Journal: Stem Cell Reviews

Article Title: Human Menstrual Blood-Derived Stromal Cells Promote Recovery of Premature Ovarian Insufficiency Via Regulating the ECM-Dependent FAK/AKT Signaling

doi: 10.1007/s12015-018-9867-0

Figure Lengend Snippet: Physiological changes to evaluate ovarian status. a Serum hormone levels assessed by ELISA kits in each group for measuring FSH (left), E2 (middle) and AMH (right), n = 6 (data were represented as mean ± SEM, * P < 0.05, ** P < 0.01). b Representative images of parturition in each group after mating with male mice. c Records of live births after CTX injection followed by MenSCs transplantation, n = 6 (data were represented as mean ± SEM, ** P < 0.01). d Apoptosis evaluation using Hoechst kit. Yellow arrows pointed to apoptotic cells with fragmented or condensed nuclei of apoptotic cells. Original magnification, ×100. e Apoptosis evaluation performed by TUNEL assay. White arrows pointed to FITC-labeled apoptotic cells. Original magnification, ×100. f Relative mRNA expression by q-RT PCR analysis for AMH, DDX4 and VEGFA controlled to GAPDH with fold change measured by 2 -ΔΔCT , n = 6. g Relative expression at protein levels of AMH, DDX4 and VEGFA (upper panel) and quantitative analysis (lower panel)

Article Snippet: TUNEL assay was performed using TUNEL FITC Apoptosis Detection Kit (Vazyme, Nanjing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Transplantation Assay, TUNEL Assay, Labeling, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Degradation of Transcription Factor Rfx5 during the Inhibition of Both Constitutive and Interferon γ–Inducible Major Histocompatibility Complex Class I Expression in Chlamydia-Infected Cells

doi:

Figure Lengend Snippet: Chlamydia inhibits both constitutive and IFN-γ–inducible MHC class I expression in infected cells. HeLa cells with or without chlamydial infection were stimulated with IFN-γ or unstimulated and collected for flow cytometry (A), Western blot (B and C), and RT-PCR (E) analysis. Chlamydia prevents both constitutive and IFN-γ–inducible HLA-A, -B, and -C heavy chain and β2M surface expression (A). Chlamydia suppresses the total cellular protein level of both constitutive and IFN-γ–inducible β2M (B) and HLA-A and -B heavy chains (C) in various human cell lines. Chlamydia inhibits the mRNA expression of both MHC class I heavy chains and β2M (E). For pulse–chase labeling experiment (D), HL cells with or without chlamydial infection were metabolically labeled with S 35 –methionine/cysteine for 30 min, and the pulsed cell samples were aliquoted and chased for various times as indicated. Mature or immature bands correspond to proteins resistant or sensitive to EndoH digestion as determined in a separate experiment (data not shown).

Article Snippet: Mouse antibodies were used to detect HLA-A and -B (HB296; ATCC), β2M (HB149; ATCC), β1 integrin (provided by Dr. J. Wilkins, University of Manitoba, Manitoba, Canada), 20S proteasome α subunit HC2 (MCP20; AFFINITI), 20S proteasome α subunit HC3 (MCP21; AFFINITI), 20S proteasome subunits α1, 2, 3, 5, 6, and 7 (MCP231; AFFINITI), 20S proteasome subunit β7 (MCP205; AFFINITI), and a chlamydial major outer membrane protein (MOMP; clone MC22; our unpublished data).

Techniques: Expressing, Infection, Flow Cytometry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Pulse Chase, Labeling, Metabolic Labelling

Journal: The Journal of Experimental Medicine

Article Title: Degradation of Transcription Factor Rfx5 during the Inhibition of Both Constitutive and Interferon γ–Inducible Major Histocompatibility Complex Class I Expression in Chlamydia-Infected Cells

doi:

Figure Lengend Snippet: Inhibition of MHC class I expression correlates with RFX5 degradation. (A) HeLa cells with or without chlamydial infection and with or without IFN-γ stimulation were analyzed on Western blot for the levels of RFX5, HLA-A and -B heavy chains, β2M, β1 integrin (as a control membrane protein), and a chlamydial MOMP. Chlamydia-infected cell samples showed reduced levels of HLA heavy chain and β2M and complete loss of RFX5. (B) Cell-free assay analysis of HLA-A and -B heavy chain, β2M, and RFX5 degradation. Cytosolic S100 fractions from either normal HeLa (HeLa S100) or chlamydia-infected HeLa cells (chlamydia S100) were incubated with the corresponding target proteins (either a membrane protein extract as the source of HLA-A and -B heavy chains and β2M or a nuclear protein extract as the source of RFX5). The incubated mixtures were analyzed on Western blot for the levels of the residual target proteins with the corresponding antibodies. Chlamydia S100 selectively degrades RFX5 but not HLA-A and -B heavy chains and β2M.

Article Snippet: Mouse antibodies were used to detect HLA-A and -B (HB296; ATCC), β2M (HB149; ATCC), β1 integrin (provided by Dr. J. Wilkins, University of Manitoba, Manitoba, Canada), 20S proteasome α subunit HC2 (MCP20; AFFINITI), 20S proteasome α subunit HC3 (MCP21; AFFINITI), 20S proteasome subunits α1, 2, 3, 5, 6, and 7 (MCP231; AFFINITI), 20S proteasome subunit β7 (MCP205; AFFINITI), and a chlamydial major outer membrane protein (MOMP; clone MC22; our unpublished data).

Techniques: Inhibition, Expressing, Infection, Western Blot, Control, Membrane, Cell-Free Assay, Incubation

Journal: The Journal of Experimental Medicine

Article Title: Degradation of Transcription Factor Rfx5 during the Inhibition of Both Constitutive and Interferon γ–Inducible Major Histocompatibility Complex Class I Expression in Chlamydia-Infected Cells

doi:

Figure Lengend Snippet: Chlamydial but not host protein synthesis is required for both the degradation of RFX5 and suppression of HLA-A and -B heavy chains and β2M. (A) Correlation between infection dose and RFX5 degradation. 30 h after chlamydial infection at various MOI, HeLa cells were analyzed for the levels of chlamydial MOMP and host RFX5, USF-1, and USF-2 proteins on Western blot. The anti–USF-2 antibody used in this experiment can bind to both USF-1 and -2. These two USF isoforms can be separated on blots if gels are run far enough. Since we have previously shown that USF-1 is degraded in chlamydia-infected cells , USF-1 is used as a positive control. Since USF-2 protein level is usually not altered by chlamydial infection, it serves as a negative control. ns denotes nonspecific binding. (B) Time course relationship between chlamydial growth and RFX5 degradation. At various time points after infection, HeLa cell samples were analyzed on Western blot as described in A. (C) Inhibition of chlamydial but not host protein synthesis prevents RFX5 degradation and restores HLA-A and -B heavy chain and β2M expression. Rifampin (RF; final concentration = 0.1 μg/ml), chloramphenicol (CH; 60 μg/ml) and penicillin (PG; 100 μg/ml) were added at the beginning of chlamydial infection and maintained throughout the culture. Cycloheximide (CY; 10 μg/ml) was added to the culture 10 h before IFN-γ treatment and maintained during the IFN-γ stimulation. The treated HeLa cells were analyzed for protein levels of HLA-A and -B heavy chains, β2M, RFX5, USF-1, and chlamydial MOMP on Western blot. ns denotes nonspecific binding.

Article Snippet: Mouse antibodies were used to detect HLA-A and -B (HB296; ATCC), β2M (HB149; ATCC), β1 integrin (provided by Dr. J. Wilkins, University of Manitoba, Manitoba, Canada), 20S proteasome α subunit HC2 (MCP20; AFFINITI), 20S proteasome α subunit HC3 (MCP21; AFFINITI), 20S proteasome subunits α1, 2, 3, 5, 6, and 7 (MCP231; AFFINITI), 20S proteasome subunit β7 (MCP205; AFFINITI), and a chlamydial major outer membrane protein (MOMP; clone MC22; our unpublished data).

Techniques: Infection, Western Blot, Positive Control, Negative Control, Binding Assay, Inhibition, Expressing, Concentration Assay

Journal: American Journal of Cancer Research

Article Title: DcR3 promotes proliferation and invasion of pancreatic cancer via a DcR3/STAT1/IRF1 feedback loop

doi:

Figure Lengend Snippet: The relationship between DcR3 and CEACAM1. A. Cellular component analysis showed that 8 plasma membrane proteins were correlated with DcR3. B. TCGA database showed that CEACAM1 and CDH11 were upregulated in pancreatic cancer tissues (n=179) compared with control (n=171) (***P<0.001) while TNFRSF10B showed no significant difference between the two groups (P>0.05). Mann-Whitney test was used to analyze the differences between groups, data were presented as the median with range. C. Western blot assays showed changes in the levels of CEACAM1 and CDH11 in DcR3 knockdown or DcR3-overexpressing cells. D. DcR3 TPM was associated with CEACAM1 TPM (*P=0.037) but not with CDH11 (P=0.27) in TCGA database. E. ChIP assay showed PCR products targeting P1-P2 of the CEACAM1 promoter. Specific anti-IRF1 or control normal mouse IgG was used for immunoprecipitations, whereas genomic DNA was used as the input control. F. Luciferase assay showed that IRF1 could bind to the two CEACAM1 promoter sites. G. Western blot was used to examine the expression of DcR3 and CEACAM1 after manipulating IRF1 expression. The data were represented as the mean ± S.D. of three independent experiments in vitro. Significant differences are indicated as follows: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Article Snippet: The antibodies against DcR3 (Ab57956), CEACAM1 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab108397","term_id":"41224950","term_text":"AB108397"}} Ab108397 ) and CDH11 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab151302","term_id":"62172120","term_text":"AB151302"}} Ab151302 ) were purchased from Abcam (Cambridge, UK).

Techniques: MANN-WHITNEY, Western Blot, Luciferase, Expressing, In Vitro

Journal: American Journal of Cancer Research

Article Title: DcR3 promotes proliferation and invasion of pancreatic cancer via a DcR3/STAT1/IRF1 feedback loop

doi:

Figure Lengend Snippet: Relationship between DcR3/STAT1/IRF1 and CEACAM1 in pancreatic cancer tissues. A. IHC for p-STAT1, IRF1 and CEACAM1 was performed between DcR3 negative group (left panel) and DcR3 positive group (right panel) in 64 pancreatic cancer tissue samples. Representative cases show stains of the same cohorts of pancreatic cancer sections. Magnification ×100. B-D. Correlation analysis of the expression of DcR3 with that of p-STAT1 (r=0.3904, **P=0.0014), IRF1 (r=0.2998, *P=0.0161) and CEACAM1 (r=0.3523, **P=0.0043). E. Schematic illustration showing that DcR3/STAT1/IRF1 forms a feedback loop to reinforce DcR3 and CEACAM1 expression.

Article Snippet: The antibodies against DcR3 (Ab57956), CEACAM1 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab108397","term_id":"41224950","term_text":"AB108397"}} Ab108397 ) and CDH11 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab151302","term_id":"62172120","term_text":"AB151302"}} Ab151302 ) were purchased from Abcam (Cambridge, UK).

Techniques: Expressing

Journal: BioMed Research International

Article Title: Angiopoietin-1 Promotes the Integrity of Neovascularization in the Subcutaneous Matrigel of Type 1 Diabetic Rats

doi: 10.1155/2019/2016972

Figure Lengend Snippet: Real-time PCR primer sequences.

Article Snippet: After hydration, dropping blocking antibody, a panel of primary antibodies against rat ZO-1 (1:100 ZSBIO China), occludin (1:400 Abcam Britain), Connexin40 (1:400 Abcam Britain), and VE-cadherin (1:400 Abcam Britain) were used.

Techniques: Real-time Polymerase Chain Reaction

Journal: BioMed Research International

Article Title: Angiopoietin-1 Promotes the Integrity of Neovascularization in the Subcutaneous Matrigel of Type 1 Diabetic Rats

doi: 10.1155/2019/2016972

Figure Lengend Snippet: Vascular maturity-related protein expression after Ang-1 treatment. (a) Immunohistochemical staining of ZO-1, occludin, VE-cadherin, and Connexin40 in the four groups was performed. Several proteins are expressed on the envelope of endothelial cells (black arrow), × 200. (b) The relative quantification of ZO-1, occludin, VE-cadherin, and Connexin40 in four groups. △ represents P <0.05 compared with the control group, ∗ represents P <0.05 compared with the Ang-1 treatment group, and N/D represents no expression.

Article Snippet: After hydration, dropping blocking antibody, a panel of primary antibodies against rat ZO-1 (1:100 ZSBIO China), occludin (1:400 Abcam Britain), Connexin40 (1:400 Abcam Britain), and VE-cadherin (1:400 Abcam Britain) were used.

Techniques: Expressing, Immunohistochemical staining, Staining, Quantitative Proteomics, Control

Journal: BioMed Research International

Article Title: Angiopoietin-1 Promotes the Integrity of Neovascularization in the Subcutaneous Matrigel of Type 1 Diabetic Rats

doi: 10.1155/2019/2016972

Figure Lengend Snippet: Analysis of Vascular maturity-related protein expression by RT-PCR. The mRNA expression of α -SMA, desmin, ZO-1, VE-cadherin, and Connexin40 was detected. △ represents P <0.05 compared with the control group, ∗ represents P <0.05 compared with the Ang-1 treatment group, and N/D represents no expression.

Article Snippet: After hydration, dropping blocking antibody, a panel of primary antibodies against rat ZO-1 (1:100 ZSBIO China), occludin (1:400 Abcam Britain), Connexin40 (1:400 Abcam Britain), and VE-cadherin (1:400 Abcam Britain) were used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Genome Biology

Article Title: Functions, structure, and read-through alternative splicing of feline APOBEC3 genes

doi: 10.1186/gb-2008-9-3-r48

Figure Lengend Snippet: Expression analysis of feline A3C, A3H and A3CH. (a,b) Analysis of expression of feline A3C, A3H and A3CH by RT-PCR of total RNA from feline cell lines (CrFK, MYA-1, KE-R) and feline PBMCs (a) and expression of A3CH in PBMCs of lion, puma, Sumatra-tiger (tiger), and lynx (b). H 2 O indicates PCRs using primers specific for the A3s without template cDNA added. (c) Analysis of expression of cat A3CH by immunoblot using rabbit serum against the sequence flanked by the C- and H-domains in cat A3C (linker) using 293T cells transfected with A3 expression plasmid or empty vector as indicated and CrFK and MyA-1 cells (two independent cultures each).

Article Snippet: Human cell line 293T, and feline cell lines CrFK (ATCC CCL-94, feline kidney cells) and KE-R (feline embryonic fibroblast cells, a gift of Roland Riebe, Friedrich-Loeffler Institut, Riems) were maintained in Dulbecco's high glucose modified Eagle's medium (Biochrom, Berlin, Germany; Dulbecco's modified Eagle's medium complete) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 0.29 mg/ml L-glutamine, and 100 units/ml penicillin/streptomycin.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Sequencing, Transfection, Plasmid Preparation