Journal: Cell Reports Medicine

Article Title: Intra-channel bi-epitopic crosslinking unleashes ultrapotent antibodies targeting Na V 1.7 for pain alleviation

doi: 10.1016/j.xcrm.2024.101800

Figure Lengend Snippet: Characterization of the pharmaceutical properties of 1080-PEG 7 -ACDTB (A) In vitro serum stability of 1080-PEG 7 -ACDTB. The cartoon on the left shows ring-opening hydrolysis of 1080-PEG 7 -ACDTB. The plot is the percentage change of the light chain and the ring-opening product of 1080-PEG 7 -ACDTB in serum over time. (B) Pharmacokinetic study in C57BL/J6 mice. 1080-PEG 7 -ACDTB was administered at 30 mg/kg (mpk) by intravenous injection, and 1080-PEG 7 -ACDTB concentrations in serum samples were determined by LC-MS/MS. Data are presented as mean ± SEM ( n = 3). (C) Immunofluorescence of mouse plantar frozen sections 2 days after subcutaneous injection of 1080-PEG 7 -ACDTB or H2L3-PEG 7 -ACDTB. The tissue sections were stained with anti-PGP9.5 antibodies and DAPI. The scale bars are 50 μm. (D) Inhibitory effect of 1080-PEG 7 -ACDTB on A-803467-insensitive currents in mouse DRG neurons. Data are presented as mean ± SD. A two-tailed unpaired Student's t test was performed for statistical analysis, ∗∗∗ p < 0.001.

Article Snippet: DRG neurons were held at a potential of −60 mV, and the half-inactivated state current was measured using a 0 mV pulse for 20 ms following a −120 mV pulse for 20 ms. Before the test, A-803467 (MedChemExpress) was applied at a concentration of 100 nM to selectively block Na V 1.8 currents, and BSA was added at a concentration of 5 μg/mL to prevent adsorption.

Techniques: In Vitro, Injection, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Staining, Two Tailed Test

Journal: Oncology Letters

Article Title: Smac/DIABLO expression in human gastrointestinal carcinoma: Association with clinicopathological parameters and survivin expression

doi: 10.3892/ol.2014.2598

Figure Lengend Snippet: Immunhistochemical staining of Smac/DIABLO in gastrointestinal carcinomas. Smac/DIABLO immunoreactivity was predominantly observed in the cytoplasm. (A) High expression of Smac/DIABLO in gastric carcinoma. (B) Low expression of Smac/DIABLO in gastric carcinoma. (C) High expression of Smac/DIABLO in rectal carcinoma. (D) Low expression of Smac/DIABLO in rectal carcinoma. Smac/DIABLO, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low pI. (A–D, magnification, ×200).

Article Snippet: Following blocking with 0.25% casein in PBS (Dako, Glostrup, Denmark), the sections were incubated with a mouse monoclonal antibody against Smac/DIABLO (1:1,000; Cell Signaling Technology, Inc., Danvers, USA) overnight at room temperature.

Techniques: Staining, Expressing, Derivative Assay, Binding Assay

Journal: Epigenomics

Article Title: Transient epigenomic changes during pregnancy and early postpartum in women with and without type 2 diabetes

doi: 10.2217/epi-2017-0129

Figure Lengend Snippet: Dimethylation levels on five histones, expressed as a percentage of total histone H3 levels, were measured in peripheral blood mononuclear cells of never-pregnant women, women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. (A) Dimethylation levels of H3K4 in never-pregnant women compared with women at 30 weeks pregnancy; and dimethylation levels in never-pregnant women compared with women at 20 weeks postpartum. (B) Dimethylation levels of H3K4 women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. (C) Dimethylation levels of H3K9 in never-pregnant women compared with women at 30 weeks pregnancy; dimethylation levels in never-pregnant women compared with 20 weeks postpartum women. (D) Dimethylation levels of H3K9 women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. (E) Dimethylation levels of H3K27 in never-pregnant women compared with women at 30 weeks pregnancy; dimethylation levels in never-pregnant women compared with 20 weeks postpartum women. (F) Dimethylation levels of H3K27 women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. (G) Dimethylation levels of H3K36 in never-pregnant women compared with women at 30 weeks pregnancy; dimethylation levels in never-pregnant women compared with 20 weeks postpartum women. (H) Dimethylation levels of H3K36 women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. (I) Dimethylation levels of H3K79 in never-pregnant women compared with women at 30 weeks pregnancy; dimethylation levels in never-pregnant women compared with 20 weeks postpartum women. (J) Dimethylation levels of H3K79 women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. Values are expressed as a mean percentage of arbitrary units ± standard deviation normalized against total H3 levels.

Article Snippet: After 1-h blocking in 5% (w/v) milk powder in TBS at room temperature, membranes were exposed overnight at 4°C to histone H3 antibodies (Methyl-Histone H3 Antibody Kit #9847, Cell Signalling Technology distributed by Australian Biosearch, Queensland, Australia) diluted in 1/1000 in 1% bovine serum albumin in TBS.

Techniques: Standard Deviation

Journal: Epigenomics

Article Title: Transient epigenomic changes during pregnancy and early postpartum in women with and without type 2 diabetes

doi: 10.2217/epi-2017-0129

Figure Lengend Snippet: Dimethylation levels on five histones, expressed as a percentage of total histone H3 levels, were measured in never-pregnant women, women with type 2 diabetes mellitus (T2DM), women with T2DM at 30 weeks pregnancy, women with T2DM at 8–10 weeks postpartum and women with T2DM at 20 weeks postpartum. (A) Dimethylation levels of H3K4 in never-pregnant women compared with women at 30 weeks pregnancy; dimethylation levels in never-pregnant women compared with women at 20 weeks postpartum. (B) Dimethylation levels of H3K4 women at 30 weeks pregnancy, 8–10 weeks postpartum women and 20 weeks postpartum women. (C) Dimethylation levels of H3K9 in never-pregnant women compared with women at 30 weeks pregnancy; dimethylation levels in never-pregnant women compared with 20 weeks postpartum women. (D) Dimethylation levels of H3K9 women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. (E) Dimethylation levels of H3K27 in never-pregnant women compared with women at 30 weeks pregnancy; dimethylation levels in never-pregnant women compared with 20 weeks postpartum women. (F) Dimethylation levels of H3K27 women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. (G) Dimethylation levels of H3K36 in never-pregnant women compared with women at 30 weeks pregnancy; dimethylation levels in never-pregnant women compared with 20 weeks postpartum women. (H) Dimethylation levels of H3K36 women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. (I) Dimethylation levels of H3K79 in never-pregnant women compared with women at 30 weeks pregnancy; dimethylation levels in never-pregnant women compared with 20 weeks postpartum women. (J) Dimethylation levels of H3K79 women at 30 weeks pregnancy, women at 8–10 weeks postpartum and women at 20 weeks postpartum. Values are expressed as a mean percentage of arbitrary units ± standard deviation normalized against total H3 levels.

Article Snippet: After 1-h blocking in 5% (w/v) milk powder in TBS at room temperature, membranes were exposed overnight at 4°C to histone H3 antibodies (Methyl-Histone H3 Antibody Kit #9847, Cell Signalling Technology distributed by Australian Biosearch, Queensland, Australia) diluted in 1/1000 in 1% bovine serum albumin in TBS.

Techniques: Standard Deviation

Journal: Epigenomics

Article Title: Transient epigenomic changes during pregnancy and early postpartum in women with and without type 2 diabetes

doi: 10.2217/epi-2017-0129

Figure Lengend Snippet: The dimethylation levels are shown as fingers and the fingernail lengths represent the standard deviation. (A) Dimethylation levels of H3 in nondiabetic never-pregnant women. (B) Dimethylation levels of H3 in type 2 diabetic never-pregnant women. (C) Dimethylation levels of H3 in nondiabetic women at 30 weeks pregnancy. (D) Dimethylation levels of H3 in type 2 diabetic women at 30 weeks pregnancy. (E) Dimethylation levels of H3 in nondiabetic women compared with women at 30 weeks pregnancy; dimethylation levels in 8–10 weeks postpartum women at 8 weeks postpartum. (F) Dimethylation levels of H3 in nondiabetic women at 8 weeks postpartum. (G) Dimethylation levels of H3 in nonpregnant women at 20 weeks postpartum. (H) Dimethylation levels of H3 in type 2 diabetic women at 20 weeks postpartum.

Article Snippet: After 1-h blocking in 5% (w/v) milk powder in TBS at room temperature, membranes were exposed overnight at 4°C to histone H3 antibodies (Methyl-Histone H3 Antibody Kit #9847, Cell Signalling Technology distributed by Australian Biosearch, Queensland, Australia) diluted in 1/1000 in 1% bovine serum albumin in TBS.

Techniques: Standard Deviation

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: DOT1L facilitates PARPi resistance via H3K79 methylation. A . Heatmaps of H3K79me2 levels detected by CUT&Tag around gene body regions in control (shNC) and DOT1L knockdown (shDOT1L) SKOV-3 cells treated with Olaparib 10µM for 48 h. 3 kb windows spanning the TSS to TES of all genes were plotted. Genes were arranged by their enrichment of H3K79me2 in shNC and shDOT1L cells. B . The distributions of H3K79me2-binding regions are shown in the pie charts. C . Venn diagram showing the overlap between RNA-seq data and CUT&Tag data. The KEEP analysis revealed the significantly enriched items based on H3K79me2 signature. D . IGV tracks showing the enrichment of H3K79me2 in ABCB1 and PLCG2 gene regions in control (shNC) and DOT1L knockdown (shDOT1L) SKOV-3 cells treated with Olaparib 10µM for 48 h. E - F . ChIP–qPCR showing the level of the indicated proteins recruited to the PLCG2 ( E ) and ABCB1 ( F ) promoter regions in DOT1L-overexpressed OVCAR8 cells. The data represent the means ± SD ( n = 3). * p < 0.05. three independent sets of PLCG2 and ABCB1 primers were used. G . RT-qPCR was performed in DOT1L overexpressed OVCAR8 cells to determine PLCG2 and ABCB1 mRNA levels. H. PLCG2 and ABCB1 (P-gly) expression was measured by western blotting in DOT1L overexpressed OVCAR8 cells. I . An H3K79me2-ChIP assay was performed in DOT1L knockdown SKOV-3 cells to examine H3K79me2 occupancy at PLCG2 and ABCB1. J . RT-qPCR was performed in DOT1L knockdown SKOV-3 cells to determine PLCG2 and ABCB1 mRNA levels. K . PLCG2 and ABCB1 (P-gly) expression was measured by western blotting in DOT1L knockdown SKOV-3 cells

Article Snippet: The beads were washed with washing buffer (20 mM HEPES-KOH, pH 7.5, 0.5 mM spermidine, 150 mM NaCl, 0.1% BSA) and resuspended in blocking buffer (20 mM HEPES-KOH, pH 7.5, 0.5 mM spermidine, 150 mM NaCl, 0.1% BSA, 2 mM EDTA) at room temperature for 5 min. Primary antibodies (Rabbit monoclonal anti-Histone H3K79me2, CST, 5427 S) were added by 1:100 dilution and incubated at room temperature for 2 h. After being washed with washing buffer, secondary antibodies were added by 1:100 dilution and incubated at room temperature for 30 min. 1.2 µl PA-Tn5 transposomes were added to each sample and incubated at room temperature for 30 min. Beads were resuspended in 30 µl washing buffer with 10 mM MgCl2 and incubated at 37 °C for 1 h. Reactions were stopped by adding 5.5 µl stop buffer (2.25 µL of 0.5 M EDTA, 2.75 µL of 10% SDS and 0.5 µL of 20 mg/ ml Proteinase K) and incubated at 55 °C for 30 min, and then 70 °C for 20 min to inactivate Proteinase K. 0.9X of VAHTS DNA clean beads (VAHTS, Cat. #N411-03) were added to each sample to extract the tagmentated DNA.

Techniques: Methylation, Binding Assay, RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: PARP1-DOT1L-PLCG2/ABCB1 axis contributes to PARPi resistance. A . H3K79me2-ChIP assay was performed with OVCAR8 Olaparib-resistant and parent OVCAR8 cell lines to determine H3K79me2 occupancy at PLCG2 and ABCB1. B . PLCG2 and ABCB1 mRNA levels were determined in R8 OlaR and parent OVCAR8 cells by RT-qPCR. C . Western blotting was performed in Olaparib-resistant OVCAR8 and parent OVCAR8 cell lines to examine PLCG2 and ABCB1 (P-gly) protein expression levels. D . Western blotting was performed in R8 OlaR and parent OVCAR8 cells which were transfected with shNC and DOT1L shRNA respectively with the indicated antibodies. E . R8 OlaR and parent OVCAR8 cells were transfected with shNC, ABCB1 shRNA and PLCG2 shRNA. After 72 h of transfection, cells were collected and analyzed by western blotting with the indicated antibodies. F – G . Colony formation ( F ) and cell apoptosis assay ( G ) were performed in R8 OlaR and parent OVCAR8 stably transfected cell lines. H . Immunohistochemistry (IHC) staining of DOT1L, PARP1, ABCB1 (P-gly), and PLCG2 in PARP inhibitor-resistant human ovarian carcinomas (OC) tissues and sensitive tissues. Representative images are shown. Scale bars: 400 μm (upper); 160 μm (lower). I – K . Correlation analysis between PARP1 and DOT1L(I), DOT1L and P-gly ( J ), and DOT1L and PLCG2 ( K ) were analyzed. L . Quantification of P-gly (right) and PLCG2 (left) expression in PARP inhibitor-resistant OC tissues ( n = 6) and sensitive tissues ( n = 9), ** p < 0.01

Article Snippet: The beads were washed with washing buffer (20 mM HEPES-KOH, pH 7.5, 0.5 mM spermidine, 150 mM NaCl, 0.1% BSA) and resuspended in blocking buffer (20 mM HEPES-KOH, pH 7.5, 0.5 mM spermidine, 150 mM NaCl, 0.1% BSA, 2 mM EDTA) at room temperature for 5 min. Primary antibodies (Rabbit monoclonal anti-Histone H3K79me2, CST, 5427 S) were added by 1:100 dilution and incubated at room temperature for 2 h. After being washed with washing buffer, secondary antibodies were added by 1:100 dilution and incubated at room temperature for 30 min. 1.2 µl PA-Tn5 transposomes were added to each sample and incubated at room temperature for 30 min. Beads were resuspended in 30 µl washing buffer with 10 mM MgCl2 and incubated at 37 °C for 1 h. Reactions were stopped by adding 5.5 µl stop buffer (2.25 µL of 0.5 M EDTA, 2.75 µL of 10% SDS and 0.5 µL of 20 mg/ ml Proteinase K) and incubated at 55 °C for 30 min, and then 70 °C for 20 min to inactivate Proteinase K. 0.9X of VAHTS DNA clean beads (VAHTS, Cat. #N411-03) were added to each sample to extract the tagmentated DNA.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Apoptosis Assay, Stable Transfection, Immunohistochemistry