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: Molecular Psychiatry

Article Title: SSRIs target prefrontal to raphe circuits during development modulating synaptic connectivity and emotional behavior

doi: 10.1038/s41380-018-0260-9

Figure Lengend Snippet: Lack of SERT increases the number of functional PFC-to-DRN synapses. a rAAV-CAG-hChR2(H134R)-mCherry was bilaterally injected into the PFC of P4–P5 control or SERT-KO mice. Photograph showing mCherry expression after the PFC AAV injection (upper left). Optogenetic stimulation and electrophysiological patch clamp recordings were made starting at P28 in coronal sections containing the DRN, as shown by the photograph of the immunolabeling of PFC mCherry+ axons innervating to DRN 5-HT neurons, identified by the presence of the enzyme TPH2 (upper right). b Amplitude of optogenetically evoked EPSCs (oEPSCs) at synapses from PFC terminals onto DRN putative 5-HT neurons (left) and non-5-HT neurons (right) at various light stimulation intensities. In control (SERT Cre/+ ) (5-HT: n = 10 cells/5 animals; non-5-HT: n = 7 cells/4 animals); in SERT-KO (SERT Cre/Cre ) (5-HT: n = 10 cells/3 animals; non-5-HT: n = 6 cells/3 animals). Top: example traces at 9.8 mW (black/gray) and at 2 mW (red) stimulation); Bottom: input/output curves. Two-way ANOVA on 9.8 mW intensity: genotype x cell-type interaction (F 1,29 = 0.003, p = 0.95); Genotype main effect (F 1,29 = 9.32, * p < 0.01); Cell-type main effect (F 1,29 = 0.51, p = 0.48). c AMPAR/NMDAR ratios at synapses from PFC-to-DRN 5-HT neurons (left) and non-5-HT neurons (right) in control (5-HT: n = 10 cells/4 animals; non-5-HT: n = 7 cells/3 animals), and SERT-KO (5-HT: n = 11 cells/3 animals; non-5-HT: n = 6 cells; 3 animals). The AMPAR responses were calculated at the peak of −50 mV, whereas NMDAR responses were determined at + 40 mV, 50 ms after stimulation. Top: example traces; bottom: bar graphs. Two-ways ANOVA: Genotype x Cell-type interaction (F 1,30 = 0.007, p = 0.94); Genotype main effect (F 1,30 = 0.16, p = 0.69); Cell-type main effect (F 1,30 = 4.51, p < 0.05). Blue bars indicate blue light stimulation. Error bars represent SEM

Article Snippet: Subsequently, after a few hours of fixation in 4% PFA at 4 °C, brain slices were processed for immunohistochemistry using a rabbit polyclonal antibody against TPH2 (1:2000, Novus Biologicals, NB100-74555) to identify 5-HT neurons.

Techniques: Functional Assay, Injection, Control, Expressing, Patch Clamp, Immunolabeling

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Acute effects of AβOs on total levels of proteins that regulate mitochondrial dynamics. (A) Mfn1 and (B) DRP1 western blot of lysates from PC12 cells treated with FCCP (10 μM, 2 h) and AβOs (0.5 μM, 1 and 2 h). A dotted line on the western blot indicates different regions of the same gel. (C, D) Quantification of Mfn1 and DRP1 levels normalized to beta actin. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 compared between the control group. One-way ANOVA with the Dunnett's multiple comparisons test was used for all statistical analyses. Mfn1, mitofusin 1; DRP1, dynamin-related protein 1. (n = 4–5 for each group) (original gel blot are provided on ).

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Acute effects of AβOs on immunoreactivity of proteins that regulate mitochondrial dynamics. Representative epifluorescence images of (A) Mfn1 and (B) DRP1 immunoreactivity in PC-12 cells control and treated with AβOs (0.5 μM) for 1 and 2 h. Quantification of (C) Mfn1 and (D) DRP1 immunoreactivity (intensity), under the same experimental conditions. Scale bars: 20 μm. Data are represented as mean ± SEM. *p < 0.05, ***p < 0.001 compared between the control group. One-way ANOVA with the Dunn's multiple comparisons test was used for all statistical analyses. Mfn1, mitofusin 1; DRP1, dynamin-related protein 1. (n=3–6 for each group, N= 56–103) (entire inmunocytochemistry for Mfn1 and DRP1 with control are provided on ).

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Model of alterations induced on SIRT1/PGC-1α pathway by AβOs. (A) . Healthy neuronal conditions were energy depletion and/or decreased catabolic rates can be sensed by SIRT1 promoting the PGC-1α deacetylation (1). Transcription and co-activates of factors like NRF- 1/2 for the expression of nuclear-encoded mitochondrial genes and dynamic mitochondrial proteins, requires of PGC-1α translocation (2). Coordinated mitochondrial dynamics (3, fission/fusion), depends of adequate expression of Mfn1, Drp1. (B) . In AβOs treated neurons, the PGC-1α is unable to be deacetylated and to translocate to the nucleus (4). The expression of key genes is loss (5), and imbalance between fusion and fission to promote the granular mitochondrial phenotype (6) and neuronal death.

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Expressing, Translocation Assay

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of Drosha, DGCR8, and β-actin in the LM2-DRR (expressing the pLCN DSB Repair Reporter) cell line transduced with DGCR8 shRNA. b Knockdown of DGCR8 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the DGCR8-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. c MYC-DGCR8-overexpressing LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by pulldown with MYC beads and immunoblotting with the indicated antibodies. d Control and DGCR8-knockdown LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by immunoprecipitation with an antibody against RNF168 or RNF8 and immunoblotting with the indicated antibodies. e Chromatin was extracted from LM2 cells that were treated with IR (8 Gy) and cultured for 1 h. The chromatin fractions, with or without MNase treatment, were immunoprecipitated with a DGCR8-specific antibody and immunoblotted with the indicated antibodies. f Quantification of MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DGCR8-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and DGCR8-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Statistical significance in b and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. n.s . not statistically significant. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Western Blot, Expressing, Transduction, shRNA, Knockdown, Cotransfection, Flow Cytometry, Control, Cell Culture, Immunoprecipitation, Incubation, Over Expression, Transfection, Ubiquitin Proteomics, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of USP36, USP51, and β-actin in parental and radioresistant LM2 cells with and without IR treatment (8 Gy followed by 24-h incubation). b Immunoblotting of DGCR8, USP36, USP51, and β-actin in USP36-knockdown and USP51-knockdown LM2 cells with or without IR treatment (8 Gy followed by 24-h incubation). c Co-IP of endogenous DGCR8 with endogenous USP51. LM2 and LM2-R cells were treated with 8-Gy IR. After 8 h, cells were lysed, immunoprecipitated with a DGCR8-specific antibody, and immunoblotted with antibodies against USP51 and DGCR8. SE short exposure, LE long exposure. d HEK293T cells with stable overexpression of MYC-DGCR8 were co-transfected with SFB-USP51 (wild-type or the C372S mutant) and HA-tagged ubiquitin or the lysine-specific mutant (K48 or K63), and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. e Knockdown of USP51 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the USP51-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. f Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in USP51-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and USP51-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. Statistical significance in e and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Western Blot, Incubation, Knockdown, Co-Immunoprecipitation Assay, Immunoprecipitation, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Cotransfection, Flow Cytometry, Expressing, Control, Cell Culture, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a , b MYC-GFP-, WT DGCR8-, S677A-DGCR8-, and S677D-DGCR8-overexpressing LM2 cells with or without IR treatment ( a , 8 Gy followed by 1-h incubation; b , 8 Gy followed by 8-h incubation) were subjected to pulldown with MYC beads and immunoblotting with the indicated antibodies. c HEK293T cells with stable overexpression of MYC-tagged WT DGCR8, S677A-DGCR8, or S677D-DGCR8 were co-transfected with SFB-USP51 (WT or the C372S mutant) and HA-tagged ubiquitin, and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. d Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8, S677A-DGCR8, or S677D-DGCR8. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. e DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8 or the S677A mutant were transduced with FLAG-H2A and RNF8 or RNF168. The cells were then transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Incubation, Western Blot, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation, Knockdown, Expressing, Two Tailed Test, Transduction, Cell Culture, Lysis, Sonication

Journal: PLoS ONE

Article Title: Substrate-Favored Lysosomal and Proteasomal Pathways Participate in the Normal Balance Control of Insulin Precursor Maturation and Disposal in β-Cells

doi: 10.1371/journal.pone.0027647

Figure Lengend Snippet: The Ins2 +/+ β-cells were cultured under the 5.5 mM glucose concentration for a 24-hour pre-experimental period until treatment. (A) The Ins2 +/+ β-cells were treated with cycloheximide (Chx; 100 µg/mL), Chx (100 µg/mL) and 3-MA (5 mM), Baf A1 (5 µM), or chloroquine (Chl; 100 µg/mL) for 30 minutes with an untreated control. Cellular proteins (30 µg) were separated by 16.5% non-reduced (upper panels) or reduced (lower panels) tricine SDS-PAGE and then examined by immunoblotting. (B) The upper panel, the immunoreactive LC3-I/II (%) in individual treatments in (A); the lower panel, the percentages of proinsulin levels on reduced gels (that were normalized by tubulin) in individual treatments compared to the untreated controls. (C) The Ins2 +/+ β-cells subjected to the same treatments described in (A) were immunostained with antibodies against LC3, C-peptide, and insulin as described in the Materials and Methods. Fluorescent Cy2 (for LC3), Cy3 (for C-peptide and insulin), and their merged images were shown. The scale of bar in (C), 10 µm. (D) The relative levels (%) of the LC3 and (pro)insulin and/or C-peptide positive dots per cell of individual treatments in (C). The data in (B) or (D) were reported as mean ± SD. * P <0.05; ** P <0.01, n = 4.

Article Snippet: In this study, we applied antibodies against mouse C-peptide II (made in the lab with help from Proteintech Group, Inc., Chicago, IL, USA), rat C-peptide II (Linco Research Inc., St. Louis, MO, USA), insulin (Dako North America, Inc., Carpinteria, CA, USA), and tubulin (Sigma-Aldrich, St. Louis, Mi, USA), ubiqutin (Sc-8017; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), LC3 (NB100-2331; Novus Biologicals Inc., Littleton, CO), PC2 (kindly provided by Dr. Donald F. Steiner, Chicago), and IAPP antibodies (kindly provided by Dr. P. Westermark, Linköping, Sweden).

Techniques: Cell Culture, Concentration Assay, Control, SDS Page, Western Blot

Journal: Cancer Research

Article Title: Breast Cancer Migration and Invasion Depend on Proteasome Degradation of Regulator of G-Protein Signaling 4

doi: 10.1158/0008-5472.can-08-3564

Figure Lengend Snippet: Figure 6. Endogenous RGS4 protein inhibits breast cancer cell migration and invasion. Columns, mean; bars, SE with *, P < 0.01 and **, P < 0.001. A, quantitative real-time PCR analysis of RGS4 mRNA levels (n = 3). Inset, representative Western blot (n = 5) of RGS4 protein levels. MDA-MB-231 cell lysate plus 0.25 ng purified RGS4 protein as positive control. MDA-MB-231 cells (B) and MDA-MB-436 cells (C) treated for 4 h with or without MG132 (20 Amol/L) F cycloheximide (CHX; 0.1 mmol/L; n = 5) before Western blot analysis (insets) or Transwell migration and invasion assays. D, silencing endogenous RGS4 expression reverses the proteasome blockade-induced inhibition of MDA-MB-231 cell migration and invasion. Cells stably expressing RGS4 shRNA or GFP shRNA were treated with or without MG132 (n = 3) and then subjected to Western blot analysis (top) or Transwell migration and invasion assays (bottom). Untreated cells expressing GFP shRNA were set as 100%.

Article Snippet: Cell-permeable C3 transferase and F-actin visualization kit (Cytoskeleton); NSC23766, MG132, PSI, and chloroquine (Calbiochem); epidermal growth factor (EGF) and fibronectin (BD Biosciences); CXCL12 (R&D Systems); BMS-200261 and TFLLR (Peptides International); SCH79797 (Tocris); cycloheximide (Sigma); rabbit anti-PAR1 antibody (Santa Cruz Biotechnology); and monoclonal anti-hemagglutinin (HA) antibody (Covance).

Techniques: Migration, Real-time Polymerase Chain Reaction, Western Blot, Purification, Positive Control, Expressing, Inhibition, Stable Transfection, shRNA

Journal: Journal of cellular physiology

Article Title: LPA(1) -induced migration requires nonmuscle myosin II light chain phosphorylation in breast cancer cells.

doi: 10.1002/jcp.22631

Figure Lengend Snippet: Fig. 2. The LPA1 receptor is involved in breast cancer cell migration. A: The silencing effects of siRNAs for LPA1, LPA2, and LPA3 receptors. 4T1 cellsweretransfectedwiththeindicatedsiRNAsforLPA1,LPA2,orLPA3receptors(siR)orforluciferaseasacontrol(con).After48 htransfection, RT-PCR was performed as described in Materials and Methods Section. B: Effects of siRNA for LPA1, LPA2, and LPA3 receptors on LPA-induced migrationinwound-healingassays(upperpart).MeasurementofdistanceforLPA-inducedmigrationaftersilencingoftheindicatedLPAreceptor isotypes (lower part). 4T1 cells were transfected with siRNAs for LPA1, LPA2, or LPA3 receptors or LPA1,2,3 receptors together or control siRNA (luciferase). Twenty-four hours after transfection, cells were serum starved for 24 h and then wounded using pipette tips in the presence of LPA. After 12 h, wound size was measured as described in Materials and Methods Section. The size of the wound was measured and expressed as a percentageoftheinitialwoundsizeattime0 h.C:EffectsofsiRNAsforLPA1,LPA2,orLPA3receptorsorLPA1,2,3receptorsoncellmigrationusing transwell chambers. 4T1 cells were transfected with siRNAs for the indicated receptors or control siRNA (luciferase). Twenty-four hours after transfection,cellswereserumstarvedfor24 h.CellmigrationwasperformedinthepresenceorabsenceofLPA.After12 h,LPA-inducedmigrating cellswerecounted.Thesevaluesarerepresentativeofthreeindependentexperiments.D:EffectofLPA1receptorknockdownoncellviability.4T1 cellsweretransfectedwithsiRNAsfortheindicatedLPA1receptorsorcontrolsiRNA(luciferase).After24 hoftransfection,thesecellswereserum starved for 24 h. Cell viability was measured in the presence or absence of LPA for 12 h.

Article Snippet: Protease inhibitor cocktail, phosphatase inhibitor cocktail, MLCK siRNA anti-phospho MYPT (Ser-695) antibody, ROCK siRNA, ROCK antibody, immobilized protein A, horseradish peroxidaseconjugated goat anti-rabbit IgG, and goat anti-mouse IgA, IgM, and IgG were from Santa Cruz (Santa Cruz, CA).

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Luciferase, Transferring

Journal: Journal of cellular physiology

Article Title: LPA(1) -induced migration requires nonmuscle myosin II light chain phosphorylation in breast cancer cells.

doi: 10.1002/jcp.22631

Figure Lengend Snippet: Fig. 6. The effects of ML-7, Y27632 or MYPT-1 on LPA-induced migration. A: After cells were serum starved for 24 h, cells were treated with control (vehicle), Y27632 (5 mM), or ML-7 (25 mM) in the presence or absence of LPA (1 mM). After 12 h, cell migration was counted. Bar graph quantifies LPA-induced migration in the presence or absence of LPA for each condition. These values represent the mean W SE. MP < 0.05 versus vehicle-treated cells. B: 4T1 cells were transfected with the indicated siRNAs for MLCK or luciferase (control). Twenty-four hours after transfection, cells were serum starved for 24 h and then incubated in the presence or absence of LPA. After 12 h, LPA-induced migration was measured. The values in the bar graph represent the mean W SE. MMP < 0.01 versus control (luciferase) siRNA-transfected cells. C: Cells were transfected with the indicated siRNAs for ROCK and luciferase (control). Twenty-four hours after transfection, cells were serum starved for 24 h andthenincubatedinthepresenceorabsenceofLPA.After12 h,LPA-inducedmigrationwasmeasured.Thevaluesinthebargraphrepresentthe mean W SE. MMMP < 0.01 versus control (luciferase) siRNA-transfected cells. D: After 4T1 cells were serum starved for 24 h, cells were treated with serum (10%) or LPA (1 mM) for 10 min. Cells were harvested, lysed, centrifuged, and the supernatants were subjected to SDS 4–12% PAGE. The membranes were probed with the indicated antibodies to pMYPT (Ser-695, Thr-853), MYPT-1, NMHC II-A, II-B and actin. Actin was used as a loading control. Relative intensity of signal was determined using ImageJ (NIH).

Article Snippet: Protease inhibitor cocktail, phosphatase inhibitor cocktail, MLCK siRNA anti-phospho MYPT (Ser-695) antibody, ROCK siRNA, ROCK antibody, immobilized protein A, horseradish peroxidaseconjugated goat anti-rabbit IgG, and goat anti-mouse IgA, IgM, and IgG were from Santa Cruz (Santa Cruz, CA).

Techniques: Migration, Control, Transfection, Luciferase, Incubation

Journal: Journal of cellular physiology

Article Title: LPA(1) -induced migration requires nonmuscle myosin II light chain phosphorylation in breast cancer cells.

doi: 10.1002/jcp.22631

Figure Lengend Snippet: Fig. 7. Both NM II-A and NM II-B play a role in 4T1 cell migration. A: The effects shRNA silencing of NMHC II-A or NMHC II-B in 4T1 cells. After infection with lentivirus, equal amounts of protein in cell lysates were loaded and subsequently immunoblotted with the indicated antibodies to NMHCII-A,II-B,oractinasaloadingcontrol.B:4T1cellswereplatedonpoly-D-lysine-coatedcultureslides.Afterstarvinginserum-freemediafor 24 h, cells were incubated in the presence or absence of LPA for 10 min. NMHC II-A or II-B depleted 4T1 cells were fixed and then immunostained with specific antibodies to NMHC II-A (green) and II-B (red) to detect the myosin isoforms. C: A wound was generated by scratching at time point 0 h. After 12 h, the image of the wound is shown in 4T1 cells stably infected with the indicated shRNAs or control, scrambled shRNA. D: Quantification of LPA-induced migration in 4T1 cells stably infected as described for (C), above. These values represent the mean W SE. E: LPA- mediated invasion using matrigel-coated membranes. These values represent the mean W SE.

Article Snippet: Protease inhibitor cocktail, phosphatase inhibitor cocktail, MLCK siRNA anti-phospho MYPT (Ser-695) antibody, ROCK siRNA, ROCK antibody, immobilized protein A, horseradish peroxidaseconjugated goat anti-rabbit IgG, and goat anti-mouse IgA, IgM, and IgG were from Santa Cruz (Santa Cruz, CA).

Techniques: Migration, shRNA, Infection, Incubation, Generated, Stable Transfection, Control

Journal: Journal of cellular physiology

Article Title: LPA(1) -induced migration requires nonmuscle myosin II light chain phosphorylation in breast cancer cells.

doi: 10.1002/jcp.22631

Figure Lengend Snippet: Fig. 10. The effects of decreasing NM II-A or II-B on LPA-induced migration in MDA-MB-231 cells. A: Measurement of LPA-induced migration afterNMHCII-Aor II-BsiRNA treatment inMDA-MB-231 cells.Migration ofNMHCII-Aor II-BsiRNA treatedcells wasmeasured inthepresence or absence of LPA. These values represent the mean W SE. B: LPA-mediated invasion of NMHC II-A, or II-B siRNA treated cells or control (luciferase) siRNA treated cells using matrigel-coated membranes. These values represent the mean W SE. C: After transfection with indicated siRNAs for control (luciferase), NMHC II-A, and NMHC II-B, cells were treated with LPA for 10 min, harvested and then lysed. The supernatants were subjected to SDS 4–12% PAGE. The membraneswere probed with the indicated antibodies to NMHC II-A, II-B and actin. Actin was used as a loading control.

Article Snippet: Protease inhibitor cocktail, phosphatase inhibitor cocktail, MLCK siRNA anti-phospho MYPT (Ser-695) antibody, ROCK siRNA, ROCK antibody, immobilized protein A, horseradish peroxidaseconjugated goat anti-rabbit IgG, and goat anti-mouse IgA, IgM, and IgG were from Santa Cruz (Santa Cruz, CA).

Techniques: Migration, Control, Luciferase, Transfection

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 2. Effects of integrin β4 knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co-culture system for 24 h. Then, the levels of integrin β4 in the VECs were examined using the immunofluorescence assay. The bar graph shows the relative intensity of integrin β4 in the single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of integrin β4 in VECs. The levels of integrin β4 were determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L integrin β4-specific siRNA or with scramble siRNA for 48 h. After the addition of SPC, the effects of the integrin β4 knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed by the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as the ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 3. Effects of Fyn knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co- culture system for 24 h. Then, the levels of Fyn in the VECs were examined using the immunofluorescence assay. The bar shows the relative intensity of Fyn in single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of Fyn in VECs. The value of Fyn was determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), the cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L Fyn-specific siRNA or with scramble siRNA for 48 h. After the SPC was added, the effects of the Fyn knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed using the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as a ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 4. Effects of integrin β4 knockdown on the changes in Fyn levels in VECs. (A) VECs were treated with 10–40 nmol/L integrin β4 siRNA or scramble siRNA for 48 h. The levels of integrin β4 in the VECs were determined by the immunofluorescence assay, and the bar graph shows the relative fluorescent intensity of integrin β4 per cell as determined by laser scanning confocal microscopy (bP<0.05 vs scramble ctrl, n=3). (B) After 10–40 nmol/L integrin β4 siRNA or scramble siRNA were treated for 48 h, the Fyn levels in the VECs were assessed using western blotting (cP<0.01 vs scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Immunofluorescence, Confocal Microscopy, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 5. Effects of integrin β4 knockdown on NO production in VECs. (A) VECs were cultured under normal conditions or with siRNA for 48 h and stimulated with SPC for 0, 3, 15, or 30 min, and the supernatant was used for the NO level assay using the NO Detection Kit. In the control group (ctrl), the cells were cultured in M199 medium with 0.3% (v/v) ethanol instead of SPC. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA in the presence of SPC. The bar graph shows the changes in NO levels in VECs that had been treated with SPC (bP<0.05 vs ctrl at 3 min, eP<0.05 vs ctrl at 15 min, n=5) or with siRNA in the presence of SPC (hP<0.05 vs scramble ctrl at 3 min, kP<0.05 vs scramble ctrl at 15 min, n=5). (B) The viabilities of VECs were measured by MTT, and no significant changes were observed (n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Transfection

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 6. Effects of integrin β4 knockdown in VECs on changes in ROCK levels in co-cultured VSMCs. VSMCs that had been co-cultured with VECs that were treated with or without integrin β4-specific siRNA (β4) or scramble control siRNA in the presence of SPC were immunostained for ROCK, α-SMA, or both. The bar graph shows the relative fluorescence intensity of ROCK per VSMC as determined by laser scanning confocal microscopy (bP<0.05 vs SPC-non-stimulated VSMCs; eP<0.05 vs SPC- stimulated scramble ctrl in co-cultured system, n=4).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Fluorescence, Confocal Microscopy

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Sequences for all the siRNAs

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Control

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Angptl3 affected the PAN-induced podocyte detachment. (a) The detachment was determined in the podocytes at different doses (50,100,200,250 and 500 ng/ml) of rm-Angptl3 with or without PAN (50 μg/ml, 10 hrs) pretreatment for 24 hrs, it shown that rm-Angptl3 without PAN could not change the attached cells in normal podocytes significantly, while, in podocytes pretreated with PAN, rm-Angptl3 reduced the attached cells markedly, and the more the doses (100-500 ng/ml), the less the attached cells. (b) The rates of attached cells podocytes treated with rm-Angptl3 for different times after PAN pretreatment; the cells decreased from 9 h to 48 h, and the rate of 24 h was the lowest. (c) ELISA results showing weaker secretion of Angptl3 in the medium of Angptl3 siRNA group (lowered by 69.50%); (d) Western blot confirmed that Angptl3 was knocked down by siRNA successfully (reduced by 86.89 ± 3.58%). (e) There was no statistical difference between cell number in the Wt group and control siRNA group with or without PAN pretreatment. Cell number in the control siRNA group and Angptl3 siRNA group were almost the same, while, after PAN pretreatment, cell number in the Angptl3 siRNA group is more than in the control siRNA group. (Wt: wide type; Control siRNA: podocytes transfected with control siRNA; Angptl3 siRNA: podocytes transfected with Angptl3 siRNA. * P <0.05, ** P <0.01, *** P <0.0001) Angptl3, angiopoietin-like3; ELISA, enzyme-linked immunosorbent assay; PAN, puromycin aminonucleoside; siRNA, small interfering RNA.

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Transfection, Small Interfering RNA

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Angptl3 affected apoptosis in PAN-induced podocyte injury model. (a) Flow cytometry analysis of the rate of apoptosis in the cultured podocytes with different treatments. No significant differences in the number of apoptotic cells were observed among control, rm-Angptl3, control siRNA and Angptl3 siRNA groups. After PAN pretreatment, the rate of apoptosis was significantly increased in the rm-Angptl3 group compared to the control group (34.80±7.9 vs. 1.89±0.92 3%, P <0.0001; n=6). And in comparison to the control siRNA group, apoptosis rate in the podocytes treated with Angptl3 siRNA was a little lower (9.29±1.27vs. 17.42±2.61%, P <0.05; n=6). (b, c) This finding was confirmed using TUNEL assay for apoptosis in cultured podocytes. In PAN-induced podocyte injury model, TUNEL positive cells in rm-Angptl3-treating group are significantly more than that without rm-Angptl3 treating (56±9 vs. 8±10 %, P <0.0001; n=6), and TUNEL positive cells in the Angptl3 siRNA group are significantly less than that in control siRNA group (32±4 vs. 54±9 %, P <0.05; n=6) (magnification ×400). (Control: podocytes treated without rm-ANGPTL3; Rm-ANGPTL3: podocytes treated with rm-ANGPTL3. *P<0.05, ***P<0.0001). Angptl3, angiopoietin-like3; PAN, puromycin aminonucleoside; siRNA, small interfering RNA; TUNEL. deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Flow Cytometry, Cell Culture, Control, Comparison, TUNEL Assay, Small Interfering RNA, End Labeling

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Angptl3 influenced the F-actin rearrangement in podocytes. (a) Phalloidin fluorescence labelling of F-actin showed podocyte stress fibers. Confocal microscopy showed no difference in the F-actin staining pattern between control group and Angptl3 siRNA group. However, F-actin rearrangement in Angptl3 group, characterized by the formation of lamellipodia (arrowhead) and increase in cell spikes (arrow). In PAN induced podocyte injury model,cells became retracted and F-actin seemed partial disrupted,moreover, F-actin almost completely disrupted in Angptl3 protein group. However, disruption did not occur in Angptl3 knockdown group. (b) The percentage of stress fibers also showed that Angptl3 protein could significantly aggravate the effect of PAN on decreaseing podocyte stress fibers while Angptl3 siRNA could prevent PAN from decreasing podocyte stress fibers. (c) Quantitative analysis showed the MFI of F-actin in different groups under confocal microscopy. After PAN treatment, MFI value in the Angptl3 siRNA group is higher than in the control siRNA group. (Scale bar, 5 μm * P <0.05, ** P <0.01. n=100). Angptl3, angiopoietin-like3; MFI, mean fluorescence intensity; PAN, puromycin-aminonucleoside; siRNA, small interfering RNA.

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Fluorescence, Confocal Microscopy, Staining, Control, Disruption, Knockdown, Small Interfering RNA

Journal: BMC Nephrology

Article Title: A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro

doi: 10.1186/s12882-015-0034-4

Figure Lengend Snippet: Angptl3 orchestrated integrin α3β1, ILK and p53, rather than caspase 3 in podocytes on treatment with PAN. (a) Western blots were performed on the expression of integrin α3, total integrin β1, phospho-integrinβ1, and ILK in podocytes with different treatment, it was showed that, there was no significant difference of in the observed molecular expression in podocytes without PAN pretreatment, while, after treatment with PAN, expression of integrin α3 and total integrin β1 became lower (ILK and phospho- integrin β1, higher) in the rm-Angptl3 group than that in the control group, and Angptl3 siRNA group showed higher expression of integrin α3, total integrin β1 (ILK, lower) than in control siRNA group, while, phospho- integrinβ1 in the two group had no statistical difference. (b, c, d, e) Relative levels of integrin α3, total integrin β1, phospho- integrinβ1 and ILK. (f, g) Western blots suggesting that there was rarely no activated caspase 3 in any observed groups; with PAN pretreatment, p53 protein level was markedly increased in rm-Angptl3 group with comparison to the control group, while, p53 protein level in Angptl3 siRNA group was a little lower than that in the control siRNA group, no p53 protein was detected in any other groups. (*P<0.05, **P<0.01, ***P<0.0001 n=6). Angptl3, angiopoietin-like3; ILK, integrin-linked kinase; PAN, puromycin aminonucleoside; siRNA, small interfering RNA.

Article Snippet: The antibodies and reagents used in the present study are listed with their sources in parentheses: mouse monoclonal antibody (mAb) to glyceraldehyde-phosphate dehydrogenase (GAPDH), rabbit polyclonal antibody to Angptl3, integrin alpha 3, total integrin beta 1, phospho-integrin beta 1 (phospho T789) and ILK; anti-active + pro- caspase 3 antibody (Abcam, Cambridge, UK); p53 mouse mAb (Cell Signaling Technology, Beverly, USA); recombinant mouse Angptl3 protein (rm-Angptl3) (R&D Systems, Minneapolis, USA); PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, USA); In Situ Cell Death Detection kit (Roche, Mannheim, Germany); and fluorescein-labelled phalloidin (Sigma Aldrich, St. Louis, USA).

Techniques: Western Blot, Expressing, Control, Comparison, Small Interfering RNA