fl - Alb - Cre mice were determined and compared. Data are represented as mean ± SEM; ns, not significant; ** p < 0.01; *** p < 0.001. Scale bar=5μm.

Article Snippet: The cells were labeled with 1:500 goat anti-UIS4 (Sicgen, Cantanhede, Portugal) overnight at 4°C and 1:100 Green donkey anti-goat secondary antibody (Abbkine, Wuhan, China) for 1 h at room temperature.

Techniques: Transfection, Infection, Plasmid Preparation, Incubation, Staining, Confocal Microscopy, Mutagenesis, Western Blot, Injection

Journal: Journal of Clinical Medicine

Article Title: Potential Effects of CXCL9 and CCL20 on Cardiac Fibrosis in Patients with Myocardial Infarction and Isoproterenol-Treated Rats

doi: 10.3390/jcm8050659

Figure Lengend Snippet: Protein array analysis evaluating the expression of CXCL9 and CCL20 among various detected protein in patients with MI (patient #2 and #3) and normal subjects (healthy volunteers in our laboratory). ( A ) The expression of CXCL9 and CCL20 consistently increased in samples of short- and long-term exposures. ( B ) Fold change of CXCL9 expression in patient #2 and #3. ( C ) Fold change of CCL20 expression in patient #2 and #3. MI = myocardial infarction; PCI = percutaneous coronary intervention; Post-PCI = 3 h after coronary reperfusion by PCI; Pre-PCI = before PCI.

Article Snippet: ELISA was used to detect the expression of CXCL9 (#ELH-MIG, RayBiotech, Peachtree Corners, GA, USA), CCL20 (#ELH-MIP3a, RayBiotech), TNF-α (#ELH-TNFa-1, RayBiotech), and TGF-β (#ELH-TGFb1-1, RayBiotech) in human plasma or culture medium and used to detect the expression of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) (#HEA485Ra, Cloud-Clone, Katy, TX, USA), CXCL9 (#MBS452489, MyBioSource, San Diego, CA, USA), and CCL20 (#MBS175952, MyBioSource) in rat plasma.

Techniques: Protein Array, Expressing

Journal: Journal of Clinical Medicine

Article Title: Potential Effects of CXCL9 and CCL20 on Cardiac Fibrosis in Patients with Myocardial Infarction and Isoproterenol-Treated Rats

doi: 10.3390/jcm8050659

Figure Lengend Snippet: Expression of CXCL9 and CCL20 before (pre-PCI) and 3 h after coronary reperfusion by PCI (post-PCI) in patients with MI ( n = 39–50) compared to normal controls (healthy volunteers in our laboratory) ( n = 11). CXCL9 and CCL20 were measured by ELISA. ( A ) Expression of CXCL9 in normal controls and patients with MI. ( B ) Serum CXCL9 levels post-PCI in patients with MI remained more elevated than pre-PCI. ( C ) Serum CCL20 levels in normal controls and patients with MI. ( D ) Serum CCL20 levels change significantly after PCI. ( E ) Serum TNF-α levels in normal controls and patients with MI. ( F ) Serum TNF-α levels did not change significantly after PCI; ( G ) Serum TGF-β levels in normal controls and patients with MI. ( H ) Serum TGF-β levels in post-PCI MI patients remained more elevated than pre-PCI. ELISA = enzyme-linked immunosorbent assay; MI = myocardial infarction; TGF-β = transforming growth factor-β; TNF-α = tumor necrosis factor-α; PCI = percutaneous coronary intervention. Results are presented as mean ± SEM.

Article Snippet: ELISA was used to detect the expression of CXCL9 (#ELH-MIG, RayBiotech, Peachtree Corners, GA, USA), CCL20 (#ELH-MIP3a, RayBiotech), TNF-α (#ELH-TNFa-1, RayBiotech), and TGF-β (#ELH-TGFb1-1, RayBiotech) in human plasma or culture medium and used to detect the expression of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) (#HEA485Ra, Cloud-Clone, Katy, TX, USA), CXCL9 (#MBS452489, MyBioSource, San Diego, CA, USA), and CCL20 (#MBS175952, MyBioSource) in rat plasma.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Medicine

Article Title: Potential Effects of CXCL9 and CCL20 on Cardiac Fibrosis in Patients with Myocardial Infarction and Isoproterenol-Treated Rats

doi: 10.3390/jcm8050659

Figure Lengend Snippet: Expression of CXCL9 and CCL20 in PBMCs and H9c2 cells. ( A ) TNF-α and TGF-β upregulated the expression of CXCL9 in PBMCs. ( B ) TNF-α enhanced the expression of CCL20 in PBMCs. ( C ) H9c2 cells did not show an increased expression of CXCL9 and CCL20 under hypoxic stress. ( D ) Hypothetical illustration of the expression of CXCL9 and CCL20 after the onset of hypoxia. PBMCs = peripheral blood mononuclear cells; TGF-β = transforming growth factor-β; TNF-α = tumor necrosis factor-α. Each experiment was conducted at least 3 times independently. Results are presented as mean ± SEM.

Article Snippet: ELISA was used to detect the expression of CXCL9 (#ELH-MIG, RayBiotech, Peachtree Corners, GA, USA), CCL20 (#ELH-MIP3a, RayBiotech), TNF-α (#ELH-TNFa-1, RayBiotech), and TGF-β (#ELH-TGFb1-1, RayBiotech) in human plasma or culture medium and used to detect the expression of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) (#HEA485Ra, Cloud-Clone, Katy, TX, USA), CXCL9 (#MBS452489, MyBioSource, San Diego, CA, USA), and CCL20 (#MBS175952, MyBioSource) in rat plasma.

Techniques: Expressing

Journal: Journal of Clinical Medicine

Article Title: Potential Effects of CXCL9 and CCL20 on Cardiac Fibrosis in Patients with Myocardial Infarction and Isoproterenol-Treated Rats

doi: 10.3390/jcm8050659

Figure Lengend Snippet: Effects of CXCL9 and CCL20 on the growth and migration of cardiac fibroblast. ( A ) A 96-hour incubation with CXCL9 (10 and 100 ng/mL) promoted the growth of NHCFs. ( B ) A 72-hour incubation with CCL20 (10 and 100 ng/mL), as well as a 96-hour incubation with CCL20 (100 ng/mL), promoted the growth of NHCFs. ( C ) CXCL9 and CCL20 promoted NHCFs migration. Detailed procedures are described in Materials and Methods. (Upper panel) Representative images of migrated NHCFs. (Lower panel) Quantitative results of the migration ability of NHCFs. CTL = control; NHCFs = Normal human cardiac fibroblasts. Each experiment was conducted at least 3 times independently. Results are presented as mean ± SEM.

Article Snippet: ELISA was used to detect the expression of CXCL9 (#ELH-MIG, RayBiotech, Peachtree Corners, GA, USA), CCL20 (#ELH-MIP3a, RayBiotech), TNF-α (#ELH-TNFa-1, RayBiotech), and TGF-β (#ELH-TGFb1-1, RayBiotech) in human plasma or culture medium and used to detect the expression of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) (#HEA485Ra, Cloud-Clone, Katy, TX, USA), CXCL9 (#MBS452489, MyBioSource, San Diego, CA, USA), and CCL20 (#MBS175952, MyBioSource) in rat plasma.

Techniques: Migration, Incubation, Control

Journal: Journal of Clinical Medicine

Article Title: Potential Effects of CXCL9 and CCL20 on Cardiac Fibrosis in Patients with Myocardial Infarction and Isoproterenol-Treated Rats

doi: 10.3390/jcm8050659

Figure Lengend Snippet: ISO-induced MI and post-MI cardiac fibrosis of rats. ( A ) Protocol of ISO-induced MI in rats. ( B ) HW/BW ratios in ISO-treated and control rats. ( C ) Serum NT-proBNP levels are increased in ISO-treated rats. ( D ) Heart sections of ISO-treated rats exhibited more collagen areas than control rats. ( E ) Serum CXCL9 levels are increased in ISO-treated rats. ( F ) Serum CCL20 levels in ISO-treated rats are not significantly increased compared to control rats. BW = body weight; CTL = control; HW = heart weight; IP = intraperitoneal; ISO = isoproterenol; MI = myocardial infarction; NT-proBNP = N-terminal prohormone of brain natriuretic peptide. Results are presented as mean ± SEM.

Article Snippet: ELISA was used to detect the expression of CXCL9 (#ELH-MIG, RayBiotech, Peachtree Corners, GA, USA), CCL20 (#ELH-MIP3a, RayBiotech), TNF-α (#ELH-TNFa-1, RayBiotech), and TGF-β (#ELH-TGFb1-1, RayBiotech) in human plasma or culture medium and used to detect the expression of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) (#HEA485Ra, Cloud-Clone, Katy, TX, USA), CXCL9 (#MBS452489, MyBioSource, San Diego, CA, USA), and CCL20 (#MBS175952, MyBioSource) in rat plasma.

Techniques: Control

Journal: Nanoscale Research Letters

Article Title: A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells

doi: 10.1186/s11671-015-0972-6

Figure Lengend Snippet: Fluorescence microscopy images of hNSCs cultured with PCL conduit for 14 days: a DAPI and b β-III tubulin. DAPI staining is indicated in blue and β-III tubulin staining is indicated in green. Arrow indicates the direction of aligned nanofibers

Article Snippet: After cryosectioning (20-μm slices), each section was washed twice in PBS for 10 min. Then, the samples were treated with blocking buffer (3 % goat serum PBS with 0.01 % Triton X-100) for 1 h at room temperature before adding β-III tubulin goat anti-mouse primary antibody (1:200 dilution, Promega, Madison, WI, USA) overnight at 4 °C.

Techniques: Fluorescence, Microscopy, Cell Culture, Staining

Journal: Nanoscale Research Letters

Article Title: A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells

doi: 10.1186/s11671-015-0972-6

Figure Lengend Snippet: Confocal microscopy images of hNSCs cultured with PSHU-RGD/PCL conduit for 14 days: representative images a , b along the center and e , f inner wall of microchannels, a , d DAPI, b , e β-III tubulin, and c , f combined DAPI and β-III tubulin images. DAPI staining is indicated in blue and β-III tubulin staining is indicated in green . Images show that hNSCs migrated far into the conduit with extensive neurite extension along the nanofiber direction ( arrows )

Article Snippet: After cryosectioning (20-μm slices), each section was washed twice in PBS for 10 min. Then, the samples were treated with blocking buffer (3 % goat serum PBS with 0.01 % Triton X-100) for 1 h at room temperature before adding β-III tubulin goat anti-mouse primary antibody (1:200 dilution, Promega, Madison, WI, USA) overnight at 4 °C.

Techniques: Confocal Microscopy, Cell Culture, Staining