Journal: Cancers

Article Title: Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

doi: 10.3390/cancers11030291

Figure Lengend Snippet: Effect of GA101 on intracellular Ca 2+ concentration in SU-DHL-4 ( A ) and BL2 ( B ) cell lines. Ca 2+ responses to GA101 (10 µg/mL) were measured using Fluo2-Leak Resistant-Acetoxy Methyl ester (Fluo2-LR-AM) Ca 2+ dye and recorded by videomicroscopy (Zeiss LSM 510) using a 25× objective. Black arrows indicate GA101 addition. Each trace represents the response of one cell and data are representative of at least three independent experiments. Data were processed using OriginPro 7.5 (Origin Lab) or GraphPad prism. Cells were recorded in extracellular Hank’s Balanced Salt Solution (HBSS) containing 2 mM Ca 2+ (2Ca) or in Ca 2+ -free HBSS (0Ca). Cells were preincubated with 100 nM thapsigargin (TG) for 45 min and recorded in Ca 2+ -free HBSS (0Ca + TG) or with 10 µM Ned-19 for 1 h and recorded in Ca 2+ -free HBSS (0Ca + Ned19). Calcium responses to GA101 in cells expressing Non Targeting shRNA (sh NT) or sh Orai1 were recorded in HBSS containing 2 mM Ca 2+ . Histograms represent areas under curves (AUC) calculated, under various recording conditions, between the application time of GA101 and t = 2000 s; * p < 0.05.

Article Snippet: Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Concentration Assay, Expressing, shRNA

Journal: Cancers

Article Title: Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

doi: 10.3390/cancers11030291

Figure Lengend Snippet: Involvement of store-operated Ca 2+ entry (SOCE) in GA101-induced cell death. ( A ) BL2 cells. ( B ) SU-DHL-4 cells. Left panels: Cells were incubated with GA101 in the presence or absence of BTP2 (10 µM) for 24 h. Right panels: Cells expressing sh NT or sh Orai1 were treated with GA101 for 24 h. Cell death was assessed by measuring the loss of mitochondrial membrane potential (Δψm), using tetramethylrhodamine methyl ester (TMRM) as a fluorescent dye, or by caspase 3 activation, measured by the FAM-FLICA in vitro caspase detection kit and both analyzed by flow cytometry; * p < 0.05.

Article Snippet: Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Incubation, Expressing, Activation Assay, In Vitro, Flow Cytometry

Journal: Cancers

Article Title: Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

doi: 10.3390/cancers11030291

Figure Lengend Snippet: Differential activation of endoplasmic reticulum (ER) stress in SU-DHL-4 and BL2 cell lines. ( A ) Cells expressing sh NT or sh Orai1 were treated with GA101 (1 µg/mL) for varying lengths of time. After lysis, phosphorylated eukaryotic translation initiation factor alpha (P-eIF2α), eIF2α, and BIM expression levels were assessed by immunoblot analysis. GAPDH was used as a loading control. ( B ) Quantification of Western blots is given as means ± SE of three to five independent experiments; * p < 0.05.

Article Snippet: Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Activation Assay, Expressing, Lysis, Western Blot

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-knockdown in RF/6A cells by siIGFBP-rP1. (A) Reverse transcription-quantitative PCR analysis of IGFBP-rP1 transcript expression in RF/6A cells. GAPDH served as an internal reference for control. Both siIGFBP-rP1 duplex 1 and 2 significantly inhibited the expression IGFBP-rP1 compared with controls ( # P<0.05). Furthermore, the inhibitory effect of siIGFBP-rP1 duplex 2 was significantly increased compared with siIGFBP-rP1 duplex 1 (*P<0.05). (B) IGFBP-rP1 expression was measured by western blotting and normalized to that of β-actin. The inhibitory effect of siIGFBP-rP1 on the IGFBP-rP1 protein expression was consistent with that of the RNA expression. The membranes were stripped off and probed for the proteins. Data are presented as the mean ± standard deviation of three independent experiments with similar results and calculated as the integrated optical density of IGFBP-rP1 relative to the internal reference. # P<0.01 vs. the blank control group. *P<0.05 vs. the siIGFBP-rP1 duplex 1 group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane M, marker; lane 1, blank control; lane 2, transfection reagent; lane 3, scrambled control siRNA; lane 4, siRNA duplex 1; lane 5, siRNA duplex 2.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, RNA Expression, Standard Deviation, Binding Assay, Marker, Transfection

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected cells and untransfected cells cultured in normoxic conditions for 6, 12, 24, 48 and 72 h. The OD values of transfected cells at 12, 24 and 48 h were significantly higher compared with untransfected cells (*P<0.01). Furthermore, the OD value of siIGFBP-rP1 duplex 2-transfected cells was significantly higher compared with siIGFBP-rP1 duplex 1-transfected cells at 24 h ( # P<0.01). No significant differences were observed among the groups at 6 and 72 h. OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 6, 12, 24, 48 and 72 h, as detected by MTS colorimetric assays. The OD values of hypoxic groups (CoCl 2 and 1% O 2 ) decreased significantly at 12, 24, 48 and 72 h compared with the control group ( # P<0.01). There was no significant difference between the hypoxic groups (P>0.05). The OD value of the siRNA group were significantly increased at 12, 24 and 48 h compared with controls (**P<0.01). Furthermore, the OD values of the transfected cells cultured in hypoxic conditions (CoCl 2 + siRNA group and 1% O 2 + siRNA group) for 12, 24, 48 and 72 h were significantly lower compared with the siRNA group; additionally, the values were significantly higher compared with the hypoxic groups (*P<0.01). No significant differences were identified between the CoCl 2 + siRNA, 1% O 2 + siRNA and control groups (P>0.05). OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; CoCl 2 , cobalt chloride; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Control, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell motility of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 24 h is detected by wound and Transwell assays. (A) Representative images showing that RF/6A cells migrated across the wound boundary to the blank area (light microscopy; magnification, ×400) and (B) passed across the filter toward the lower surface (light microscopy; magnification, ×200). Hypoxia significantly promoted cell mobility compared with the controls ( # P<0.01). siIGFBP-rP1 transfection (siIGFBP-rP1 group) further enhanced cell migration, as compared with untransfected cells cultured in hypoxic conditions (hypoxia group; *P<0.01). The transfected cells cultured in hypoxic conditions (hypoxia + siRNA group) exhibited a significantly increased migration ability compared with the transfected cells cultured in normoxic conditions (siIGFBP-rP1 group; **P<0.01). Values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Light Microscopy, Migration, Standard Deviation, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing stimulates hypoxia-induced tube formation of RF/6A cells. Representative images (inverted phase contrast microscopy; magnification, ×50) demonstrating that RF/6A cells formed capillary-like tube structures within the Matrigel layer in different mediums. RF/6A cells in hypoxic conditions or siIGFBP-rP1-transfected cells significantly formed completely enclosed capillary-like tubes compared with the controls ( # P<0.01 the hypoxia group vs. the control group; *P<0.01 the siIGFBP-rP1 group vs. the control group). Moreover, siIGFBP-rP1 transfection further promoted tube formation in RF/6A cells in the hypoxia + siIGFBP-rP1 group compared with the siIGFBP-rP1 group (**P<0.01). The values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific small interfering RNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Microscopy, Transfection, Control, Standard Deviation, Binding Assay, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing upregulates the hypoxia-induced RAF/MEK/ERK signaling pathway activation and VEGF expression. Representative images and quantified data demonstrated that hypoxic stress upregulated B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A cells compared with controls ( # P<0.05). siIGFBP-rP1 transfection significantly promoted hypoxia-induced B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A compared with the hypoxia group ( # P<0.05). IGFBP-rP1 restoration significantly downregulated the expression of B-RAF, p-MEK, p-ERK and VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions, compared with the siIGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, respectively (*P<0.01). The membranes were stripped off and probed for the proteins. Values are presented as the mean ± SD of 3 independent experiments with similar results and are presented as the integrated optical density of studied proteins relative to GAPDH. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane 1, control; lane 2, siIGFBP-rP1; lane 3, siIGFBP-rP1 + IGFBP-rP1; lane 4, hypoxia; lane 5, hypoxia + siIGFBP-rP1; lane 6, hypoxia + siIGFBP-rP1 + IGFBP-rP1.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Activation Assay, Expressing, Transfection, Binding Assay, Control

Journal: Annals of Translational Medicine

Article Title: High-fat diet-induced obesity primes fatty acid β-oxidation impairment and consequent ovarian dysfunction during early pregnancy

doi: 10.21037/atm-21-2027

Figure Lengend Snippet: Antibodies used for western blotting and immunohistochemistry (IHC)

Article Snippet: The light density and grey level were analyzed and calculated by using ImageJ (Bio-Rad, USA), with β-actin used as a loading control. table ft1 table-wrap mode="anchored" t5 caption a7 Peptide/protein target Species Manufacturer, catalog The concentration of IHC The concentration of Western Blotting Aromatase (CYP19A1) Rabbit AB clonal, ab18995 1:200 1:1,000 Cytochrome P450 family 11 subfamily A member 1 (CYP11A1/P450Scc) Rabbit Boster, BA2120 1:100 1:500 Steroidogenic acute regulatory protein (StAR) Rabbit Bioss, bs-3570R 1:100 1:1,000 Hypoxia-inducible factor 1-alpha (HIF-1α) Rabbit AB clonal, A11945 1:100 1:1,000 Pyruvate dehydrogenase phosphatase1 (PDP1) Rabbit AB clonal, A6332 1:50 1:500 Phosphoinositide-dependent kinase1 (PDK1) Rabbit Boster, BA4499 1:100 1:1,000 Pyruvate dehydrogenase E1 alpha 1 (PDHA1) Rabbit AB clonal, A1895 1:500 1:5,000 Carnitine palmitoyltransferase 1A (CPT1A) Rabbit Proteintech, 15184-1-AP 1:1,000 β-Actin Mouse Boster, BM0005 1:1,000 Open in a separate window caption a8 Antibodies used for western blotting and immunohistochemistry (IHC) Biochemical analyses Blood was centrifuged at 3,000 rotations per minute for 10 minutes under 4 °C to obtain the serum.

Techniques: Western Blot, Immunohistochemistry, Concentration Assay

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Differential location of HMGB1 expression in HDFs and KFs. ( a ) HMGB1 expression levels by immunofluorescence. HMGB1 expression (red) was mainly detected in the HDF nuclei. ( b ) HMGB1 expression was observed in KFs, but was more abundant in the cytosol and extracellular space. ( c and d ) HMGB1 protein expression. The levels of proteins were assessed by western blot. α-tubulin was used as a loading control for cytosol; histone was as a nuclei protein control. Relative levels of HMGB1 in the cytoplasmic fraction were significantly elevated in the KFs. ( e and f ) Stimulation of HDFs with LPS (100 ng/mL) for 24 h and 48 h resulted in increased time-dependent expression of HMGB1 protein in the cell lysate (** p < 0.01). ( g ) Whole cell lysates were fractionated and the levels of HMGB1 in the cytoplasmic and nuclear fractions were determined by western blotting to determine whether LPS treatment induced HMGB1 translocation. HMGB1 translocated from nucleus to cytoplasm in HDFs under LPS treatment (500 ng). Dermal fibroblasts cell line was purchased as primary cell line from the ATCC (American Type Culture Collection, Manassas, VA, USA). Primary keloid fibroblast cell line were obtained from human keloid explant under IRB protocol. Both cells were used for experiment after 2~3 passage.

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Expressing, Immunofluorescence, Western Blot, Translocation Assay

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Role of HMGB1 as a profibrotic molecule. ( a ) An MTT assay revealed a significant increase in time-dependent proliferation activity in the HMGB1-treated HDFs (50 and 100 ng; ** p < 0.01). ( b ) Type I collagen mRNA expression was equivalently increased in the HDFs treated with HMGB1 (100 ng) or TGF-β1 (10 ng) (* p < 0.05, ** p < 0.01). There was also an additive increase of type I collagen mRNA expression after simultaneous treatment with HMGB1 and TGF-β1 ( § p < 0.05). ( c ) Levels of collagen types I and III were detected by western blotting in HDF lysates treated with HMGB1 (100 ng) for 24 and 48 h. ( D ) The protein expression levels of collagen types I and III increased significantly after 24 h (** p < 0.01). ( e) The protein expression level of α-SMA was assessed by western blotting in HDF cells treated with HMGB1 (50 and 100 ng). ( f ) Stimulation of HDFs with HMGB1 resulted in increased protein expression of α-SMA (** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: MTT Assay, Activity Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: HMGB1 induced TGF-β1, Smads, Erk 1/2, Akt, and NF-κb in HDFs. ( a – c ) Effects of HMGB1 on TGF-β1 signaling pathway in vitro . Significant increases in the expression levels of TGF-β1, Smad 2, and Smad 3 mRNA were observed in the HMGB1 (50 ng)-treated HDFs (** p < 0.01). ( d ) Effects of HMGB1 on TGF-β and phosphor-Smad 2/3 complex protein expressions. ( e , f ) TGF-β and phosphor-Smad 2/3 complex protein levels were significantly increased in the HMGB1 (100 ng)-treated HDFs (** p < 0.01). ( g ) Western blotting analysis in normal dermal spheroids. Increased expression of TGF-β and phosphor-Smad 2/3 complex protein were observed in normal dermal spheroids treated with HMGB1 (400 ng and 1000 ng). ( h ) The levels of Erk 1/2, Akt, and NF-κb protein were detected by western blotting in HDFs lysates treated with HMGB1 (50 ng and 100 ng). ( i to k ) Erk 1/2, Akt, and NF-κb protein levels in HMGB1 (100 ng)-treated HDFs were significantly increased by 1.5-, 2.0-, and 2.3-fold, respectively, versus non-treated HDFs (** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: In Vitro, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: HMGB1 reduced MMP 1 and TIMP-1 mRNA expression. HDFs were treated with 50 ng/mL of HMGB1 for various times, and examined using real-time RT-PCR. ( a and c ) MMP-1 mRNA levels were significantly reduced, but TIMP1 mRNA level was increased in 48 hr after treatment with HMGB1 (** p < 0.01). ( b ) MMP-2 mRNA expression increased up to 24 h, but the increase was not statistically significant.

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Effect of HMGB1 on normal dermal spheroids. ( a ) Picrosirius red staining of HMGB1-treated normal dermal spheroids. Collagen deposition increased. The HMGB1 dose dependency of dense and coarse collagen bundles was investigated. ( b ) Semi-quantitative analysis revealed that treatment with 400 and 1000 ng of HMGB1 significantly increased collagen deposition in normal dermal spheroids by 2.9- and 2-fold, respectively, versus non-treated dermal spheroids (** p < 0.01). ( c ) Western blotting analysis of HMGB1-treated normal dermal spheroids for the detection of type-I and -III collagen protein expression level ( d ) Immunohistochemical staining of HMGB1-treated normal spheroid sections for the detection of types I and III collagen, elastin, and fibronectin protein. Increased expression of ECM components (collagen types I and III, elastin, and fibronectin protein) was examined versus nontreated spheroids. Original magnification: 400×. ( e to h ) Semi-quantitative image analysis of ECM protein expression. The expression levels of type I collagen, type III collagen, elastin, and fibronectin increased significantly versus nontreated spheroids (* p < 0.05; ** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Staining, Western Blot, Expressing, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: Immunohistochemical analysis of HMGB1 and its receptors (RAGE and TLR4) in human keloid tissues. ( a ) Higher expression levels of HMGB1 and its receptors (RAGE and TLR4) were observed in the region of the keloid tissues than in the adjacent normal tissue. Original magnification: 400×. ( b to d ) The comparison of HMGB1, RAGE, and TLR4 expression levels between keloid tissues and extra-lesional normal tissue (** p < 0.01).

Article Snippet: The proteins on the gel were electrotransferred to a polyvinylidene fluoride membrane, incubated with the primary mouse anti-HMGB1 monoclonal Ab (ab11354; Abcam, Cambridge, UK), rabbit anti-histone monoclonal Ab (#7631; Cell Signaling Technology, Beverly, MA, USA), or mouse anti-α-tubulin monoclonal Ab (#3873; Cell Signaling Technology, Beverly, MA, USA), and then incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody (6160–05; Southern Bio Technology Associates, Inc., Birmingham, AL, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: Chromosoma

Article Title: COORDINATION OF THE RECRUITMENT OF THE FANCD2 AND PALB2 FANCONI ANEMIA PROTEINS BY A UBIQUITIN SIGNALING NETWORK

doi: 10.1007/s00412-016-0602-9

Figure Lengend Snippet: a Representative immunoblot showing knockdown of MDC1. b–c Representative images (b) and quantification (c) of FANCD2 focus formation in HeLa cells transfected with a control siRNA or two distinct siRNAs targeting MDC1 and treated with 0.5 µM MMC for 16 hr. FANCD2 foci (red) are also shown in merged images in b with DAPI signal in blue to indicate the position of nuclei. d–e Representative images of MMC-induced PALB2 foci (d) in cells transfected with either siLacZ or MDC1 siRNAs and quantification (e) of the percentage of HeLa cells with five or more PALB2 foci. PALB2 foci (red) are also shown in merged images in d with DAPI signal in blue. f Immunoblot showing FANCD2 monoubiquitination status in cells treated with a siRNA directed against RNF8 or MDC1. The intensity of the upper monoubiquitinated band divided by the intensity of the lower unubiquitinated band is shown for each lane. g Quantification of the percentage of HeLa cells with five or more ubiquitin foci following transfection with siRNAs and treatment with MMC. Values in c,e,g represent the mean of three independent counts of at least 150 cells each ± standard deviation; * indicates p<0.005.

Article Snippet: Antibodies Primary antibodies utilized for immunofluorescence microscopy and immunoblotting were as follows: FK2 (EMD Millipore, 04-263), FANCD2 (E35) ( Garcia-Higuera et al. 2001 ), PALB2 ( Zhang et al. 2009a ), γH2AX (EMDMillipore, JBW301), RNF8 (Santa Cruz, sc271462), MDC1 (Novus Biologicals, NB100-395), FAAP20 ( Ali et al. 2012 ), RAP80 (Bethyl Laboratory, A300-763A), and anti-HA (Covance, 16B12).

Techniques: Western Blot, Knockdown, Transfection, Control, Ubiquitin Proteomics, Standard Deviation

Journal: Genes & Development

Article Title: ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair

doi: 10.1101/gad.292516.116

Figure Lengend Snippet: ZMYM3 antagonizes the BRCA1-A complex to promote HR. ( A ) ZMYM3 promotes HR and opposes RAP80 and ABRA1 inhibition of HR. DR-GFP reporter assays were performed after depletion or codepletion of the indicated proteins by siRNAs. Data represent mean ± SD. n = 3. ( B ) Confirmation of knockdown efficiency by Western blotting from experiments performed in A . ( C ) 53BP1 depletion rescues HR defects in ZMYM3-depleted cells. Experiments were performed as in A . ( D ) ZMYM3 knockout cells are sensitive to IR and PARP inhibitors compared with parental U2OS cells. Cells were challenged with IR or PARP inhibitor as indicated and were analyzed by colony formation assays. Data represent mean ± SD. n = 3. ( E ) Western blotting analysis of knockdown efficiency in cells transfected with BRCA1 siRNA. ( F ) Epistasis analysis of ZMYM3 and BRCA1. Wild-type and ZMYM3 knockout cells either alone or in combination with siBRCA1 were challenged with IR and PARP inhibitor followed by colony formation assays. ( G ) Chromosome aberration analyses in ZMYM3 knockout and BRCA1 knockdown cells. Experiments were performed as in E. ( H ) Complementation assay of ZMYM3 knockout cells. ZMYM3 knockout cells with empty vector or wild-type ZMYM3 were analyzed as in D . (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 versus same treatment with control cells, Student's t -test.

Article Snippet: Primary antibodies used in this study were Flag M2 (Sigma, F1804), Myc (Santa Cruz Biotechnology, SC-40), H2AX (Millipore, 07-627), γH2AX (Millipore, 05-636), H2AZ (Cell Signaling, 2718S), macroH2A (Abcam, AB37264), H2A.Bbd (Millipore, 06-1319), ZMYM3 (Abcam, AB106626), ATM (Santa Cruz Biotechnology, SC-135663), pATM S1981 (Abcam, AB81292), β-tubulin (Abcam, AB6046), RAD51 (Abcam, AB88572), RAP80 (Bethyl Laboratories, A300-763A), ABRA1 (Abcam, AB139191), BRCA1 (Santa Cruz Biotechnology, SC-6954), HRP-linked anti-GST (Sigma, A7340), MBP (Abcam, AB9084), phospho-H3 S10 (Cell Signaling, 3377), 53BP1 (Novus Biologicals, NB100-304), RAD18 (Cell Signaling, 9040), RPA2 (Abcam, AB2175), pRPA2 S33 (Bethyl Laboratories, A300-246), pRPA2 S4/S8 (Bethyl Laboratories, A300-245), Chk1 (Santa Cruz Biotechnology, SC-8408), and pChk1 (Cell Signaling, 2348).

Techniques: Inhibition, Knockdown, Western Blot, Knock-Out, Transfection, Plasmid Preparation, Control