Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Kidney international

Article Title: Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

doi: 10.1046/j.1523-1755.1999.00626.x

Figure Lengend Snippet: Fig. 1. Leptin receptor expression in glomerular endothelial cells (GER). (A) Ligand binding of [125I]-recombinant murine leptin to GER monolayers. Data are presented as Scatchard blot. Each point is the mean of four independent experiments performed in duplicate. Unspecific binding was performed in the presence of 1026 m nonradioactive murine leptin and is subtracted from the binding curve. GERs exhibit high-affinity receptors for leptin with a Kd of 4 nm and Bmax of 9700 receptors/cell. (B) Binding of 5 nm of [125I]-recombinant murine leptin in the presence of 5 mg of a goat polyclonal antimouse leptin receptor antibody (a-ObR Ab) or the same concentration of normal goat IgG. Total binding of leptin in the presence of normal goat IgG was considered as 100%. The a-ObR antibody, but not nonimmune goat IgG, significantly reduced binding of leptin to its putative receptor (P , 0.01, N 5 5 independent binding experiments). (C) Expression of the short rat isoform (r-Ob-Ra) but not the long form (r-Ob-Rb) in cultured GERs. cDNA amplification of reverse transcribed total RNA. A specific 487 bp band is detected in the r-Ob-Ra, but not the predicted 370 bp band in the r-Ob-Rb lane. This experiment was independently repeated five times with similar results. However, the r-Ob-Rb isoform could be easily amplified from rat whole brain cDNA using the same concentrations of primers and amplification conditions (data not shown).

Article Snippet: Additional cells were treated with binant murine leptin in the presence of 5 mg of an affinity- recombinant mouse leptin from two additional suppliers purified goat polyclonal antibody (Santa Cruz Biotech- (Biomol, Hamburg, Germany; and Pepro Tech, Rocky nology, Santa Cruz, CA, USA) raised against a peptide Hill, CT, USA), to insure that the results were not depencorresponding to amino acids 32 to 51 mapping at the dent on the source of the leptin.

Techniques: Expressing, Ligand Binding Assay, Recombinant, Binding Assay, Concentration Assay, Cell Culture, Reverse Transcription

Journal: Kidney international

Article Title: Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

doi: 10.1046/j.1523-1755.1999.00626.x

Figure Lengend Snippet: Fig. 4. Leptin stimulates expression of transforming growth factor-b1 (TGF-b1) in glomerular endothelial cells (GERs). (A) TGF-b1 protein secretion in the cell culture supernatant was also significantly stimulated by a single dose of leptin (6.25 to 625 nm) for 48 hours (N 5 3 to 4 independent stimulation experiments, *P , 0.05 vs. unstimulated controls). (B) Northern blot. A single dose of 0.62 to 625 nm leptin for 48 hours increased TGF-b1 mRNA expression. This blot is representative of three independent experiments with qualitatively similar results.

Article Snippet: Additional cells were treated with binant murine leptin in the presence of 5 mg of an affinity- recombinant mouse leptin from two additional suppliers purified goat polyclonal antibody (Santa Cruz Biotech- (Biomol, Hamburg, Germany; and Pepro Tech, Rocky nology, Santa Cruz, CA, USA) raised against a peptide Hill, CT, USA), to insure that the results were not depencorresponding to amino acids 32 to 51 mapping at the dent on the source of the leptin.

Techniques: Expressing, Cell Culture, Northern Blot

Journal: Kidney international

Article Title: Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

doi: 10.1046/j.1523-1755.1999.00626.x

Figure Lengend Snippet: Fig. 6. Glomerular mRNA expression in leptin-infused rats. (A) Intraperitoneal infusion of murine leptin with osmotic minipumps for 72 hours stimulated glomerular mRNA expression for PCNA and TGF-b1, but not for a1(IV) collagen. (B) Continuous infusion for three weeks increased glomerular transcripts for TGF-b1 and a1(IV) collagen, whereas PCNA mRNA returned to baseline. This blot is representative of three independent experiments with qualitatively similar results.

Article Snippet: Additional cells were treated with binant murine leptin in the presence of 5 mg of an affinity- recombinant mouse leptin from two additional suppliers purified goat polyclonal antibody (Santa Cruz Biotech- (Biomol, Hamburg, Germany; and Pepro Tech, Rocky nology, Santa Cruz, CA, USA) raised against a peptide Hill, CT, USA), to insure that the results were not depencorresponding to amino acids 32 to 51 mapping at the dent on the source of the leptin.

Techniques: Expressing

Journal: Kidney international

Article Title: Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

doi: 10.1046/j.1523-1755.1999.00626.x

Figure Lengend Snippet: Fig. 7. Immunohistological staining of renal sections from in vivo infusion experiments. (A and B) Staining of kidney sections of control or leptin- infused rats (72 hr) with a specific anti-PCNA antibody to assess in vivo proliferation of renal cells. Kidney section of rats infused with solvent for 72 hours revealed no glomerular PCNA staining, suggesting very low basal proliferation in normal glomeruli (A). In contrast, in leptin-infused animals PCNA-expressing cells are found in glomeruli (arrow; B). (C–F) Staining for collagen type IV in rats infused for three weeks with solvent or leptin. There was a light collagen type IV staining in mesangial matrix and tubular basal membranes of control-infused rats (C). However, an increase in glomerular collagen type IV staining was detected in rats infused with leptin for three weeks (D). A higher magnification reveals glomerular collagen type IV staining restricted to the mesangial area of control-infused animals (E), whereas leptin-infused animals exhibit a segmental increase in collagen type IV deposition (F). Magnifications 3150 for A–D, 3 300 for E and F.

Article Snippet: Additional cells were treated with binant murine leptin in the presence of 5 mg of an affinity- recombinant mouse leptin from two additional suppliers purified goat polyclonal antibody (Santa Cruz Biotech- (Biomol, Hamburg, Germany; and Pepro Tech, Rocky nology, Santa Cruz, CA, USA) raised against a peptide Hill, CT, USA), to insure that the results were not depencorresponding to amino acids 32 to 51 mapping at the dent on the source of the leptin.

Techniques: Staining, In Vivo, Control, Solvent, Expressing

Journal: Pharmaceutical biology

Article Title: Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl 4 /2-AAF.

doi: 10.1080/13880209.2021.1961820

Figure Lengend Snippet: Figure 3. YGJ inhibits the activation of M1 macrophages and non-canonical Wnt signalling pathways and inhibits the differentiation of hepatic progenitor cells into myofibroblasts in vivo. (a) STAT1, IRF3, IRF5, IRF8, and SOCS3 protein bands were depicted in the immunoblot images, and (b) the densitometric quantification of the protein bands presented as a histogram (n ¼ 5 per group). (c) The mRNA expressions of Wnt-4, -5 A, -5B, FZD-2, -3, and -6 in liver were measured by RT-PCR and nor- malized to GAPDH mRNA (n ¼ 5 per group). (d) Wnt5A, Wnt5B, and FZD2 protein bands were depicted in the immunoblot images, and (e) the densitometric quantifi- cation of the protein bands presented as a histogram (n ¼ 5 per group). (f) OV6, SOX9, EpCAM, CK19, and Hep mRNA expressions were measured by RT-PCR and normalized to GAPDH mRNA (n ¼ 5 per group). (g) EpCAM protein bands were depicted in the immunoblot images, and (h) the densitometric quantification of the protein bands presented as a histogram (n ¼ 5 per group). p < 0.05 and p < 0.01. N: untreated group (control); 2-AAF/CCl4: 2-acetylaminofluorene/carbon tetra- chloride-treated group; YGJ: 2-AAF/CCl4 þ Yiguanjian decoction-treated group; SORA: 2-AAF/CCl4 þ sorafenib-treated group.

Article Snippet: Rabbit polyclonal antibodies against Oval Cell Marker (OV6), cytokeratin (CK) 19, signal transducer and activator of transcription (STAT) 5A, STAT5B, STAT6, interferon regulatory factor (IRF) 3, IRF5, IRF8, frizzled (FZD) 2, and LDL receptor-related protein (LRP) 5 were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Activation Assay, In Vivo, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Pharmaceutical biology

Article Title: Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl 4 /2-AAF.

doi: 10.1080/13880209.2021.1961820

Figure Lengend Snippet: Figure 4. YGJ inhibits the differentiation of WB-F344 cells into myofibroblasts through inhibiting the activation of M1 macrophages. (a) a-SMA immunofluorescent staining (green) in WB-F344 cells (200) (the presented in vitro experiments were conducted in the same batch, the normal and model groups used the same pictures as published articles. The figures of WB-F344 co-cultured with inactivated macrophages and WB-F344 co-cultured with M1 macrophages were reused form a previous study [Ying Xu et al. 2018] and are reproduced with permission here). (b) a-SMA mRNA expression in WB-F344 cells was measured by RT-PCR and normalized to GAPDH mRNA (n ¼ 3 per group). (c) STAT1, NF-jB, IRF3, IRF5, IRF8, and SOCS3 mRNA expressions in RAW264.7 cells were measured by RT-PCR and normalized to GAPDH mRNA (n ¼ 3 per group); (d) STAT1, NF-jB, IRF3, IRF5, and SOCS3 protein bands were depicted in the immunoblot images, and (e) the densitometric quantification of the protein bands presented as a histogram (n ¼ 3 per group). p < 0.05 and p < 0.01. N: WB-F344 cells co-cultured with inactivated RAW264.7 cells; M: WB-F344 cells co-cultured with LPS (100ng/mL)-activated RAW264.7 cells (referred to as LPS-RAW264.7); YGJ: WB-F344 cells co-cultured with LPS-RAW264.7 treated with Yiguanjian decoction; WIF-1: WB-F344 cells co-cultured with LPS-RAW264.7 treated with Wnt inhibitory factor-1.

Article Snippet: Rabbit polyclonal antibodies against Oval Cell Marker (OV6), cytokeratin (CK) 19, signal transducer and activator of transcription (STAT) 5A, STAT5B, STAT6, interferon regulatory factor (IRF) 3, IRF5, IRF8, frizzled (FZD) 2, and LDL receptor-related protein (LRP) 5 were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Activation Assay, Staining, In Vitro, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot