Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Expression and functional analysis of TRPV1 in BMMs and Osteoclasts. (a) BMMs stained for the macrophage marker CD11b (red) and TRPV1 (green) depict the latter’s expression in these cells, both in the absence (upper panel) and presence (lower panel) of RANKL. (b) Expression of TRPV1 (red) in phalloidin-stained osteoclasts (green, upper panel) is confirmed by a peptide segment against anti-TRPV1 antibody (lower panel) that reduces the specific fluorescence signal intensity of the TRPV1 channel.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Expressing, Functional Assay, Staining, Marker, Fluorescence

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Functional analysis of TRPV1 in BMMs. (a) BMMs were assessed for intracellular Ca 2+ levels upon TRPV1 modulation. Representative intensity profiles of Fluo4-AM intensity at different frames are indicated. (b) Time series graphs of intracellular Fluo4-AM intensities across 200 frames of live imaging. The arrow at the x-axis signifies the time of addition of the respective drugs (20th frame). Gray traces are of individual cells, and the black trace represents the average of 50 cells. (c) Compiled average of different treatments of BMMs, individual cell traces omitted.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Functional Assay, Imaging

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Functional analysis of TRPV1 in BMMs grown on the CMT:HEMA hydrogel. (a) BMMs grown on hydrogels to check for the endogenous levels of Ca 2+ using Fluo4-AM Ca 2+ -sensitive dye. TRPV1 activation elevates the intracellular Ca 2+ levels, as is quantified in (b); n = 100 cells; one-way ANOVA; ns: non-significant, **** p < 0.0001. (c) Correlation representation of the area of cells and per unit area intensity of Fluo4-AM depicts strong positive correlations under basal and TRPV1-activated conditions but not upon inhibition of the channel.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Functional Assay, Activation Assay, Inhibition

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Morphological analysis of BMMs grown on the hydrogel. (a) Representative images of BMMs grown on glass or hydrogel in the presence of RANKL and TRPV1 modulators. Right panels denote marked inset of respective images. Phalloidin intensity (b) and morphometric analyses of BMM’s area (c), perimeter (d), length (e), width (f), and LWR (g). n = 18–51 cells per group; one-way ANOVA; ns: non-significant, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques:

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Differentiation propensities of BMMs into osteoclasts grown on hydrogel. (a) Representative TRAP assay of BMMs grown on the hydrogel in the presence of the TRPV1 activator (RTX) and inhibitor (5′-IRTX) under differentiating conditions (MCSF + RANKL). (b,c) Quantitation of TRAP-positive cells and multinucleated cells in the presence of capsaicin (b) and RTX (c) shows elevated osteoclastogenesis as compared to MCSF and CMT:HEMA control groups. n = 5–10; one-way ANOVA; ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: TRAP Assay, Quantitation Assay

Journal: iScience

Article Title: Loss of histone reader Phf7 leads to immune pathways activation via endogenous retroviruses during spermiogenesis

doi: 10.1016/j.isci.2023.108030

Figure Lengend Snippet:

Article Snippet: Rabbit Lamin B1 Polyclonal , bioss , bs-1840R.

Techniques: ROS Assay, TUNEL Assay, Apoptosis Assay, Bicinchoninic Acid Protein Assay, Recombinant, SYBR Green Assay, Gene Knockout, Software

Journal: Molecular Medicine Reports

Article Title: E2F1 promotes Warburg effect and cancer progression via upregulating ENO2 expression in Ewing sarcoma

doi: 10.3892/mmr.2022.12753

Figure Lengend Snippet: E2F1 involvement in aerobic glycolysis in EWS. (A) (left and right panel) Venn diagrams showing different expressed glycolytic genes and transcription factors (P<0.05) in EWS (GSE17679) associated with the status of mortality and progression, respectively; (middle panel) overlapping analysis with 15 transcription factors regulating three glycolytic genes analyzed by ChIP-X. (B) The relative ENO2 (upper panel), PFKM (middle panel) and TPI1 (lower panel) levels in EWS (GSE17679) with the mortality and progression. (C) Kaplan-Meier curves showing the overall survival in EWS (GSE17679) with high or low levels of ENO2, PFKM and TPI1 (cutoff values=7.474, 9.177 and 10.900). Student's t-test compared the difference in panel B. Log-rank test for survival comparison in panel C. E2F1, E2F transcription factor 1; EWS, Ewing sarcoma.

Article Snippet: Then the membrane was blocked with PBS containing 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated with primary antibodies against rabbit anti-human antibodies against E2F1 (1:1,000; cat. no. A19579, ABclonal), rabbit anti-human antibodies against ENO2 (1:1,000; A3118, ABclonal), rabbit anti-human antibodies against PFKM (1:1,000; cat. no. A5477, ABclonal), rabbit anti-human antibodies against TPI1 (1:1,000; cat. no. A2579, ABclonal), or rabbit anti-human antibodies against β-actin (1:50,000; cat. no. AC026, ABclonal) at 4°C overnight.

Techniques: Comparison

Journal: Molecular Medicine Reports

Article Title: E2F1 promotes Warburg effect and cancer progression via upregulating ENO2 expression in Ewing sarcoma

doi: 10.3892/mmr.2022.12753

Figure Lengend Snippet: Expression of E2F1 and glycolytic genes in tumor tissues. (A) The relative E2F1 levels in EWS (GSE17679) with the mortality and progression. (B) Kaplan-Meier curves showing the overall survival in EWS (GSE17679) with high or low expression of E2F1 (cutoff values=5.998). (C) Kaplan-Meier curves showing the overall survival in EWS (GSE17679) with high or low expression of ENO2, PFKM, TPI1 , and E2F1 (cutoff values=7.347, 9.177, 10.900 and 5.998). Student's t-test compared the difference in panel A. Log-rank test for survival comparison in panels B and C. E2F1, E2F transcription factor 1; EWS, Ewing sarcoma.

Article Snippet: Then the membrane was blocked with PBS containing 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated with primary antibodies against rabbit anti-human antibodies against E2F1 (1:1,000; cat. no. A19579, ABclonal), rabbit anti-human antibodies against ENO2 (1:1,000; A3118, ABclonal), rabbit anti-human antibodies against PFKM (1:1,000; cat. no. A5477, ABclonal), rabbit anti-human antibodies against TPI1 (1:1,000; cat. no. A2579, ABclonal), or rabbit anti-human antibodies against β-actin (1:50,000; cat. no. AC026, ABclonal) at 4°C overnight.

Techniques: Expressing, Comparison

Journal: Molecular Medicine Reports

Article Title: E2F1 promotes Warburg effect and cancer progression via upregulating ENO2 expression in Ewing sarcoma

doi: 10.3892/mmr.2022.12753

Figure Lengend Snippet: E2F1 regulates aerobic glycolysis. The levels of (A) glucose uptake, (B) lactate production and (C) cellular ATP levels were detected in RDES cells upon the transfection of the indicated plasmids. Reverse transcription-quantitative PCR (normalized to β-actin , n=3) revealing the transcript levels of (D) E2F1 , (E) ENO2 , (F) PFKM and (G) TPI11 in RDES cells treated with stably transfected as indicated. Student's t-test compared the difference in Mock vs. E2F1. One-way ANOVA followed by post-hoc Bonferroni's test was used to compare the difference in sh-Scb vs. sh-E2F1 #1 or sh-E2F1 #2. ***P<0.001 vs. Mock or sh-Scb; ns, no significance; E2F1, E2F transcription factor 1; sh, short hairpin.

Article Snippet: Then the membrane was blocked with PBS containing 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated with primary antibodies against rabbit anti-human antibodies against E2F1 (1:1,000; cat. no. A19579, ABclonal), rabbit anti-human antibodies against ENO2 (1:1,000; A3118, ABclonal), rabbit anti-human antibodies against PFKM (1:1,000; cat. no. A5477, ABclonal), rabbit anti-human antibodies against TPI1 (1:1,000; cat. no. A2579, ABclonal), or rabbit anti-human antibodies against β-actin (1:50,000; cat. no. AC026, ABclonal) at 4°C overnight.

Techniques: Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Stable Transfection

Journal: Molecular Medicine Reports

Article Title: E2F1 promotes Warburg effect and cancer progression via upregulating ENO2 expression in Ewing sarcoma

doi: 10.3892/mmr.2022.12753

Figure Lengend Snippet: E2F1 regulates aerobic glycolysis gene expression of ENO2 . (A) Western blot assays revealed the protein levels of E2F1, ENO2, PFKM and TPI11 in RDES cells treated with stably transfected as indicated. (B-E) Protein expression was quantified by densitometry using Image-Pro Plus with β-actin as the loading control and statistics were performed in GraphPad Prism 9. (F-H) The positive gene expression correlation between E2F1 and (F) ENO2 , (G) PFKM and (H) TPI1 . Student's t-test compared the difference in Mock vs. E2F1. One-way ANOVA followed by post-hoc Bonferroni's test was used to compare the difference in sh-Scb vs. sh-E2F1 #1 or sh-E2F1 #2. ***P<0.001 vs. Mock or sh-Scb; ns, no significance; E2F1, E2F transcription factor 1; sh, short hairpin.

Article Snippet: Then the membrane was blocked with PBS containing 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated with primary antibodies against rabbit anti-human antibodies against E2F1 (1:1,000; cat. no. A19579, ABclonal), rabbit anti-human antibodies against ENO2 (1:1,000; A3118, ABclonal), rabbit anti-human antibodies against PFKM (1:1,000; cat. no. A5477, ABclonal), rabbit anti-human antibodies against TPI1 (1:1,000; cat. no. A2579, ABclonal), or rabbit anti-human antibodies against β-actin (1:50,000; cat. no. AC026, ABclonal) at 4°C overnight.

Techniques: Gene Expression, Western Blot, Stable Transfection, Transfection, Expressing, Control

Journal: Molecular Medicine Reports

Article Title: E2F1 promotes Warburg effect and cancer progression via upregulating ENO2 expression in Ewing sarcoma

doi: 10.3892/mmr.2022.12753

Figure Lengend Snippet: E2F1 regulates EWS progression via ENO2 . (A) MTT colorimetric assay indicating the viability of RDES cells treated stably transfected as indicated (n=3). (B) MTT colorimetric assay indicating the viability of SK-ES-1 cells treated with stably transfected as indicated (n=3). (C) Representative images (left panel) and quantification (right panel) of Matrigel invasion assays showing the invasion of RDES cells treated with stably transfected as indicated (n=3). (D) Representative images (left panel) and quantification (right panel) of Matrigel invasion assays showing the invasion of SK-ES-1 cells treated with stably transfected as indicated (n=3). Scale bars in images: ×200 magnification scale bar, 100 µm. Student's t-test compared the difference in panel A and B. One-way ANOVA with Bonferroni's multiple comparison post hoc test compared the difference in panel C and D. **P<0.01, ***P<0.001 vs. Mock + sh-Scb; ns, no significance; E2F1, E2F transcription factor 1; sh, short hairpin.

Article Snippet: Then the membrane was blocked with PBS containing 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated with primary antibodies against rabbit anti-human antibodies against E2F1 (1:1,000; cat. no. A19579, ABclonal), rabbit anti-human antibodies against ENO2 (1:1,000; A3118, ABclonal), rabbit anti-human antibodies against PFKM (1:1,000; cat. no. A5477, ABclonal), rabbit anti-human antibodies against TPI1 (1:1,000; cat. no. A2579, ABclonal), or rabbit anti-human antibodies against β-actin (1:50,000; cat. no. AC026, ABclonal) at 4°C overnight.

Techniques: Colorimetric Assay, Stable Transfection, Transfection, Comparison

Journal: Molecular Medicine Reports

Article Title: E2F1 promotes Warburg effect and cancer progression via upregulating ENO2 expression in Ewing sarcoma

doi: 10.3892/mmr.2022.12753

Figure Lengend Snippet: Schematic diagram of the mechanism. Schematic depicting the mechanisms underlying E2F1-promoted Warburg effect and cancer progression: as a transcription factor, E2F1 promotes expression of ENO2, resulting in enhanced Warburg effect and cancer progression in EWS. E2F1, E2F transcription factor 1.

Article Snippet: Then the membrane was blocked with PBS containing 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated with primary antibodies against rabbit anti-human antibodies against E2F1 (1:1,000; cat. no. A19579, ABclonal), rabbit anti-human antibodies against ENO2 (1:1,000; A3118, ABclonal), rabbit anti-human antibodies against PFKM (1:1,000; cat. no. A5477, ABclonal), rabbit anti-human antibodies against TPI1 (1:1,000; cat. no. A2579, ABclonal), or rabbit anti-human antibodies against β-actin (1:50,000; cat. no. AC026, ABclonal) at 4°C overnight.

Techniques: Expressing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Quantitative real-time PCR primer sequences

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: GATA6 is expressed in human hepatocytes during early gestation but not in normal perinatal or adult hepatocytes. A–C: immunohistochemical staining of GATA6 protein in normal human liver. Brown indicates positive staining in nuclei. In fetal liver (FL) at gestational week (GW) 13 (FL GW13), GATA6 protein is expressed in all hepatocytes (arrowhead) and other cell types of the liver (A). At GW37, GATA6 protein expression is diminished in hepatocytes (arrowhead) and restricted to cholangiocytes lining the bile ducts (arrow) (B). The expression pattern of GATA6 protein in adult liver (AL) is similar to that of late FL with positive cholangiocytes (arrow) and negative hepatocytes (arrowhead) (C). D–G: in situ hybridization of GATA6 mRNA in FL GW37 (D and E) and AL (F and G). Green indicates positive signal in cytoplasm. Cholangiocytes show positive signal (arrow); hepatocytes show weak or negative signal (arrowhead). Scale bar = 200 µm (A–C) and 20 µm (D–G).

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Immunohistochemical staining, Staining, Expressing, In Situ Hybridization

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Hepatocyte expression of GATA6 is elevated in biliary atresia (BA) and decreased after portoenterostomy (PE). A: boxplot showing the relative GATA6 mRNA expression, as determined by qPCR, in different liver sample groups. Dots represent individual samples, the box represents the interquartile range, and the whiskers represent the 1st and 4th quartile. The line inside the box is the median, and the dashed line represents the mean. *P < 0.05, **P < 0.01 compared with BA group. The P values of other paired comparisons are indicated in Table 2. B–E: in situ hybridization of GATA6 in BA samples demonstrates strongly positive hepatocytes (arrowhead) (B and C), whereas in situ hybridization in BA-post-PE samples shows weak or negligible signal in hepatocytes (arrowhead) (D and E). F–H: GATA6 immunohistochemistry from normal adult liver (AL) with negative hepatocytes and positive cholangiocytes (F), BA with strong immunoreactivity in hepatocyte nuclei (G), and BA-post-PE with less immunoreactivity in hepatocytes compared with BA (H). I: Western blotting of GATA6 protein in AL, BA, BA-post-PE, and disease control (DC) samples. NBI, normalized band intensity. J: paired sample analysis of GATA6 protein expression from patients before BA and after PE (BA-post-PE). Green (in situ hybridization) and brown (immunohistochemistry) indicate positive staining. Scale bars = 150 µm (B and C), 20 µm (C and E), and 200 µm (F and H).

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Western Blot, Staining

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: P values from comparisons of mRNA levels of different liver sample types (in Figs. 2A and 7A, C, and E) using Wilcoxon method

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: GATA6 immunoreactivity in liver sample groups

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Double in situ hybridization in biliary atresia (BA) liver. A and B: double in situ hybridization was performed on 2 different samples of BA liver. GATA6 expression is high in both ductular reaction (DR) area and liver parenchyma (hepatocytes); these 2 histological compartments are denoted in the figures by dashed lines. C and D: CK7 (C) is strongly expressed in DR area, whereas its expression is weaker in hepatocytes. CFTR (D) expression is limited to DR area. E and F: merged images of DAPI, GATA6, and CK7 (E) or DAPI, GATA6, and CFTR (F). Bile duct epithelium (arrow) is positive for both GATA6 and CK7/CFTR, but hepatocytes (arrowhead) express only GATA6. G and I: higher-magnification images from E. H and J: higher-magnification images from F. Green indicates positive signal for GATA6, and red indicates positive signal for CK7 or CFTR. Blue indicates DAPI staining in the nuclei of all cell types. Scale bars = 50 µm (A–F) and 10 µm (G–J).

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: In Situ Hybridization, Expressing, Staining

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: GATA6 is overexpressed in hepatocytes in 2 mouse models of biliary obstruction. A: GATA6 protein is expressed in bile duct epithelium (arrow) but not in hepatocytes (arrowhead) of normal murine liver (4 wk after sham surgery). B: in mice subjected to bile duct ligation (BDL), hepatocytes (arrowhead) in the periportal area strongly express GATA6. C and D: at postnatal day 15 (P15), before the onset of ductular reaction (DR), there remains weak expression of GATA6 in normal hepatocytes (arrowhead), and there is no difference in GATA6 expression between control (Ctrl) and Alb-Cre;Rbpjflox/flox;Hnf6flox/flox double knockout (DKO) mice. E and F: at P30 Ctrl hepatocytes are GATA6 negative, whereas the Alb-Cre;Rbpjflox/flox;Hnf6flox/flox liver with severe DR shows strong GATA6 immunoreactivity in hepatocytes. G and H: at P120 in Alb-Cre;Rbpjflox/flox;Hnf6flox/flox liver, the DR has diminished, and liver histology, as well as GATA6 expression, is similar to Ctrl liver with immunoreactivity only in bile duct epithelial cells (arrow). n = 3 in each group. Scale bars = 50 µm.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Ligation, Expressing, Double Knockout

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: In patients with biliary atresia (BA), GATA6 protein expression in hepatocytes correlates to bile duct expansion (BDE), age at portoenterostomy (PE), and the liver injury marker alanine aminotransferase. Hepatocyte GATA6 protein expression was divided into 2 groups (<70% positive nuclei = low/intermediate vs. >70% positive nuclei = high) and correlated to BDE rate (P = 0.0094) (A), age at PE (B), and plasma (P)-alanine aminotransferase (C). A: contingency tabling coupled with χ2 test was employed to test the statistical significance. B and C: dots represent individual samples, the box represents the interquartile range, and the whiskers represent the 1st and 4th quartile. The line inside the box is the median, and the dashed line represents the mean. *P < 0.05.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing, Marker

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Enforced expression of GATA6 causes alterations in expression of genes regulating cholangiocyte and hepatocyte differentiation. The human hepatocellular cell line HepG2 and primary human hepatocytes were transiently transfected with pCDNA3-GATA6 or pCDNA3 plasmid alone. After 48 h, RNA was harvested and subjected to qRT-PCR analysis. A: in HepG2 cells, 3 genes related to cholangiocyte differentiation (HNF1β, HNF6, and JAG1) were significantly upregulated in cells overexpressing GATA6 compared with control (Ctrl) cells. B: in primary human hepatocytes, the relative expression of 4 genes related to cholangiocyte differentiation (HNF1β, HNF6, JAG1, and DKK1) and 1 gene related to hepatocyte differentiation (HNF4α) were upregulated in cells with GATA6 overexpression compared with control cells. For both cell types, 5 independent experiments were performed in triplicate. Each bar depicts the logarithm of the ratio of mRNA expression GATA6 overexpression vs. control cells. *P < 0.05, **P < 0.01.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Expression of HNF1β, HNF6, and JAG1 is elevated in biliary atresia (BA) liver, decreases after successful portoenterostomy (PE), and correlates to GATA6. A, C, and E: boxplots showing relative mRNA expression of HNF1β (A), HNF6 (C), and JAG1 (E), as measured by qPCR, in BA vs. other liver specimens. Dots represent individual samples, the box represents the interquartile range, and the whiskers represent the 1st and 4th quartile. The line inside the box is the median, and the dashed line represents the mean. *P < 0.05, **P < 0.01. The P values of other paired comparisons are indicated in Table 2. B, D, and F: linear regression analyses of HNF1β (B), HNF6 (D), and JAG1 (F) mRNA expression vs. GATA6 mRNA expression in BA-post-PE samples. FL, fetal liver; AL, adult liver; DC, disease control.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing

Journal: bioRxiv

Article Title: SMPD3 -mediated extracellular vesicle biogenesis inhibits oligodendroglioma growth

doi: 10.1101/2020.07.14.202200

Figure Lengend Snippet: A-C . Kaplan–Meier survival curves for correlation between SMPD3 (A) , TSG101 (B) , STAM1 (C) expression and survival in low-grade glioma patients (Log-rank (Mantel-Cox) test; p <0.0001 ). D. SMPD3 levels are higher in normal brain cortex compared to low-grade gliomas and GBMs. E. SMPD3 levels are higher in ODG tumors versus astrocytoma. p <0.0001 . F. Kaplan–Meier survival curves for correlation between SMPD3 expression and survival of astrocytoma patients. Log-rank (Mantel-Cox) test; χ 2 =7.601; p =0.0058 ; high SMPD3 >15.81 (n=66); low SMPD3 <15.0 (n=65). G. Kaplan–Meier survival curves for correlation between SMPD3 expression and survival of ODG patients. Log-rank (Mantel-Cox) test; χ 2 =15.27; p <0.0001 ; high SMPD3 ≥16.96 (n=66); low SMPD3 ≤16.28 (n=66). ****, p < 0.0001 .

Article Snippet: For all in vitro SMPD3 knockdown experiments, we used NSMase2 (SMPD3) Human shRNA Plasmid Kit (Origene, Locus ID 55512), which included what we termed sh SMPD3 variants A-D and shScr.

Techniques: Expressing

Journal: bioRxiv

Article Title: SMPD3 -mediated extracellular vesicle biogenesis inhibits oligodendroglioma growth

doi: 10.1101/2020.07.14.202200

Figure Lengend Snippet: A-C’ . Generation of doxycycline-inducible system to assess the effects of SMPD3 on BT088 cell growth, showing Ctrl-GFP (A) and SMPD3 -GFP (B) cells. Western blotting showing relative nSMase2 expression normalized to Actin (B,B’). Analysis of CD9 + EV particle number using nano-flow cytometry in Ctrl-GFP and SMPD3 -GFP cells (C,C’). D-H. Live cell imaging to monitor growth of Ctrl-GFP and SMPD3 -GFP cells, showing growth with and without doxycline at day 0 (D-G) and day 5 (D’-G’). Quantitation of cell growth (normalized to day 0) (H). I-M. Live cell imaging of Cytotox dye labeled Ctrl-GFP and SMPD3 -GFP cells to monitor induction of cell death, showing growth without (I-L) and with (I’-L’) doxycycline at days 1, 2, 3, and 5. Quantitation of Cytotox + cells (normalized to day 0) (M). Bars represent means ± s.e.m‥ *, p < 0.05 ; **, p < 0.01 . p -values for all points in H and M in Table S1. Scale bars: 200 μm.

Article Snippet: For all in vitro SMPD3 knockdown experiments, we used NSMase2 (SMPD3) Human shRNA Plasmid Kit (Origene, Locus ID 55512), which included what we termed sh SMPD3 variants A-D and shScr.

Techniques: Western Blot, Expressing, Flow Cytometry, Live Cell Imaging, Quantitation Assay, Labeling

Journal: bioRxiv

Article Title: SMPD3 -mediated extracellular vesicle biogenesis inhibits oligodendroglioma growth

doi: 10.1101/2020.07.14.202200

Figure Lengend Snippet: A-C’. Use of shRNA constructs to assess the effects of SMPD3 knockdown on BT088 cell growth, depicting shScr and sh SMPD3 (variant A,B,C,D) constructs (A). Western blotting showing relative nSMase2 expression normalized to Actin (B,B’). Analysis of CD9 + EV particle number using nano-flow cytometry in shScr and sh SMPD3 (variant A,B,C,D) BT088 cells (C,C’). D-H. Live cell imaging to monitor growth of shScr and sh SMPD3 (variant A,B,C,D) BT088 cells at day 0 (D-G) and day 5 (D’-G’). Quantitation of cell growth (normalized to day 0) (H). I-M. Live cell imaging of Cytotox dye labeled shScr and sh SMPD3 (variant A,B,C,D) BT088 cells to monitor induction of cell death, showing Cytotox + cells at day 0 (I-L) and day 5 (I’-L’). Quantitation of Cytotox + cells (normalized to day 0) (M). Bars represent means ± s.e.m‥ *, p < 0.05 ; **, p < 0.01 . p -values for all points in H and M in Table S1. Scale bars: 200 μm.

Article Snippet: For all in vitro SMPD3 knockdown experiments, we used NSMase2 (SMPD3) Human shRNA Plasmid Kit (Origene, Locus ID 55512), which included what we termed sh SMPD3 variants A-D and shScr.

Techniques: shRNA, Construct, Variant Assay, Western Blot, Expressing, Flow Cytometry, Live Cell Imaging, Quantitation Assay, Labeling

Journal: bioRxiv

Article Title: SMPD3 -mediated extracellular vesicle biogenesis inhibits oligodendroglioma growth

doi: 10.1101/2020.07.14.202200

Figure Lengend Snippet: A. Schematic of xenografting protocol, showing NSG mice were xenografted with shScr and SMPD3 -KD BT088 cells and tumor growth was monitored. B. Kaplan–Meier survival curves associated with shScr and SMPD3 KD BT088 cell xenografts. Log-rank (Mantel-Cox) test; χ 2 =7.162; p=0.0074. C-J. Co-immunostaining of shScr (C,E,G,I) and SMPD3 KD (D,F,H,J) BT088 cell xenografted tumors with HNA (red) and Olig2 (green, C,D), Ki-67 (green, E,F), Isolectin (green, G,H), and Gfap (green, I,J). Blue is DAPI counterstain. Insets present split channel images of regions marked by a dotted box. Br, normal brain; xe, xenograft. Scale bars: 50 μm in (C-J).

Article Snippet: For all in vitro SMPD3 knockdown experiments, we used NSMase2 (SMPD3) Human shRNA Plasmid Kit (Origene, Locus ID 55512), which included what we termed sh SMPD3 variants A-D and shScr.

Techniques: Immunostaining

Journal: bioRxiv

Article Title: SMPD3 -mediated extracellular vesicle biogenesis inhibits oligodendroglioma growth

doi: 10.1101/2020.07.14.202200

Figure Lengend Snippet: A-A’. Schematic of cerebral organoid (CO)-BT088 co-culture assay, depicting 30-day old hESC-derived COs co-cultured with BT088 cells (shScr, sh SMPD3 -B/D) for 7 DIV (A). Quantitation method, depicting division of each CO into 7 zones spanning organoid periphery to the core (0-350 μm) (A’). B-E’’’. Immunostaining of COs grown alone or in CO-BT088 co-cultures with SOX2 (red, B-E, white, B’-E’) and turbo GFP (t-GFP; green, B-E, white, B’’-E’’). Blue is DAPI counterstain (B-E, white, B’’’-E’’’). F. Percentage of SOX2 + cells in each of 7 zones. G. Lines of best-fit from (F) plotted for the three conditions. shScr: slope= −0.04789 ± 0.003408; sh SMPD3 -B: slope= −0.02941 ± 0.007366, p = 0.1234 ; sh SMPD3 -D: slope= −0.03215 ± 0.003388, p = 0.2205 H. Percentage of t-GFP intensity per zone per section. I. Lines of best-fit from (H) plotted for shScr: slope= −0.01259 ± 0.003080; sh SMPD3 -B: slope= −0.02711 ± 0.001588, p = 0.003646 ; sh SMPD3 -D: slope= −0.03269 ± 0.004513, p = 0.000118 . J. Cumulative t-GFP intensity/unit area in shScr and sh SMPD3 -CO sections. K. Summary of major findings: SMPD3 expression in the brain is high under normal healthy conditions. ODG cells with high SMPD3 expression produce more EVs and grow slower. ODG cells with low SMPD3 expression produce less EVs and grow faster. *, p < 0.05 ; ***, p < 0.005 . Scale bars: 100 μm.

Article Snippet: For all in vitro SMPD3 knockdown experiments, we used NSMase2 (SMPD3) Human shRNA Plasmid Kit (Origene, Locus ID 55512), which included what we termed sh SMPD3 variants A-D and shScr.

Techniques: Co-culture Assay, Derivative Assay, Cell Culture, Quantitation Assay, Immunostaining, Expressing

Journal: The World Journal of Men's Health

Article Title: Docetaxel Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Prostate Cancer Cells via Epigenetic Gene Regulation by Enhancer of Zeste Homolog 2

doi: 10.5534/wjmh.220073

Figure Lengend Snippet: Expressions of EZH2 and E2F1 in DU 145 cells. WB assay shows decreases of EZH2 and E2F1 expressions after docetaxel treatment (A). Using knockdown of E2F1, the expressions of E2F1 and EZH2 are decreased (B), and TRAIL (100 ng/mL) treatment causes significant cell death in siE2F-1 DU 145 cells ( *** p<0.001) (C). DR4: death receptor 4, DR5: death receptor 5, EZH2: enhancer of zeste homolog 2, ns: not significant, TRAIL: tumor necrosis factor-related apoptosis-inducing ligand, WB: western blot.

Article Snippet: Small interfering RNA (siRNA) against human E2F1 (siE2F1) (sc-29297) and control siRNA (scRNA) (sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Western Blot

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: A, B, Prevention and reversal of paclitaxel-induced hyperalgesia by intrathecal injection of a TRPV1 antagonist (AMG9810). The baseline (BL) behavioral test in A and B were collected before paclitaxel (Pac) or vehicle (Veh) treatments. In A, the gray shading indicates the time of treatment with 15 μg of AMG9810 (intrathecal) or vehicle solution. In B, paclitaxel-induced mechanical hypersensitivity was confirmed as significant from Veh–Veh-treated rats (open squares, n = 5) at 14 d after treatment (P) in two groups (open and filled circles, n = 5 each); rats were then treated with 15 μg of the TRPV1 antagonist AMG9810 intrathecally (filled circles) or vehicle solution (open circles) as indicated by the arrow. *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA followed by Bonferroni post hoc test. The representative image in C shows the baseline staining of TRPV1 (red) in the DRG in vehicle-treated rats that was not different from naive rats (data not shown); TRPV1 staining becomes elevated by day 7 after paclitaxel treatment (D). Cotreatment of rats with PBS (intrathecally) with paclitaxel did not affect the increased staining of TRPV1 (E), whereas cotreatment with LPS-RS (intrathecally) completely prevented the increase in TRPV1 (F). As indicated by the bar graphs in G, the increase in TRPV1+ neurons by paclitaxel was statistically significant (**p < 0.01), whereas in H, this was significantly less in the LPS-RS-treated rats versus the PBS-treated rats. Scale bar, 100 μm. **p < 0.01.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Injection, Staining

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: TRPV1 is colocalized with TLR4 in DRG neurons and afferent terminals in the spinal cord. TRPV1 alone is shown in red in the left column for DRG neurons in A and in spinal terminals in D. TLR4 staining in subsets of DRG neurons is shown in green in B and spinal terminals in E (center column). Colocalization of the two are shown in the merged image by yellow for DRG in the right column (C), as well as in fiber profiles in the superficial spinal dorsal horn (F). Scale bar, 100 μm. The representative recording in G shows that acute application of paclitaxel (12.5 μm) evoked spontaneous action potentials in subsets of DRG neurons from animals treated with paclitaxel. In H and I, representative action potential waveforms for the neuron in G evoked by direct current injection after 5 min of vehicle treatment and then after 5 min of acute perfusion with 12.5 μm paclitaxel are shown. The bar graphs in J show the group data for the effects of paclitaxel on several action potential properties. AP, Action potential; RMP, resting membrane potential; AHP, after-hyperpolarization; AHPxx%, interval to each percentage of maximal amplitude. *p < 0.05; **p < 0.01; ***p < 0.001 paclitaxel versus vehicle; paired t test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Staining, Injection

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: TRPV1 sensitization by paclitaxel and LPS in DRG neurons shown using calcium imaging. Representative calcium imaging results of change in 340/380 ratio in dissociated DRG neurons after perfusions of capsaicin (CAP, 200 nm) alone and in combination with paclitaxel (12.5 μm) or paclitaxel plus LPS-RS (2 μg/ml) are shown in A–D, each colored line is a single neuron and the time of each application is indicated by the bars over the traces. The bar graphs show the grouped results for experiments testing the interactions between paclitaxel on the responses to capsaicin (E–G) on the effects of LPS on DRG neurons alone (H) and the effects of LPS on DRG neurons from vehicle- and paclitaxel-treated rats (I). **p < 0.01; ***p < 0.001, vehicle rats versus paclitaxel rats; ###p < 0.001, CAP+Pac versus CAP+Pac+LPS-RS.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Imaging

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Interactions between TLR4 and TRPV1 in HEK 293 cells studied using whole-cell patch clamp. Inward currents were recorded in HEK293 cells transfected with TLR4 only (top line), TRPV1 only (second line), and both TLR4 and TRPV1 (line 3). The bar graphs at the bottom show the summarized responses with statistical differences determined by paired t tests. Capsaicin (200 nm) did not induce inward currents in cells expressing TLR4 alone, whereas the responses to repeated capsaicin showed desensitization in cells expressing TRPV1 alone. LPS (10 ng/ml) and paclitaxel (12.5 μm) sensitized the responses to capsaicin in cells expressing both TLR4 and TRPV1. *p < 0.05, LPS+capsaicin; ***p < 0.001, paclitaxel+capsaicin; *p < 0.05 vehicle solution+capsaicin versus first capsaicin response.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Patch Clamp, Transfection, Expressing

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Representative examples of whole-cell recordings for substantia gelatinosa (SG) neurons before (left) and after (right) administration of the TRPV1 antagonist AMG9810 (5 μm) in the vehicle-treated group (A, B), day 7 paclitaxel-treated group (C, D), and day 14 paclitaxel-treated group (E, F) show increased sEPSCs only in the day 7 paclitaxel-treated group that is suppressed by the AMG9810. The washout segment is not shown. Bar graphs in G and H summarize the mean (±SEM) change in amplitude (G) and frequency (H) of sEPSCs before and after 5 min of AMG9810 application. **p < 0.01, day 7 paclitaxel-treated group versus vehicle and day 14 paclitaxel-treated groups; two-way ANOVA followed by Newman–Keuls post hoc test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques:

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Paclitaxel application increased mEPSC frequency in superficial dorsal horn neurons in rat spinal cord slice. A, Native recording of mEPSC activity before and after paclitaxel (50 nm) application. B, The TRPV1 antagonist SB366791 (10 μm) did not change the mEPSC frequency but prevented its increase during coapplication with paclitaxel. C, Averaged responses demonstrate that paclitaxel treatment induced a significant increase in mEPSC frequency compared with the baseline (control, 100%) value (140.7 ± 11.1%; n = 14). This increase was prevented by the TRPV1 antagonist (SB366791+paclitaxel) treatment, whereas the antagonist alone had no effect (SB366791; n = 10). D, Paclitaxel (50 nm) application did not change the frequency or amplitude of the sEPSCs or the amplitude of the dorsal root sEPSCs (E). ***p < 0.001 versus control values; ###p < 0.001 versus paclitaxel; one-way ANOVA followed by Student–Newman–Keuls test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Activity Assay

Journal: The Journal of Neuroscience

Article Title: The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

doi: 10.1523/JNEUROSCI.1956-15.2015

Figure Lengend Snippet: Immunofluorescent double staining shows that TRPV1 (red, A) is colocalized with TLR4 (green, B) in human DRG neurons (yellow in merged image at right, C). The red arrows in the merged image indicate cells only showing TRPV1, the green arrows indicate cells only expressing TLR4, and the yellows arrow points to cells positive for both TRPV1 and TLR4. Scale bar, 200 μm. Three types of responses were observed when human DRG neurons were tested by application of capsaicin (Cap) and paclitaxel (Pac). Type 1 neurons did not respond to either capsaicin or paclitaxel (data not shown). Type 2 neurons (D) responded positively to capsaicin (left column), showed no responses to paclitaxel (center column), and then showed desensitization to a second application of capsaicin (right column). Type 3 neurons (E) showed responses to capsaicin (left column) and to paclitaxel (center column) and then showed a facilitation of response to the repeated application of capsaicin (right column). The bar graphs at the bottom show the summarized response for the type 2 and 3 neurons. The baseline response to capsaicin was not different between groups. The second response to capsaicin was significantly reduced compared with the first in the type 2 neurons. The type 3 neurons showed significantly greater responses to paclitaxel than did type 2 neurons; and type 3 neurons showed a significantly increased response to the second application of capsaicin compared with the first. *p < 0.05; Mann–Whitney U test.

Article Snippet: After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the sections were incubated overnight at 4°C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing primary antibodies against the following targets: TRPV1 (rabbit anti-rat, 1:1000; Alomone), TLR4 (mouse anti-rat, 1:200; Abcam), IB4 (1:1000 BS-Isolectin B4 FITC Conjugate; Sigma-Aldrich), and CGRP (mouse anti-rat, 1:1000; Abcam).

Techniques: Double Staining, Expressing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Generation of p53null A549 cells by CRISPR/Cas9 technology. (A) Single-guide RNA (sgRNA) sequence designed to target p53WT for gene knockout by CRISPR/Cas9 technology. (B) Western blot screening of p53 protein expression in CRISPR A549 cell clones. (C) Western blot analysis confirmed loss of p53 protein expression in p53null cell clones A549-KO1, A549-KO2, and A549-KO3. Cells were pretreated with either Nutlin-3 or 5-fluorouracil to induce p53 accumulation in A549 cells. (D) Caspase 3/7 assay indicated that all p53null CRISPR clones had lower caspase activity compared with p53WT A549 cells in response to 4 h treatment of Nutlin-3 (25 µM, left panel) or 5-fluorouracil (50 µg/ml, right panel). * p < 0.05. (E) WST-1 cell proliferation assay indicated that the three selected p53null CRISPR clones were resistant to Nutlin-3 (15 µM, left panel) or 5-fluorouracil (30 µg/ml, right panel) induced growth inhibition/cell death.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: CRISPR, Sequencing, Gene Knockout, Western Blot, Expressing, Clone Assay, Activity Assay, Proliferation Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: p53null A549 cells exhibit decreased influenza virus susceptibility compared with parental p53WT A549 cells. (A) p53null cells A549-KO1, A549-KO2, and A549-KO3 and p53WT A549 were infected with human influenza virus (IAV) (strain A/Puerto Rico/8/1934 H1N1), 24 h later, cells were fixed, permeabilized, and labeled for influenza nucleoprotein (NP) detection by flow cytometry. The population of NP-positive cells was gated, and their percentages are shown. (B) Percentages of NP-positive cells 24 h post-IAV infection were compared between A549 and A549-KO3 cells from seven individual experiments. *** p < 0.001. (C) Flow cytometry analysis of NP-positive p53WT A549 and A549-KO3 cells 24 h post-IAV infection at a range of multiplicity of infection (MOI). (D) Fluorescence imaging of A549 and A549-KO3 cells co-labeled for viral NP (green) and cellular p53 (red) 24 h post-IAV infection. Scale bar, 50 µm. (E) Real-time quantitative PCR (RT-qPCR) measurement of viral RNA encoding NP, hemagglutinin (HA), and non-structural protein 1 (NS1) in mock- and IAV-infected A549 and A549-KO3 cells at various time points. (F) RT-qPCR measurement of viral RNA encoding NP, HA, and NS1 in culture supernatants of IAV-infected A549 and A549-KO3 cells at various time points.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Virus, Infection, Labeling, Flow Cytometry, Fluorescence, Imaging, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Decreased IAV susceptibility is not mediated by blocked cell entry at the initial stage of infection or decreased caspase 3 activation in p53null cells. (A) Real-time quantitative PCR (RT-qPCR) of viral RNA detected in IAV-infected A549, A549-KO2, and A549-KO3 cells at 1.5 h post-infection [multiplicity of infection (MOI) = 0.001]. (B) Caspase 3/7 activity in mock- or IAV-infected A549 and p53null cells. * p < 0.05. (C) Caspase 3/7 activity in mock- or IAV-infected A549 and A549-KO3 cells in the presence (+) or absence (−) of the caspase 3-specific inhibitor Z-DEVD-fmk (40 µM). * p < 0.05. (D) Flow cytometry analysis of IAV nucleoprotein (NP) expression in mock- or IAV-infected A549 and A549-KO3 cells, in the presence (+) or absence (−) of the caspase 3-specific inhibitor Z-DEVD-fmk. (E) RT-qPCR of viral RNA in the culture supernatant of IAV-infected A549 and A549-KO3 cells, in the presence (+) or absence (−) of the caspase 3-specific inhibitor Z-DEVD-fmk. (F) Cellular cytotoxicity in mock- and IAV-infected p53WT and p53null A549 cells. (G) Cell viability in mock- and IAV-infected p53WT and p53null A549 cells. For panels (B–G) , all measurements were performed at 24 h post-IAV infection (MOI = 0.001).

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Transcriptome analysis of mock- and IAV-infected A549 and A549-KO3 cells. (A) Venn diagram of significantly differentially expressed genes in A549-KO3 PR8 versus A549-KO3 Mock (blue circle) and A549 PR8 versus A549 Mock (red circle). (B) Heatmap showing the 396 overlapping genes significantly differentially expressed in both A549-KO3 PR8 versus A549-KO3 Mock, and A549 PR8 versus A549 Mock. (C) Venn diagram of significantly differentially expressed genes in A549-KO3 Mock versus A549 Mock (green circle) and A549-KO3 PR8 versus A549 PR8 (yellow circle). (D) Heatmap showing the 720 overlapping genes significantly differentially expressed in both A549-KO3 Mock versus A549 Mock, and A549-KO3 PR8 versus A549 PR8. (E) Venn diagram of significantly differentially expressed genes in A549-KO3 PR8 versus A549-KO3 Mock (blue), A549 PR8 versus A549 Mock (red), A549-KO3 Mock versus A549 Mock (green), and A549-KO3 PR8 versus A549 PR8 (yellow). (F) Heatmap showing the 50 overlapping genes significantly differentially expressed in all four groups.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Interferon-induced transmembrane proteins (IFITMs) expression is highly induced by IAV infection and interferons in p53null cells compared with p53WT A549 cells. (A) Expression levels of the top 3 most differentially expressed genes (IFITM1, IFITM2, and IFITM3) from the 50 overlapping gene set (Figure F) were validated by real-time quantitative PCR (RT-qPCR) validation in IAV-infected A549 and A549-KO3 cells [multiplicity of infection (MOI) = 0.001] at the indicated time points. (B) Western blot analysis of expression of viral nucleoprotein (NP), p53, IFITM1, and IFITM2/3 in p53WT A549 and p53null A549-KO3 cells 24 h post-IAV infection at a range of MOI. β-actin blotting was used as a loading control. (C) RT-qPCR analysis of IFITM1, IFITM2, and IFITM3 expression level in mock- or IAV-infected p53WT A549, p53null A549-KO1, A549-KO2, and A549-KO3 cells (MOI = 0.001) at 24 h post-infection. * p < 0.05. (D) Immunofluorescence microscopy images of IFITM1 and IFITM2/3 expression in IAV-infected p53WT A549, p53null A549-KO1, A549-KO2, and A549-KO3 cells (MOI = 0.001) at 24 h post-infection. Scale bar, 20 µm. (E) RT-qPCR analysis of IFITMs mRNA expression level in interferon-β 1a (IFN-β-1a) treated p53WT A549 and p53null A549-KO3 cells pretreated with Nutlin-3 or DMSO solvent control. * p < 0.05. (F) Western blot analysis of IFITM1, IFITM2/3, p53, and p21 protein expression level in IFN-β-1a treated p53WT A549 and p53null A549-KO3 cells pretreated with Nutlin-3 or DMSO solvent control. β-actin blotting was used as a loading control. For panels (E,F) , both A549 and A549-KO3 cells were treated with Nutlin-3 (25 µM) or DMSO for 6 h, followed by IFN-β-1a treatment (50 U/ml) for another 24 h.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, Western Blot, Control, Immunofluorescence, Microscopy, Solvent

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Knockdown of interferon-induced transmembrane proteins (IFITMs) in p53null A549 cells restores susceptibility to IAV infection. A549-KO3 cells were transfected with either IFITM1 (si-IFITM1), IFITM2 (si-IFITM2), IFITM3 (si-IFITM3), a combination of three short interfering RNAs (siRNAs) (si-IFITM1/2/3), or scramble control siRNA (si-Ctrl). 24 h later, cells were detached using trypsin and re-plated onto cell culture plates or μ-slides and after overnight culture, cells were infected with IAV (multiplicity of infection = 0.001) or mock control. 24 h post-infection, cells were harvested for different assays and the RNA and protein lysates were also collected. A549 cells transfected with control siRNA were also included as a benchmark reference. (A) Real-time quantitative PCR analysis of IFITM1, IFITM2, IFITM3, IAV nucleoprotein (NP), IAV hemagglutinin (HA), and IAV non-structural protein 1 (NS1) gene expression level in IAV-infected cells pretreated with different siRNAs. For IFITM1, IFITM2, and IFITM3, expression data were normalized to that of the reference gene HPRT, and then to control siRNA transfected A549-KO3 cells, while for NP, HA, and NS1 gene expression, data were normalized to an HPRT control. * p < 0.05. (B) Western blot analysis of IAV NP, IFITM1, and IFITM2/3 protein expression in IAV-infected p53WT A549 and p53null A549-KO3 cells pretreated with different siRNAs. β-actin blotting was used as a loading control. (C) Flow cytometry analysis of Mock- or IAV-infected p53WT A549 and A549-KO3 cells pretreated with different siRNAs. Each population of NP-positive cells was boxed and their percentages were shown. (D) Representative immunofluorescence microscopy images of IAV-infected A549-KO3 cells pretreated with different siRNAs. IAV NP was detected using a FITC-labeled anti-NP antibody, and cell nuclei were stained with Hoechst 33342. Scale bar, 50 µm. (E) Percentages of NP-positive cells from IAV-infected A549-KO3 cells pretreated with different siRNAs. Five areas were randomly selected for image capture and the% of NP-positive cells was calculated by the number of NP-positive cells (green) as a proportion of total cell numbers, reflected by Hoechst staining (blue). * p < 0.05.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Knockdown, Infection, Transfection, Control, Cell Culture, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Western Blot, Flow Cytometry, Immunofluorescence, Microscopy, Labeling, Staining

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: Overexpressed interferon-induced transmembrane proteins (IFITMs) reduce cellular susceptibility to IAV infection. Mammalian expression plasmids encoding human IFITM1, IFITM2, or IFITM3 were constructed and transfected individually into p53WT A549 cells. After 24 h, cells were detached using trypsin and re-plated onto cell culture plates or μ-slides and after overnight culture, cells were infected with IAV (multiplicity of infection = 0.001) or mock control. At 24 h post-infection, cells were harvested for different assays and protein lysates were also collected. (A) Transfection efficiency was measured by flow cytometry analysis using IFIMT1- or IFITM2/3-specific antibodies. (B) Overexpression of IFITMs in transfected A549 cell cultures was detected by Western blot. (C) Representative immunofluorescence microscopy images of IAV-infected A549 cell cultures after transfection with IFITM1, IFITM2, or IFITM3 expression plasmids. IAV nucleoprotein (NP) is shown in green, and Hoechst 33342-stained nuclei in blue. Scale bar, 50 µm. (D) Percentage of NP-positive cells in IAV-infected A549 cell cultures that overexpressed IFITM1, IFITM2, or IFITM3 proteins, quantified from fluorescence microscopy images. Five areas were randomly selected for image capture and the percentages of NP-positive cells (green) were calculated relative to the total cell numbers, reflected by Hoechst staining (blue). ** p < 0.01. (E) Immunofluorescence microscopy images of IAV-infected A549 cells transfected with IFITM1, IFITM2, or IFITM3 expression plasmids. IFITMs are shown in red, IAV NP in green, and Hoechst 33342-stained nuclei in blue. Scale bar: 20 µm.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Infection, Expressing, Construct, Transfection, Cell Culture, Control, Flow Cytometry, Over Expression, Western Blot, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: Influenza A Virus Facilitates Its Infectivity by Activating p53 to Inhibit the Expression of Interferon-Induced Transmembrane Proteins

doi: 10.3389/fimmu.2018.01193

Figure Lengend Snippet: p53 regulates interferon-induced transmembrane proteins (IFITMs) independent of its transcriptional activity. (A) Schematic presentation of modular structures of full-length p53 and short isoform Δ40p53 proteins. (B) Western blot analysis of p53 (detected by antibody FL393, sc-6243), IFITM1, and IFITM2/3 expression levels in interferon-β 1a (IFN-β-1a) treated p53WT A549, A549-KO3, and A549-Δ40 cells. β-actin blotting was used as a loading control. (C) Real-time quantitative PCR (RT-qPCR) analysis of IFITMs mRNA expression in IFN-β-1a treated p53WT A549, A549-KO3, and A549-Δ40 cells. * p < 0.05. (D) A549, A549-KO3, and A549-Δ40 were infected with IAV [multiplicity of infection (MOI) = 0.001] and after 24 h were analyzed for IAV nucleoprotein (NP) expression by flow cytometry. The populations of NP-positive cells were boxed and their percentages shown. (E) Percentage of NP-positive cells in IAV- (MOI = 0.001) -infected p53WT A549, A549-KO3, and A549-Δ40 cells quantified from fluorescence microscopy images: between 4 and 6 areas were randomly selected for image capture and the percentages of NP-positive cells within the total cell population were calculated. * p < 0.05. (F) Western blot analysis of p53 (detected by both DO-1, sc-126 and FL393, sc-6243), IAV NP, IFITM1, and IFITM2/3 protein expression in p53WT A549, A549-KO3, and A549-Δ40 cells following IAV infection (MOI = 0.001). β-actin blotting was used as a loading control. (G) RT-qPCR measurement of viral RNAs encoding NP, hemagglutinin (HA), and non-structural protein 1 (NS1) genes detected either in IAV- (MOI = 0.001) infected cells (top) or from the culture supernatant (bottom). * p < 0.05. (H) RT-qPCR analysis of IFITM1, IFITM2, and IFITM3 gene expression in mock- or IAV-infected p53WT A549, A549-KO3, and A549-Δ40 cells. * p < 0.05.

Article Snippet: The human lung carcinoma cell line A549 (CCL-185) was purchased from ATCC (Manassas, VA, USA).

Techniques: Activity Assay, Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Flow Cytometry, Fluorescence, Microscopy, Gene Expression

Journal: Nature Communications

Article Title: Programmed cell removal by calreticulin in tissue homeostasis and cancer

doi: 10.1038/s41467-018-05211-7

Figure Lengend Snippet: Cell surface asialoglycans regulates CRT-mediated PrCR. a , b Treatment with neuraminidase led to the removal of sialic acids from the cell surface of HL60 cells. HL60 cells were treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu). Cell surface sialic acids were examined by staining with EBL (a) and MAL (b) by flow cytometry analysis. EBL, Elderberry Bark Lectin; MAL, Maackia Amurensis Lectin II. c , d Examination of cell surface CRT and PHA-L binding sites on cancer cells. HL60 cells were treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu). Recombinant CRT ( c ) and PHA-L ( d ) binding after treatment were measured by flow cytometry. e , f Phagocytosis of cancer cells with neuraminidase treatment. In vitro Phagocytosis assays were performed with HL60, K562, DLD-1, and SW620 cells treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu) as target cells. Mouse bone marrow-derived ( e ) and human peripheral blood monocyte-derived ( f ) macrophages were used for the assay. Phagocytosis was normalized to the maximal response in the experiments. n = 3. * P < 0.05, ** P < 0.01 ( t -test) for phagocytosis between heat inactivated neuraminidase (Δneu)- and neuraminidase (neu)-treated groups. Error bars represent standard deviation. g , h Effects of suppressing the expression of endogenous neuraminidases in cancer cells. Neu1–Neu4 gene knockout were performed with CRISPR in HL60 cells. In vitro Phagocytosis assays were performed with HL60 cells as target cells and mouse bone marrow-derived macrophages ( h ). Macrophages were treated with PBS (ctrl) or lipopolysaccharide (LPS). Neu4 knockout led to the decrease of cell surface CRT-binding sites and inhibited cancer cell phagocytosis. Phagocytosis was normalized to the maximal response in the experiments. n = 3. * P < 0.05, ** P < 0.01 ( t -test) for phagocytosis between ctrl and Neu4 KO groups. Error bars represent standard deviation

Article Snippet: Human cancer derived cell lines SW620, DLD1, HL60, K562, and murine macrophage/monocyte cell line J774 were obtained from ATCC.

Techniques: Staining, Flow Cytometry, Binding Assay, Recombinant, In Vitro, Derivative Assay, Standard Deviation, Expressing, Gene Knockout, CRISPR, Knock-Out