Journal: Cells

Article Title: The Role of GLP1 in Rat Steatotic and Non-Steatotic Liver Transplantation from Cardiocirculatory Death Donors.

doi: 10.3390/cells8121599

Figure Lengend Snippet: Figure 5. Effect of GLP1 on inflammatory responses in LT with DCDs. (A) Protein levels of suppressors of cytokine signaling 1 (SOCS1) (A1), SOCS2 (A2), and SOCS3 (A3) in liver. (B) MPO (B1) and MDA (B2) levels in liver, Von Willebrand Factor (vWF) in plasma (B3), and nitrate, nitrite (B4), and nitrotyrosine (B5) levels in liver. For A1–A3 and B1–B5, six transplants per group were performed for each measurement. Representative Western blots for the proteins SOCS1, SOCS2, and SOCS3 and the housekeeping protein β-actin are shown at the top, and densitometric quantification of SOCS1, SOCS2, and SOCS3 protein expression normalized to β-actin (arbitrary units) is shown at the bottom. + p < 0.05 vs. LT; # p < 0.05 vs. CD+LT.

Article Snippet: After assessing transfer, the membranes were saturated in 4 mM Tris-HCl, pH 7.6, 30 mM NaCl, and 0.1% Tween 20 (TBST) containing 5% non-fat milk and incubated overnight at 4 ◦C using antibodies against GLP1 (ab23468), cyclin A (sc-239), CD-26 (also known as DPP4; sc-52642), SOCS3 (sc-9023) and transferrin (sc-52256) (Santa Cruz Biotechnology, Dallas, TX, USA), SOCS1 (ARP42148) and SOCS2 (ARP63434) (Aviva Systems Biology, San Diego, CA, USA), cyclin D1 (SAB5500090), and β-Actin (A2228) (Sigma-Aldrich).

Techniques: Clinical Proteomics, Western Blot, Expressing

Journal: Frontiers in oncology

Article Title: Mitochondrion-Localized SND1 Promotes Mitophagy and Liver Cancer Progression Through PGAM5.

doi: 10.3389/fonc.2022.857968

Figure Lengend Snippet: FIGURE 5 | SND1 promotes mitophagy through PGAM5. (A, B) Hep3B cells stably expressing shSND1 (A) or shPGAM5 (B) were treated with 10 mM FCCP for 6 h. Samples were collected for immunoblotting to analyze the expression of SND1, PGAM5, MFN1, TIM23, COX4, p-DRP1S637, and DRP1. Actin served as loading control. LC3 protein levels were detected in Hep3B-shSND1 cells treated with FCCP. (C, D) Hep3B cells stably expressing shSND1 (C) or shPGAM5 (D) were treated with glucose-free medium for 24 h. Samples were collected for immunoblotting to analyze the expression of SND1, PGAM5, MFN1, TIM23, COX4, and p- DRP1S637. Actin served as loading control. LC3 protein levels were detected in Hep3B-shSND1 cells treated with glucose-free medium. (E, F) Hep3B cells stably overexpressing SND1 with further PGAM5 knockdown by shRNAs were treated with FCCP for 6 h (E) or cultured with glucose-free medium for 24 h (F), followed by immunoblotting analysis with antibodies against SND1, PGAM5, MFN1, TIM23, COX4, and p-DRP1S637. Actin served as loading control. (G, H) Hep3B cells stably overexpressing MTS-GFP with further SND1 knockdown (G) or PGAM5 knockdown (H) were cultured with FCCP for 6 h, followed by immunoblotting analysis with antibodies against SND1, PGAM5, MFN1, TIM23, COX4, GFP, and p-DRP1S637. Actin served as loading control. (I) Hep3B cells with endogenous SND1 knockdown were further infected with viruses expressing Flag-EV, Flag-SND1, or Flag-SND1D1-63. These cells were treated with FCCP for 6 h followed by immunoblotting analysis with antibodies against SND1, MFN1, TIM23, COX4, and p-DRP1S637. Actin served as loading control. (J, K) Hep3B cells were infected virus expressing HA-DRP1 together with Flag-PGAM5 plasmids, and the binding activity of PGAM5 and DRP1 under FCCP treatment (J) or glucose deprivation conditions (K) was determined when we further knocked down SND1. Cell lysates were immunoprecipitated with anti-Flag antibody or IgG, followed by immunoblotting analysis with antibodies against HA, Flag, and SND1. Actin served as loading control.

Article Snippet: Primary antibodies against the following proteins were used: SND1 (1:2,000, ab65078, Abcam), PGAM5 (1:1,000, sc-515880, Santa Cruz Biotechnology), TOM20 (1:1,000, 11802-1-AP, Proteintech), TOM70 (1:1,000, 14528-1-AP, Proteintech), COX4 (1:1,000, 11242-1-AP, Proteintech), TIM23 (1:1,000, sc514463, Santa Cruz Biotechnology), MFN1(1:1,000, 13798-1-AP, Proteintech), LC3 (1:1,000, NB100-2220, Novus Biologicals), Phospho-DRP1 (Ser637) (1:1,000, 4867S, CST), DRP1 (1:1,000, 12957-1-AP, Proteintech), Lamin B1 (1:2,000, 12987-1-AP, Proteintech), Tubulin (1:5,000, 66031-1-Ig, Proteintech), bActin (1:2,000, 66009-1-Ig, Proteintech), GAPDH (1:5,000, 60004-1-Ig, Proteintech), TFAM (1:2,000, 22586-1-AP, Proteintech), Flag-M2 (1:2,000, F1804, Sigma-Aldrich), HA (1:1,000, 51064-2-AP, Proteintech), HA-HRP (1:1,000, 2999S, CST), GST (1:5,000, 10000-0-AP, Proteintech), GFP (1:4,000, 50430-2-AP, Proteintech), Calnexin (1:5,000, 10427-2-AP, Proteintech), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Bio-Rad) were used.

Techniques: Stable Transfection, Expressing, Western Blot, Control, Knockdown, Cell Culture, Infection, Virus, Binding Assay, Activity Assay, Immunoprecipitation

Journal: Frontiers in oncology

Article Title: Mitochondrion-Localized SND1 Promotes Mitophagy and Liver Cancer Progression Through PGAM5.

doi: 10.3389/fonc.2022.857968

Figure Lengend Snippet: FIGURE 6 | The promotion effect of SND1 on cell proliferation and tumor growth depends on PGAM5. (A, B) Growth curves were measured in SND1- overexpressing Hep3B cells expressing shRNA against PGAM5. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 comparing the indicated samples. (C–E) Equal numbers of Hep3B cells mentioned in panel (A) were injected subcutaneously into the flanks of BALB/c nude mice (n = 5 mice in each group). Tumor sizes were measured every 3 days using calipers (C). Photographs show xenografts (D), and tumor weights (E) were determined at the end of the experiment (Day 35). Data are presented as the mean ± SD, *P < 0.05 comparing the indicated groups. (F) Protein levels of SND1, PGAM5, MFN1, TIM23, COX4, p-DRP1S637, and DRP1 in tumor lysates of panel D were measured by immunoblotting analysis. Actin served as loading control.

Article Snippet: Primary antibodies against the following proteins were used: SND1 (1:2,000, ab65078, Abcam), PGAM5 (1:1,000, sc-515880, Santa Cruz Biotechnology), TOM20 (1:1,000, 11802-1-AP, Proteintech), TOM70 (1:1,000, 14528-1-AP, Proteintech), COX4 (1:1,000, 11242-1-AP, Proteintech), TIM23 (1:1,000, sc514463, Santa Cruz Biotechnology), MFN1(1:1,000, 13798-1-AP, Proteintech), LC3 (1:1,000, NB100-2220, Novus Biologicals), Phospho-DRP1 (Ser637) (1:1,000, 4867S, CST), DRP1 (1:1,000, 12957-1-AP, Proteintech), Lamin B1 (1:2,000, 12987-1-AP, Proteintech), Tubulin (1:5,000, 66031-1-Ig, Proteintech), bActin (1:2,000, 66009-1-Ig, Proteintech), GAPDH (1:5,000, 60004-1-Ig, Proteintech), TFAM (1:2,000, 22586-1-AP, Proteintech), Flag-M2 (1:2,000, F1804, Sigma-Aldrich), HA (1:1,000, 51064-2-AP, Proteintech), HA-HRP (1:1,000, 2999S, CST), GST (1:5,000, 10000-0-AP, Proteintech), GFP (1:4,000, 50430-2-AP, Proteintech), Calnexin (1:5,000, 10427-2-AP, Proteintech), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Bio-Rad) were used.

Techniques: Expressing, shRNA, Injection, Western Blot, Control

Journal: Frontiers in oncology

Article Title: Mitochondrion-Localized SND1 Promotes Mitophagy and Liver Cancer Progression Through PGAM5.

doi: 10.3389/fonc.2022.857968

Figure Lengend Snippet: FIGURE 7 | Mitochondrial localization is required for SND1-mediated cell proliferation and tumor growth. (A, B) Growth curves of Hep3B cells with endogenous SND1 knockdown that were expressing Flag-EV, Flag-SND1, or Flag-SND1D1-63 were determined by trypan blue counting. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 comparing the indicated samples. (C–E) Equal numbers of Hep3B cells mentioned in panel (A) were injected subcutaneously into the flanks of BALB/c nude mice (n = 5 mice in each group). Tumor sizes were measured every 3 days using calipers (C). Photographs show xenografts (D), and tumor weights (E) were determined at the end of the experiment (Day 31). Data are presented as the mean ± SD, *P < 0.05 comparing the indicated groups. (F) Protein levels of SND1, MFN1, TIM23, COX4, p-DRP1S637, and DRP1 in tumor lysates of panel (D) were measured by immunoblotting analysis. Actin served as loading control.

Article Snippet: Primary antibodies against the following proteins were used: SND1 (1:2,000, ab65078, Abcam), PGAM5 (1:1,000, sc-515880, Santa Cruz Biotechnology), TOM20 (1:1,000, 11802-1-AP, Proteintech), TOM70 (1:1,000, 14528-1-AP, Proteintech), COX4 (1:1,000, 11242-1-AP, Proteintech), TIM23 (1:1,000, sc514463, Santa Cruz Biotechnology), MFN1(1:1,000, 13798-1-AP, Proteintech), LC3 (1:1,000, NB100-2220, Novus Biologicals), Phospho-DRP1 (Ser637) (1:1,000, 4867S, CST), DRP1 (1:1,000, 12957-1-AP, Proteintech), Lamin B1 (1:2,000, 12987-1-AP, Proteintech), Tubulin (1:5,000, 66031-1-Ig, Proteintech), bActin (1:2,000, 66009-1-Ig, Proteintech), GAPDH (1:5,000, 60004-1-Ig, Proteintech), TFAM (1:2,000, 22586-1-AP, Proteintech), Flag-M2 (1:2,000, F1804, Sigma-Aldrich), HA (1:1,000, 51064-2-AP, Proteintech), HA-HRP (1:1,000, 2999S, CST), GST (1:5,000, 10000-0-AP, Proteintech), GFP (1:4,000, 50430-2-AP, Proteintech), Calnexin (1:5,000, 10427-2-AP, Proteintech), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Bio-Rad) were used.

Techniques: Knockdown, Expressing, Injection, Western Blot, Control

Journal: Developmental biology

Article Title: Dgcr8 controls neural crest cells survival in cardiovascular development.

doi: 10.1016/j.ydbio.2011.11.008

Figure Lengend Snippet: Fig. 1. Severe cardiovascular malformations observed in Dgcr8 mutants. Sagittal aortic arch sections of E10.5 Wnt1-cre;Dgcr8loxp/+;R26R-YFP controls (A, n=3) and Wnt1-cre; Dgcr8loxp/loxp;R26R-YFP mutants (B, n=4) stained for YFP (green) and DGCR8 (red). Magnified view in insets. (C–E) Gross thorax anatomy of E17.5 controls (C, n=10) and Wnt1-cre;Dgcr8loxp/loxp mutants (D–E, n=11) and respective schemes of the great vessels (C′–E′). All Wnt1-cre;Dgcr8loxp/loxp displayed persistent truncus arteriosus (PTA; D′,E′ n=11/11), while additional abnormalities included interrupted aortic arch type B (IAA; E′ n=3/11), aberrant origin of the right subclavian artery (Ab-RSA; D′ n=3/11) and cer- vical aortic arch (Cx-AA; D′ n=2/11). RSA, right subclavian artery; RCC, right common carotid; IA, inominate artery; LSA, left subclavian artery; LCC, left common carotid; AA, aortic arch; Ao, aorta; DA, ductus arteriosus. Anterior (top) view of E18.5 OFT, divided into pulmonary artery and aorta in healthy controls (F) and displays persistent truncus arteriosus in Wnt1-cre;Dgcr8loxp/loxp (G). H&E staining of representative E18.5 frontal heart sections from controls (H, n=3) and Wnt1-cre;Dgcr8loxp/loxp mutants (I, n=3), the latter manifests with persistent truncus arteriosus (PTA) and ventricular septal defect (VSD). Ao, aorta; PA, pulmonary artery; RV, right ventricle; LV, left ventricle. Frontal view of E18.5 control thymus (J, n=4) and Wnt1-cre;Dgcr8loxp/loxp thymus that is malformed and ectopically positioned, in a lateral situs in the thorax (K, n=3). LT, left thymic lobe; RT, right thymic lobe; Tr, trachea.

Article Snippet: Primary antibodies in CAS-Block were incubated overnight at 4 °C: DGCR8 (10996-1-AP, 1:100 dilution, Proteintech), GFP (ab6658, 1:300, Abcam), Smooth muscle α-actin (A 2547, 1:400, DAKO), cleaved Caspase-3 (9661, 1:300, Cell Signaling Technology).

Techniques: Staining, Control

Journal: Developmental biology

Article Title: Dgcr8 controls neural crest cells survival in cardiovascular development.

doi: 10.1016/j.ydbio.2011.11.008

Figure Lengend Snippet: Fig. 2. Normal migration but reduced number of neural crest-descendants in Dgcr8 mutant OFT. India ink-injected E10.5 pharyngeal arch arteries. Lateral view of control (A, n=24) and Wnt1-cre;Dgcr8loxp/loxp mutant (B, n=8). 3, 3rd PAA; 4, 4th PAA; 6, 6th PAA. Whole mount in situ hybridization study of Ap2-alpha (C,D, n=5, per genotype), and Crabp1 (E,F, n=5 per genotype) on E10.5 Wnt1-cre;Dgcr8loxp/loxp embryos (D,F) and control littermates (C,E). Arrows indicate the migratory streams of NCCs entering the pharyngeal arches. Whole-mount beta-gal staining of E10.5 embryos, expressing a R26R-LacZ lineage tracing reporter in control (G) and Wnt1-cre;Dgcr8loxp/loxp (H) embryos (n=4, per genotype). Higher magnification reveals seemingly-normal cNCCs migration into the pharyngeal arches and OFT of controls (I) and Wnt1-cre;Dgcr8loxp/loxp (J). PA1, 1st pharyngeal arch. A black arrowhead denotes the distal-most LacZ-positive cells in the OFT. Sagittal sections of E10.5 embryos confirm the presence of NC-derived, LacZ-positive cells, in the 1st pha- ryngeal arch and the developing cardiac OFT, of control littermates (K) and Wnt1-cre;Dgcr8loxp/loxp mutants (L). Whole-mount beta-gal staining of E12.5 embryonic hearts expres- sing R26R-LacZ, reveals reduced cNCC numbers at the OFT of Wnt1-cre;Dgcr8loxp/loxp mutants (N, n=4, denoted by a black arrowhead), relative to controls (M, n=3). Ao, aorta; PA, pulmonary artery; PTA, persistent truncus arteriosus.

Article Snippet: Primary antibodies in CAS-Block were incubated overnight at 4 °C: DGCR8 (10996-1-AP, 1:100 dilution, Proteintech), GFP (ab6658, 1:300, Abcam), Smooth muscle α-actin (A 2547, 1:400, DAKO), cleaved Caspase-3 (9661, 1:300, Cell Signaling Technology).

Techniques: Migration, Mutagenesis, Injection, Control, In Situ Hybridization, Staining, Expressing, Derivative Assay

Journal: Developmental biology

Article Title: Dgcr8 controls neural crest cells survival in cardiovascular development.

doi: 10.1016/j.ydbio.2011.11.008

Figure Lengend Snippet: Fig. 3. Dgcr8 mutant cNCC descendants exhibit normal proliferation and smooth muscle differentiation. Immunofluorescent analysis of E10.5 sagittal sections reveals co-localization of α-smooth muscle actin (SMA-α, red) and cNCC genetic fate tracer R26R-YFP (Green) in the OFT of control (A, n=3) and Wnt1-cre;Dgcr8loxp/loxp mutant (B, n=3). Yellow, merged green and red signals. Representative cells at the OFT, positive for SMA-α and for R26R-YFP, are denoted by white arrowheads in A and B, respectively. Transverse sections of E10.5 embryos at the level of the OFT reveals comparable levels of BrdU incorporation in E10.5 Wnt1-cre;Dgcr8loxp/loxp (D) relative to controls (C). Red, BrdU; Green, YFP; Blue, DAPI. (E) Quantification of the percentage of BrdU-positive cells out of the YFP-positive (cNCCs) in mutants (red) and controls (blue, n=4 per genotype). n.s. not statistically significant.

Article Snippet: Primary antibodies in CAS-Block were incubated overnight at 4 °C: DGCR8 (10996-1-AP, 1:100 dilution, Proteintech), GFP (ab6658, 1:300, Abcam), Smooth muscle α-actin (A 2547, 1:400, DAKO), cleaved Caspase-3 (9661, 1:300, Cell Signaling Technology).

Techniques: Mutagenesis, Control, BrdU Incorporation Assay

Journal: Developmental biology

Article Title: Dgcr8 controls neural crest cells survival in cardiovascular development.

doi: 10.1016/j.ydbio.2011.11.008

Figure Lengend Snippet: Fig. 4. Dgcr8 mutant cNCC descendants exhibit enhanced cell death and attenuated ERK signaling. Cleaved caspase 3 (cCasp-3) immunofluorescent analysis of E10.5 sagittal pha- ryngeal arch (A,C) or outflow tract (OFT, B,D) sections. PA1-6 denote the 1st to the 6th pharyngeal arches, respectively; (n=3 per genotype). (E) Quantification of cleaved caspase 3 (cCasp-3) immunoreactive cells reveals enhanced apoptosis in the pharyngeal arch region of Wnt1-cre;Dgcr8loxp/loxp mutant (red) relative to control (blue), but not in the OFT per-se. * pb0.05. n.s. not statistically significant. Immunofluorescent analysis of E10.5 sagittal pharyngeal arch sections reveals significant downregulation of phosphorylated Erk1/2 in Wnt1-cre;Dgcr8loxp/loxp 1st and 2nd pharyngeal arches (I–K, J,K are enlargements of insets in I), relative to controls (F–H, G,H are enlarged micrographs of insets in F. n=3, per genotype).

Article Snippet: Primary antibodies in CAS-Block were incubated overnight at 4 °C: DGCR8 (10996-1-AP, 1:100 dilution, Proteintech), GFP (ab6658, 1:300, Abcam), Smooth muscle α-actin (A 2547, 1:400, DAKO), cleaved Caspase-3 (9661, 1:300, Cell Signaling Technology).

Techniques: Mutagenesis, Control

Journal: Molecular cancer therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma.

doi: 10.1158/1535-7163.MCT-15-0583

Figure Lengend Snippet: Figure 1. Analysis of Hh pathway activation in human MPM specimens and rat MPM model in tumor and stromal fractions. A, immunohistochemical detection of GLI1, DHH, PTCH1, and HHIP in tumor (T) and stroma (S) and box plot of semiquantitative expression levels (H-score) of GLI1 and DHH in 146 MPM specimens, and PTCH1 and HHIP in 8 MPM specimens (lines indicate medians). B, histology of rat MPM model revealed cellular areas of closely packed spindled tumor cells (T) with relatively large, irregular nuclei, adjacent to stroma (S) containing fibroblasts (, organized small spindle cells), blood vessels (X), and inflammatory cells (small round cells with dense blue chromatin; arrow). Of note is the presence of some fibroblasts and inflammatory cells also in the tumor area. Scatter plot shows semiquantitative expression levels of GLI1, DHH, PTCH1, and HHIP (H-score) of 6 tumors (lines indicate medians).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Activation Assay, Immunohistochemical staining, Expressing

Journal: Molecular cancer therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma.

doi: 10.1158/1535-7163.MCT-15-0583

Figure Lengend Snippet: Figure 2. The comparison of Hh pathway activation in vitro and in vivo rat MPM model. A, expression levels (mRNA) of pathway components were monitored in IL45-luc cultured in 10% serum, primary cell culture in 3% O2 without serum compared with tumor from the rat model (in vivo). B, cell growth measurement by MTT (left) and colony formation assay (right) shows that IL45-luc and the parental line (IL45) cultured in 20% O2 with serum are identically sensitive to vismodegib (data are given in mean SD; , P < 0.05; , P < 0.001; , P < 0.0001). C and D, IL45 parental cells were exposed to increasing concentration of vismodegib and measured for the downregulation of hedgehog pathway target gene (Gli1 and Ptch1) in addition to Gli2 and Gli3. No significant suppression of all genes was observed upon the treatment course (24 and 48 hours).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Comparison, Activation Assay, In Vitro, In Vivo, Expressing, Cell Culture, Colony Assay, Concentration Assay

Journal: Molecular cancer therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma.

doi: 10.1158/1535-7163.MCT-15-0583

Figure Lengend Snippet: Figure 4. Efficient downregulation of Hh signaling and reduction of tumor cell growth following the treatment with vismodegib. A, mRNA levels of Hh target genes, Gli1 is strongly reduce in skin and tumor of vismodegib-treated rats. Expression level of other GLI1 target genes, Ptch1 and Hhip, isalso reduced in treated tumor compared with control. Dhh, Gli2, and Gli3 expression remains unchanged. B, immunohistochemical staining of GLI1 and HHIP showing reduced expression (intensity and frequency) in treated compared with control. More GLI1- and HHIP-negative stromal cells are present in the treated rat (see circles: T, tumor; S, stroma). C, quantitative analysis histo(H)-score of GLI1, HHIP, PTCH1, and DHH staining showing pronounced GLI1 and HHIP downregulation in stromal fractions. D, proliferation (Ki-67–positive), mitotic (phospho-histone H3–positive) indices are significantly reduced in the treated group. No change in necrosis and apoptosis level (TUNEL-positive nuclei) is observed between groups. (C, control, n ¼ 6; T, treated, n ¼ 6, data are given in mean SD; , P < 0.05; , P < 0.001).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Expressing, Control, Immunohistochemical staining, Staining, TUNEL Assay

Journal: Molecular cancer therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma.

doi: 10.1158/1535-7163.MCT-15-0583

Figure Lengend Snippet: Figure 5. Vismodegib dose dependently suppresses growth of mesothelioma cells but could not suppress Hh pathway in vitro. A, primary cells isolated from four different rat tumors cultured in 3% O2 without serum and IL45-luc cell line cultured 20% O2 were exposed to increasing concentration of vismodegib and measured for viability. No difference in terms of sensitivity to vismodegib was observed (data are given in mean SD). B, two lines of primary cell (isolated from control rats) were treated with vismodegib for 24 hours and analyzed for the downregulation of Gli1. No significant suppression of Gli1 was observed. C, IC50 of vismodegib determined in four cell lines after 72-hour treatment (MTT assay). D, the expression levels of Hh pathway components (Dhh, Smo, Ptch1, Gli1) of the four cell lines showing no correlation with their sensitivity to vismodegib.

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: In Vitro, Isolation, Cell Culture, Concentration Assay, Control, MTT Assay, Expressing

Journal: Molecular cancer therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma.

doi: 10.1158/1535-7163.MCT-15-0583

Figure Lengend Snippet: Figure 6. Daily treatment with vismodegib causes downregulation of previously described fibroblast Hh-responsive genes. A, mRNA levels of canonical Hh target gene such as CyclinD1, Abcg2 remained unchanged in vismodegib-treated group compared with control. Previously described fibroblast Hh-responsive genes, i.e., Fn1, Vegfa, and Lif mRNA levels were reduced in vismodegib-treated group. (C, control, n ¼ 6; T, treated, n ¼ 6; data are given in mean SD; , P < 0.05; , P < 0.001). B, mouse embryonic fibroblast NIH3T3 cells were treated with supernatant collected from primary culture of rat MPM cells. Treatment with SMO agonist (SAG) was employed as a positive control. Gli1, Ptch1, and Fn1 expression levels were analyzed after 72-hour incubation at 37C 3% O2 (data represent mean of triplicates SD).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Control, Positive Control, Expressing, Incubation

Journal: iScience

Article Title: BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

doi: 10.1016/j.isci.2022.104273

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-TDP43 (C-terminal) , Proteintech , Cat#12892-1-AP; RRID: AB_2200505.

Techniques: Magnetic Beads, Virus, Recombinant, Expressing, Plasmid Preparation, Cloning, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on tumor necrosis factor (TNF)-α protein expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. Protein expression levels of TNF-α were determined using immunohistochemistry (magnification, ×400). (A) No TNF-α expression was detected in the Schwann cells derived from the SN of a sham-operated rat. (B) Moderate protein expression levels of TNF-α were detected in the Schwann cells derived from the SN fiber of a rat in the 12 h post-reperfusion I/R subgroup. (C) Higher protein expression levels of TNF-α were detected in the Schwann cells derived from the SN of a rat in the 24 h post-reperfusion I/R subgroup. (D) Numerous inflammatory cells had infiltrated the area surrounding the Schwann cells and moderate protein expression levels of TNF-α were detected in the SN of a rat in the 72 h post-reperfusion I/R subgroup. (E) Widespread demyelination and mild-to-moderate TNF-α protein expression levels in Schwann cells were detected in the SN derived from a rat in the 7 days post-reperfusion I/R subgroup. (F) A SN from a rat in the I/R + MK-801 group at 12 h post-reperfusion exhibited mild-to-moderate TNF-α protein expression levels in Schwann cells. (G) A SN from a rat in the I/R + MK-801 group at 24 h post-reperfusion exhibited markedly fewer infiltrating cells, as compared with the SN derived from I/R rats at the same time point post-reperfusion. Moderate protein expression levels of TNF-α expression were observed. (H) A SN derived from a rat in the I/R + MK-801 group at 7 days post-reperfusion. As compared with the SNs derived from the I/R rats, the extent of demyelination was markedly reduced and Schwann cells exhibited only low protein expression levels of TNF-α.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Protein expression levels of tumor necrosis factor-α in the various treatment subgroups were quantified using the integrated optical density method, and are presented as the mean ± standard deviation (n=6). Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. I/R, ischemia reperfusion.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on TNF-α and TACE mRNA expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. TNF-α and TACE mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction and are expressed relative to β-actin. Agarose gel images showing TNF-α and TACE mRNA expression levels in the SN homogenates at (A) 0, (B) 6, (C) 12, (D) 24 and (E) 72 h and (F) 7 days post-reperfusion. β-actin=280 bp; TNF-α=402 bp; TACE=624 bp. Relative (G) TNF-α and (H) TACE mRNA expression levels are presented as the mean ± standard deviation (n=6). *P<0.05, **P<0.01 vs. the sham-operated group; Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. TNF-α, tumor necrosis factor-α; TACE, TNF-α-converting enzyme.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Standard Deviation