Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 4. mTORC1 activation is an upstream event in palmitate induced ATF4 activation. A: AML12 cells were pretreated with or without Torin1 (0.25 mM) or rapamycin (Rapa at 50 nM) for 2 h before palmitate (0.4 mM) exposure for 16 h. Total protein was extracted. Protein abundance of ATF4 and actin were detected by Western blotting. The signal of ATF4 protein band was measured by densitometry and then divided by the sig- nal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.001 vs. control). B: AML12 were pretreated with Torin1 (0.25 mM) for 2 h before a 16-h palmitate (0.4 mM) exposure. Total RNA was extracted. ATF4 mRNA levels were detected by real time-qPCR. Data are expressed as means ± SD, n = 4 sep- arated experiments. Differences between the two groups were deter- mined using Student’s t test (P < 0.001 vs. control). ATF, activating transcription factor.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Quantitative Proteomics, Western Blot, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 5. mTORC1 activation contributes to palmi- tate-induced ER stress and NNMT upregulation. A and B: AML12 cells were pretreated with Torin1 (0.25 mM) for 2 h before the palmitate (0.4 mM) exposure for 16 h. Total RNA was extracted. The gene expres- sions of Xbp1, Xbp1s, and Xbp1u were quantified by real time-qPCR and Xbp1s/Xbp1u ratio calculated. Data are expressed as means ± SD, n = 4 different experiments. Differences between the two groups were determined using Student’s t test (P < 0.001vs. control). C: AML12 cells were pretreated with Tornin1 for 2 h before tunicamycin (10 μm) treat- ment for 16 h. Protein abundance of p-S6 and actin was detected by Western blotting. The signal of p-S6 protein band was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 5 separate experiments. Student’s t test was used for statistical evaluation (P < 0.01; P < 0.0001 vs. control). D: Ten- week-old male C57BL/6N mice were injected with tunicamycin (2 mg/kg body wt ip) or isovolumic vehi- cle (150 mM dextrose) and 16 h later livers were har- vested. Protein abundance of ATF4, p-S6 and actin was detected by Western blotting. E: AML12 cells were pretreated with Torin1 (0.25 mM) for 2 h before tunicamycin (10 μm) treatment for 16 h. Protein abun- dance of ATF4 was detected by Western blotting. F: AML12 cells were pretreated with Torin1 for 2 h before tunicamycin (10 μm) treatment for 16 h. Total RNA was extracted and NNMT gene expression quantified by real time-qPCR. All data were expressed as means ± SD, n = 4 separated experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001; P < 0.0001 vs. control). NNMT, nicotinamide N-methyl- transferase; XBP1, X-box binding protein 1.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Control, Quantitative Proteomics, Western Blot, Injection, Gene Expression, Binding Assay

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 6. NNMT inhibition protects against palmitate-induced cell death. A: AML12 cells were pretreated with either JBSNF-000088 (20 mM) or II399 (20 mM) at the indicated concentrations for 4 h before palmitate (0.4 mM) exposure for 16 h. Cell viability was determined by LDH release mea- surement. Data are expressed as mean ± SD, n = 3 separated experi- ments. Bars with different character differ significantly (P < 0.05). B: AML12 cells were transfected with either scramble siRNA or NNMT siRNA for 24 h and treated with palmitate at 0.4 mM for 16 h. Cell death was determined by LDH release measurement. Data are expressed as means ± SD, n = 3 separated experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001; P < 0.001 vs. control). NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Inhibition, Transfection, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 7. Protein kinase A (PKA) inhibition compro- mises the protective effect of NNMT inhibition against palmitate-induced cell death. A and B: AML12 cells were treated with either JBSNF-000088 (25 mM) or II399 (25 mM) for 6 h. Total proteins were isolated and PKA substrates detected by Western blotting. The signal of PKS substrates was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.05; P < 0.01 vs. untreated cells). C: AML12 cells were pretreated with either JBSNF-000088 (25 mM) or II399 (25 mM) at the pres- ence/absence of PKA inhibitor, either SQ22536 (200 mM) or H89 (10 mM) for 4 h before palmitate exposure for 16 h. Cell death was determined by LDH release. All data are expressed as means ± SD, n = 3 sepa- rated experiments. Differences between the two groups were determined using Student’s t test (P < 0.05; P < 0.01; P < 0.001 vs control). D: sche- matic illustration of the role and mechanism of NNMT upregulation in palmitate-induced hepatocyte lipotox- icity. The mTORC1-ATF4 pathway activation contrib- utes to palmitate-elicited NNMT upregulation and protein kinase A (PKA) activation contributes to NNMT inhibition-conferred protection against hepatolipotox- icity. NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Inhibition, Isolation, Western Blot, Control, Activation Assay

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 1 Sema3A-overexpressing adenovirus plasmids contributed to decreased keratinocyte migration and proliferation. A Transfection efficiency was confirmed by qRT-PCR analysis in Hacat and NHEK cells. Bars indicate the mean fold changes ± SEM relative to the control; n = 4. B The effect of Sema3A adenovirus plasmids on the proliferation potential of Hacat cells was analysed by CCK-8 and Colony formation experiments. Data are shown as means ± SEM; n = 4. C The effect of Ad-Sema3A on the proliferation potential of NHEK cells was analysed by CCK-8. Data are shown as means ± SEM; n = 4. D Wound healing assays were performed in Ad-Sema3A or si-Sema3A-transfected Hacat and NHEK cells. The percentage of wound closure is displayed as the mean ± SEM; n = 3. E Transwell assays showed that transfection with adenovirus Seam3A restrained the migratory ability, while Sema3A inhibition reversed this effect. Bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. F Angiogenesis in HUVEC during the incubation with supernatant gathered from Sema3A- or si- Sema3A-transfected keratinocytes. G Western blotting analysis of EMT markers in Hacat cells transfected with Ad-Sema3A, si-Sema3A and the relative control. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Transfection, Quantitative RT-PCR, Control, CCK-8 Assay, Inhibition, Incubation, Western Blot

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 2 Sema3A transfection suppressed TGF-β1-induced keratinocyte migration in a NRP1-dependent manner. A Western blotting analysis of Sema3A and EMT markers after exposure to escalated concentrations of TGF-β1 in Hacat and NHEK cells. B Wound healing experiment of incubation with TGF-β1. Data are shown as means ± SEM; n = 3. C Sema3A adenovirus plasmids were transfected into keratinocytes in the absence or presence of TGF-β1. The expression of EMT markers and the phosphorylation of Smad2/3 were shown by western blotting. Wound healing (D) and Transwell (E) assays in transfected Ad-Sema3A keratinocytes in the absence or presence of TGF-β1. The percentage of wound closure is displayed as the mean ± SEM; n = 3. For the transwell assays, bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. F EMT-related proteins were determined in Ad-Sema3A ± si-NRP1-transfected cells with or without TGF-β1. Phenotypic alterations were verified by wound healing (G) and transwell (H) assays. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Migration, Western Blot, Incubation, Expressing, Phospho-proteomics, Control

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 3 Ad-Sema3A transfection suppressed activation of EGFR/ERK axis. A Ad-Seam3A plasmids were transfected into NHEK cells for 48 h. Then, recombinant EGF protein was added to the transfected cells for 15 min. Sema3A, p-EGFR and p-ERK were analysed by western blot. B Wound healing and Transwell assays in transfected Sema3A plasmids in the absence or presence of EGF. C Recombinant EGF protein was incubated in the Ad-Sema3A- or NC-transfected cells for 2 days, and immunoblotting analysis is displayed. D Transfection of short peptides interfering with Sema3A function in keratinocytes, treatment with the EGFR signal inhibitor erlotinib, and testing of the protein expression of EMT markers. E Cells treated with si-Sema3A ± erlotinib were plated in the chamber, and the migration capacity was assessed. Bars indicate the fold changes ± SEM relative to the negative control. F The ERK-specific inhibitor U0126 was introduced into NHEKs. Western blot analysis showed that U0126 attenuated the EMT process mediated by TGF-β1 and that Sema3A deficiency enhanced the protein expression of mesenchymal markers triggered by U0126. G qRT-PCR analysis was performed to confirm the expression level of transcriptional factors including Sema3A, NRP1, GATA-1, CEBPA, XBP1, TP53, CEBPB and TCF4 in Hacat cells. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Activation Assay, Recombinant, Western Blot, Incubation, Expressing, Migration, Negative Control, Quantitative RT-PCR

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 4 Loss of Sema3A delayed cutaneous wound healing in vivo. A Schematic representation of the wound-healing studies performed in K14-CreTM+;Sema3AL/L and K14-CreTM-;Sema3AL/L mice. B qRT-PCR analysis of epidermal Sema3A mRNA after tamoxifen induction. GAPDH served as control. Bars indicate the mean fold changes ± SEM relative to the control (K14-CreTM-;Sema3AL/L group); n = 3. C Quantification of the wound closure area at different time points after wounding in the exp (K14-CreTM+;Sema3AL/L) and con (K14-CreTM-;Sema3AL/L) groups. Data are shown as means ± SEM; n = 6. D Representative macroscopic illustration of wound healing in exp and con animals at Days 0, 4, 7 and 14. E H&E-stained sections of wounds used for morphometric analysis of the percentage of wound closure (length of newly formed epithelium (NFE)/length of NFE + length of gap between edges of wound epithelium (red dotted line) × 100) and re-epithelialization (length of NFE). White asterisk (*) indicates the proliferative connective tissue in the control group. Scale bar = 200 µm. F Quantification of the percentage of Sema3A/ZEB2 + area of the epithelial and granulation tissue at different time points. G Quantification of the percentage of wound re- epithelialization at Days 7 and 14 after wounding in K14-CreTM+;Sema3AL/L and K14-CreTM-;Sema3AL/L wounds. Data are shown as means ± SEM; n = 6. H Comparison of the healing times (scab falling off) in the days after wounding. Data are shown as means ± SEM; n = 6. I Comparison of connective tissue in control and Sema3A cKO mice. Data are shown as means ± SEM; n = 6. J Thickness of crust after injury. Bars indicate the mean fold changes relative to con (K14-CreTM-;Sema3AL/L) ± SEM; n = 6. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: In Vivo, Quantitative RT-PCR, Control, Staining, Comparison

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 5 Enhancement of keratinocyte migration upon Rb-Sema3A treatment. Epithelial cells extracted from the injured (Day 4) margin of K14-CreTM+;Sema3AL/L and K14-CreTM-;Sema3AL/L mice were cultured, and the proliferation and migration capacity was determined by CCK-8 (A), wound healing (B) and Transwell assays (C) in vitro. D Morphology of keratinocytes. Immunofluorescence staining of F-actin in cells from the K14-CreTM+;Sema3AL/L (exp) and K14-CreTM-;Sema3AL/L (con) groups. White arrows point to spindle morphological alterations in the control group. E Western blot analysis of EMT markers by Day 4 after injury. Lysates were extracted from the injured margins of sema3A cKO or control mice. F The effect of Rb-Sema3A on the proliferation potential of Hacat cells was analysed by CCK-8. Data are shown as means ± SEM; n = 4. G Wound healing assays were performed in Rb-Sema3A-incubated Hacat and NHEK cells. The percentage of wound closure is displayed as the mean ± SEM; n = 3. H Transwell assays showed that incubated with recombinant Seam3A enhanced the migratory ability of NHEK cells. Bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. I Two 8-mm excisional wounds were created on the back of each 7–8-week-old BALB/c nude mouse. Sema3A-transfected Hacat cells or recombinant Sema3A proteins as well as the relative control were injected subcutaneously in the margin (2 mm from the incision) of the wound in nude mice. Photographs were taken at Days 0, 4, 7, 14 and 21. The thickness of the connective tissues (J), time of scar falling (K) and area of wound are displayed (L, M). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Cell Culture, CCK-8 Assay, In Vitro, Staining, Control, Western Blot, Incubation, Recombinant, Transfection, Injection

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 6 Successive recruitment of Sema3A and NRP1 proteins during the process of wound healing. A Schematic representation of the wound-healing studies performed in K14-CreTM+;Sema3AL/L and K14-CreTM-;Sema3AL/L mice. B qRT-PCR analysis of epidermal Sema3A mRNA after tamoxifen induction. GAPDH served as control. Bars indicate the mean fold changes ± SEM relative to the control (K14-CreTM-;Sema3AL/L

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Control

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 7 Interaction between NRP1 and EGFR signaling in keratinocytes. A Detection of the EGFR-ERK pathway and EMT inducers by western blotting in Hacat cells incubated with recombinant Sema3A and EGF for 48 h. B Protein levels of NRP1, EGFR, p-EGFR, ERK and p-ERK in cells transfected with si-NRP1. C Localization of NRP1 and EGFR proteins in Hacat cells. Scale bar = 20 µm. D Co-IP experiment between NRP1 and EGFR. IP: NRP1. WB: EGFR. E EGFR- and NRP1-overexpressing Hacat cells were treated with cycloheximide (CHX) for the indicated time periods to inhibit de novo protein synthesis. As a control, MG132 was added to block the catalytic activity. F si-NRP1 and EGFR plasmids were cotransfected into NHEK cells for 48 h. Protein levels of NRP1, EGFR, p-EGFR, ERK and p-ERK were determined by western blot. G NHEK cells were transfected with si-NRP1 plasmids for 2 days before EGF (50 ng/ml) stimulation. Then the IF analysis of EGFR or NRP1 was showed. Scale bar = 20 µm. H NHEKs were stimulated with EGF (100 ng/ml) for the indicated periods of time. IFs were subsequently conducted in the resulting cells to monitor EGFR and NRP1 localization/expression. Scale bar = 20 µm.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Incubation, Recombinant, Transfection, Co-Immunoprecipitation Assay, Control, Blocking Assay, Activity Assay, Expressing

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 8 Synergetic effect of Rb-EGF and Rb-Sema3A through EGFR/ERK signaling regulated by NRP1. A NHEKs were serum starved and subsequently stimulated with Rb-Sema3A for 5 min to 1 h and subjected to IF analyses. B Combined treatment with Rb-EGF and Rb-Sema3A was utilized in keratinocytes at the indicated time points. EGFR and NRP1 localization/expression was shown by IF staining. Wound healing assays (C) and Transwell assays (D) were performed in Rb-Sema3A-,Rb-EGF and EGFR plasmids incubated in Hacat cells after transfected with si-NRP1 or negative control. The percentage of wound closure is displayed as the mean ± SEM; n = 3. Bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. E Western blot analysis of EGFR-ERK pathway after treatment at the indicated time points. F Immunofluorescence in Hacat cells. si-NRP1 or si-NC plasmids were transfected in EGFR-overexpressing cells. Recombinant EGF and Sema3A cytokines were added in cells to test the process of NRP1 and EGFR activation and degradation. Scale bar = 20 µm. G A schematic of the proposed mechanism. **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Staining, Incubation, Transfection, Negative Control, Control, Western Blot, Recombinant, Activation Assay

Journal: Bioactive materials

Article Title: Calcium phosphate-based materials regulate osteoclast-mediated osseointegration.

doi: 10.1016/j.bioactmat.2021.05.003

Figure Lengend Snippet: Fig. 5. Effect of Ca/P ratio in CPC on RANKL-RANK signaling. (a) Immunofluorescence analysis of RANK expression (red) in osteoclasts on surface of CPC scaffold. (b) Concentration of RANK was analysis by ELISA. The concentration of RANK was measured after inducing BMMs for 1 days. (c) Affinity between RANKL and RANK was measured in BIAcore T200 system. Human-RANK anchored on the chip was applied to interact with the human-RANKL dissolved into the CPC extract. The vehicle means MEM-α medium. (d) 1.67CPC promoted RANKL-induced phosphorylation of NF-κB p65, and degradation of IκBα. (e) p-p65 and (f) IκBα intensity of Western blot bands was calculated with normalizing to β-actin. The untreated medium was treated as control. (g–l) Relative mRNA expression of osteoclast was measured after inducing BMMs for 7 days in CPC extract. BMMs cultured with untreated medium was treated as control. (*P ＜ 0.05, **P ＜ 0.01,***P ＜ 0.001).

Article Snippet: The concentration of RANK was measured by Mouse RANK ELISA Kit (Boster Biological Technology, China), according to the manufacturer’s instructions and the total protein concentration was measured by BCA Protein Assay Kit (Beyotime, China), which was used to normalize the expression of RANK.

Techniques: Immunofluorescence, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control, Cell Culture

Journal: Frontiers in cellular neuroscience

Article Title: CB 1 Cannabinoid Receptors Stimulate Gβγ-GRK2-Mediated FAK Phosphorylation at Tyrosine 925 to Regulate ERK Activation Involving Neuronal Focal Adhesions.

doi: 10.3389/fncel.2020.00176

Figure Lengend Snippet: FIGURE 5 | CB1-stimulated FAK phosphorylation at tyrosine 925 and ERK2 phosphorylation at tyrosine 204 are mediated by Gβγ and GRK2 in N18TG2 cells. (A–D) Cells were pretreated with the Gi/o inhibitor pertussis toxin (100 ng/mL, 20 h), Gβγ inhibitor gallein (10 µM, 15 min), or GRK2 inhibitor (1 µM, 15 min) before treatment with 0.01 µM WIN55212-2 (WIN) for 1 or 2 min (m) at 37◦C. Cell lysates were analyzed using western blots and representative blot images are shown. Data are reported as mean ± SEM of (B) % of vehicle-treated FAK 925 Tyr-P (normalized to total FAK levels) at the same time point, (C) % of vehicle-treated ERK2 204 Tyr-P (normalized to total ERK2 levels) at the same time point, and (D) % of basal/time 0 ERK2 204 Tyr-P (normalized to total ERK2 levels). Significance was assessed using One Way ANOVA followed by Dunnett’s multiple comparisons posthoc test [*p < 0.01, #p < 0.05 indicates significantly different from vehicle-treated (at the same time point); **p < 0.05 indicates significantly different from basal/time 0]. (E–H) Cells (2 × 105) were transfected with no siRNA (mock transfection), GRK2-specific siRNA (100 nM), or negative control siRNA (100 nM) before treatment with 0.01 µM WIN for 2 min at 37◦C. Immunoblot analysis was performed and data are reported as mean ± SEM of the % change over basal (G) FAK 925 Tyr-P (normalized to total FAK levels) and (H) ERK2 204 Tyr-P (normalized to total ERK2 levels). Significance was assessed using One Way ANOVA followed by Dunnett’s multiple comparisons posthoc test (*p < 0.01, #p < 0.05 indicates significantly different from GRK2 siRNA at the same time point). For each dataset, cells were cultured and experiments were completed on at least three separate occasions.

Article Snippet: N18TG2 cells were transfected with 100 nM GRK2-specific siRNA (mouse) or negative control siRNA-A using siRNA transfection reagent according to the manufacturer’s protocol (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot, Transfection, Negative Control, Cell Culture

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: Autophagy

Article Title: Autophagy-mediated HMGB1 release antagonizes apoptosis of gastric cancer cells induced by vincristine via transcriptional regulation of Mcl-1.

doi: 10.4161/auto.8.1.18319

Figure Lengend Snippet: Figure 2. Vincristine-induced autophagy protects gastric cancer cells from apoptosis. (A and B) BGC-823 and SGC-7901 cells were treated with 3-MA (5 mM) or z-VAD-fmk (30 mM) for 1 h before the addition of vincristine (50 mg/ml) for 24 h (A) or 48 h (B). Cells were then stained with acridine orange for quantitation of AVO (A), or with Annexin V and PI for quantitation of apoptotic cells (B), in flow cytometry analysis. The data shown are the mean ± SE of three individual experiments. (C) BGC-823 cells were transfected with the control or BECN1 siRNA. Twenty-four hours later, whole cell lysates were subjected to western blot analysis of Beclin 1 and b-actin (as a loading control) (upper panel). Lower panel, 24 h later, cells were treated with vincristine (50 mg/ml) for 48 h. Apoptosis was quantitated by staining with Annexin V and PI in flow cytometry. The data shown are either representative (upper panel), or the mean ± SE (lower panel), of three individual experiments. (D) BGC-823 cells were transfected with the control or ATG5 siRNA. Twenty-four hours later, whole cell lysates were subjected to western blot analysis of Atg5 and b-actin (as a loading control) (upper panel). Twenty-four hours later, cells were treated with vincristine (50 mg/ml) for 48 h (lower panel). Apoptosis was quantitated by staining with Annexin V and PI in flow cytometry. The data shown are either representative (upper panel), or the mean ± SE (lower panel), of three individual experiments.

Article Snippet: Small interfering RNAs (siRNA) targeting human Beclin 1 (6222), Atg5 (6345), Mcl-1 (6315) or control siRNA (6568) were from Cell Signaling Technology. siRNAs targeting human HMGB1 (L-018981-00), TLR2 (L-005120-01), TLR4 (L-008088-01), RAGE (L-00362500) and control siRNA (D-001810-10) were from Dharmacon. siRNA duplexes were transfected into cells using INTERFERin (Polyplus-transfection, 409-10) according to the standard protocol.

Techniques: Staining, Quantitation Assay, Flow Cytometry, Transfection, Control, Western Blot

Journal: Autophagy

Article Title: Autophagy-mediated HMGB1 release antagonizes apoptosis of gastric cancer cells induced by vincristine via transcriptional regulation of Mcl-1.

doi: 10.4161/auto.8.1.18319

Figure Lengend Snippet: Figure 6. RAGE mediates HMGB1 signaling to transcriptionally upregulate Mcl-1. (A) BGC-823 cells were transfected with the control or RAGE siRNA. Twenty-four hours later, whole cell lysates were subjected to western blot analysis of RAGE and b-actin (as a loading control) (upper panel). Twenty-four hours later, cells were treated with rHMGB1 (10 mg/ml) or vincristine (50 mg/ml) for a further 24 h (left panel). Total RNA was subjected to qPCR analysis of Mcl-1 mRNA abundance. The level of Mcl-1 mRNA before treatment was arbitrarily designated as 1. The data shown are either representative (upper panel), or the mean ± SE (lower panel), of three individual experiments. (B) BGC-823 cells were transfected with the control or TLR2 siRNA. Twenty-four hours later, whole cell lysates were subjected to western blot analysis of TLR2 and b-actin (as a loading control) (upper panel). Twenty-four hours later, cells were treated with rHMGB1 (10 mg/ml) or vincristine (50 mg/ml) for a further 24 h (lower panel). Total RNA was subjected to qPCR analysis of Mcl-1 mRNA abundance. The level of Mcl-1 mRNA before treatment was arbitrarily designated as 1. The data shown are either representative (upper panel), or the mean ± SE (lower panel), of three individual experiments. (C) BGC-823 cells were transfected with the control or TLR4 siRNA. Twenty-four hours later, whole cell lysates were subjected to western blot analysis of TLR4 and b-actin (as a loading control) (left panel). Twenty-four hours later, cells were treated with rHMGB1 (10 mg/ml) or vincristine (50 mg/ml) for a further 24 h (right panel). Total RNA was subjected to qPCR analysis of Mcl-1 mRNA abundance. The level of Mcl-1 mRNA before treatment was arbitrarily designated as 1. The data shown are either representative (left panel), or the mean ± SE (right panel), of three individual experiments. (D) BGC-823 cells were transfected with the control, RAGE, TLR2 or TLR4 siRNA. Twenty-four hours later, cells were treated with vincristine (50 mg/ml) for a further 48 h (left panel) or 24 h (right panel). Apoptosis was quantitated by staining with Annexin V and PI in flow cytometry (left panel). Formation of AVO was quantitated by staining with acridine orange in flow cytometry (right panel). The data shown are the mean ± SE of three individual experiments.

Article Snippet: Small interfering RNAs (siRNA) targeting human Beclin 1 (6222), Atg5 (6345), Mcl-1 (6315) or control siRNA (6568) were from Cell Signaling Technology. siRNAs targeting human HMGB1 (L-018981-00), TLR2 (L-005120-01), TLR4 (L-008088-01), RAGE (L-00362500) and control siRNA (D-001810-10) were from Dharmacon. siRNA duplexes were transfected into cells using INTERFERin (Polyplus-transfection, 409-10) according to the standard protocol.

Techniques: Transfection, Control, Western Blot, Staining, Flow Cytometry