Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: The Cnot genes maintain self-renewal by repressing early trophectoderm (TE) transcription factors. (A): Cnot1, Cnot2, and Cnot3 knockdown did not immediately affect known self-renewal factors and pathways. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) in M15 medium. Cells were collected 48 hours after transfection, and total Stat3, Smad1, b-Catenin as well as phospho-Stat3, phospho-Smad1, phosphor-b-Catenin, Oct4, and Nanog levels were determined by Western blot. Starved: control-transfected ESCs cultured in serum-free and LIF-free medium for additional 4 hours. (B): Comparing gene expression changes caused by perturbations of known self-renewal factors: Cnot1, 2, and 3 silencing induced similar changes to those of Oct4 or Sox2 silencing. Pearson's correlation coefficients were calculated between microarray datasets and depicted in a heatmap. The self-renewal factors were clustered by unsupervised hierarchical clustering based on the correlation coefficients. Microarray datasets used for this plot are listed in Supporting Information Table 2. (C): Cnot2 or Cnot3 overexpression cannot rescue Oct4 or Sox2 silencing-induced differentiation. Oct4GiP cells and Oct4GiP cells overexpressing Cnot2 (Cnot2-Rescue, same as in Fig. 1C) or Cnot3 (Cnot3-Rescue, same as in Fig. 1C) were transfected with control, Oct4 (Oct4-KD), or Sox2 (Sox2-KD) siRNAs, and the % differentiation was determined by the Oct4GiP reporter assay. (D): Cnot1, Cnot2, and Cnot3 knockdown induced TE differentiation in the presence of sustained Oct4 expression. ZHBTc4 cells that constitu-tively express Oct4 at the normal level from a Tet-Off promoter were transfected with control or Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), Cnot3-siRNA2 (Cnot3-KD), and the expression of TE markers Cdx2 and Gata3 was determined by qRT-PCR after 4 days. (E): Cdx2 deletion partially rescued Cnot1, Cnot2, and Cnot3 silencing-induced differentiation. Oct4GiP (WT) or dKO23-5 (Cdx2-/- ) cells were transfected with Control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD), and the expression of lineage markers was determined by qRT-PCR 96-hour after transfection. Abbreviations: ESC, embryonic stem cell; KD, Knockdown; WT, wild type.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Transfection, Western Blot, Cell Culture, Expressing, Microarray, Over Expression, Reporter Assay, Quantitative RT-PCR

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Silencing Cnot1, Cnot2, or Cnot3 led to mouse embryonic stem cell (ESC) differentiation. (A): Silencing Cnot1, Cnot2, or Cnot3 resulted in ESC differentiation based on the Oct4GiP reporter assay. Oct4GiP ESCs were transfected with indicated siRNAs (two different siR NAs for each CCr4-Not complex gene) in M15 medium and cultured for 4 days. The percentage of differentiated cells (% differentiation) was determined by measuring the percentage of green fluorescent protein-negative cells by fluorescence-activated cell sorting (FACS) at the end of the culture. (B): Expression of siRNA-resistant Cnot2 or Cnot3 rescued the differentiation caused by Cnot2 or Cnot3 knockdown, respectively. Oct4GiP cells or Oct4GiP cells expressing siRNA-resistant Cnot2 (Cnot2-Rescue) or Cnot3 (Cnot3-Rescue) were transfected with Control, Cnot1-siRNA1, Cnot2-siRNA2, or Cnot3-siRNA2, and the percentage of differentiated cells was determined by the Oct4GiP reporter assays. Note that Cnot2-Rescue cells were not able to rescue the differentiation caused by Cnot1 or Cnot3 silencing, and Cnot3-Rescue cells were not able to rescue Cnot1 or Cnot2 silencing. ***, p < .001. (C): Silencing Cnot1, Cnot2, or Cnot3 resulted in morphological changes and loss of alkaline phosphatase (AP) staining in ESCs. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were stained with the AP staining kit and imaged 4 days after transfection. (D): Cnot1, Cnot2, or Cnot3 silencing led to downregulation of ESC marker and upregulation of differentiation markers. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were harvested for quantitative real-time PCR (qRT-PCR) analysis 4 days after transfection. ESC marker: Oct4; differentiation markers: Cdx2, Eomes, Gata3, Hand1, and Krt8. (E): Cnot1, Cnot2, or Cnot3 silencing reduced cell proliferation or viability in 2i medium. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) and cul tured in 2i medium. Cell numbers were counted by FACS 4 days after transfection and normalized to control-transfected cells. (F): Cnot1, Cnot2, or Cnot3 silencing led to differentiation in 2i medium. Oct4GiP cells were transfected with indicated siRNAs and cultured in 2i medium. Cells were harvested for qRT-PCR analysis 4 days after transfection. (G): Expression of C-terminally HA-tagged Cnot2 (Cnot2-HA) in E14Tg2a cells. Expression of the exogenous Cnot2-HA was detected in Western blot with the HA-antibody, and Ran was used as a loading control. Expression of total (endogenous and exogenous) Cnot2 was determined by qPCR in wild-type E14Tg2a (E14) and Cnot2-HA expressing cells. The expression of the Cnot2-HA was estimated to be ∼2-fold of the endogenous Cnot2 on the mRNA level. (H): Identification of Cnot1 and Cnot3 in Cnot2-HA immunoprecipitation. HA-pull-down was carried out in E14Tg2a cells expressing Cnot2-HA. The presence of Cnot1, Cnot2-HA, and Cnot3 in the total lysate and pull-down sample (HA-beads) were detected by Western blot. Note that Oct4 was not detected in the pull down sample. As a negative control, protein-A beads were used in an independent pull-down. Abbreviations: HA, hemagglutinin; IP, immunoprecipitation; KD, knockdown.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Reporter Assay, Transfection, Cell Culture, Fluorescence, FACS, Expressing, Staining, Marker, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Negative Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Cnot1, Cnot2, and Cnot3 are required for human embryonic stem cell (ESC) self-renewal. (A): Cnot1, Cnot2, and Cnot3 were down-regulated during human ESC differentiation. H1 human ESCs were differentiated for 7 days using 100 ng/ml human recombinant BMP4. The expression levels of Cnot1, Cnot2, and Cnot3 as well as Oct4 and differentiation markers Cdx2 and Hand1 were determined by quantitative realtime PCR (qRT-PCR). (B): Silencing of Cnot1, Cnot2, or Cnot3 led to morphological changes of human ESCs. H1 cells were imaged 6 days after transfection. Phase-contrast images highlight the undifferentiated morphology of human ESCs in the lipids-only transfected cells (mock) versus the differentiated phenotype in the Cnot1, Cnot2, or Cnot3 siRNA transfected cells. (C): Silencing of the Cnot genes led to upregulation of the Cdx2 and Gata3 proteins. H1 cells were transfected with lipids-only (mock), Oct4, Cnot2, or Cnot3 siRNAs. Cells were fixed and stained for Cdx2 or Gata3 expression by immunofluorescence staining 6 days after transfection. (D): Silencing of the Cnot genes led to downregulation of the ESC marker and upregulation of the extraembryonic markers. H1 cells were harvested 6 days after transfection and marker expression was determined by qRT-PCR. Abbreviations: BMP, bone morphogenetic protein; DAPI, 4′-6-diamidino-2-phenylindole.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Transfection, Staining, Immunofluorescence, Marker

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: 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

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Hela, HepG2, and Huh7 cells were seeded in a 12-well plate for overnight, then left untreated or treated with human IFN-α (1,000 IU/ml) for 18 or 36 h. The untreated cells (control) were harvested together with the cells treated by IFN-α for 36 h. ISG20 expression was detected by Western blot. Hela cells transfected with plasmid F-ISG20 expressing the FLAG-tagged ISG20 were used as positive control for ISG20 Western blot (lane 2). β-actin expression was presented as loading control. (B) PHHs and HepG2 cells were cultured in 12-well-plate and treated with type I IFN-α (1,000 IU/ml), type II IFN-γ (100 ng/ml) or type III IFN-λ (100 ng/ml), or left untreated (control) for 2 days. The levels of ISG20 expression were determined by Western blot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Control, Expressing, Western Blot, Transfection, Plasmid Preparation, Positive Control, Cell Culture

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with either pHBV1.3 and F-ISG20 or empty vector, or pCMVHBV and F-ISG20 or empty vector, as indicated. Cells were harvested at day 5 post-transfection, and the levels of viral RNA and DNA were determined by Northern (top) and Southern (middle) blot hybridization, respectively. For RNA analysis, each lane was loaded with 10 μg of total RNA and probed with a genome-length, plus-strand-specific HBV riboprobe. Ribosomal RNAs (28S and 18S) are presented as loading controls. The positions of HBV pgRNA (3.5kb) and subgenomic surface RNAs (2.4kb and 2.1kb) are indicated. For DNA analysis, HBV core DNA was probed with genome-length, minus-strand-specific HBV riboprobe. The positions of relaxed circular (RC) and single-stranded (SS) DNAs are indicated. The relative pgRNA, sRNA or total DNA replicative intermediate level in each sample is expressed as the percentage of RNA or DNA of the cells transfected with empty vector. ISG20 overexpression was confirmed by Western blot using monoclonal antibodies against FLAG-tag. β-actin expression was presented as protein loading control (bottom panels). (B) The same experiment was done in Huh7 cells with pHBV1.3 as HBV expression vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Northern Blot, Hybridization, Over Expression, Western Blot, Bioprocessing, FLAG-tag, Expressing, Control

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepDES19 cells were seeded in 35 mm-dish and cultured with tet-free medium to induce HBV pgRNA transcription. 24 h later, cells were transfected with 4 μg of control vector or plasmid F-ISG20 for 36 h, then tet was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of FLAG-tagged ISG20 was detected by Western blot. The results are representative of three separate trials. (B) HepG2 cells in 12-well-plate were co-transfected with 0.7 μg of pTREHBVDES and 0.1 μg of pTet-off, plus 0.7 μg of control vector or plasmid F-ISG20. Four days post transfection, tet was added back and cells were harvested at indicated time points and subjected to HBV RNA qPCR analysis. The relative levels of HBV total RNA normalized to β-actin mRNA levels in each samples were expressed as the percentage of the RNA levels from the corresponding sample at 0 h time point (Mean ± SD, n = 4). The half-life of HBV RNA was marked on the plot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Cell Culture, Transfection, Control, Plasmid Preparation, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepDES19 cells were transfected with 100 nM of control siRNA (Lane 1–3) or ISG20 siRNA (siISG20) (lane 4–6) twice with a 24 h interval after tet being withdrawn. Culture medium was replaced 12 h after the 2nd siRNA transfection, and cells were either left untreated as controls (lane 1 &4) or treated with 100 IU/ml (lanes 2 & 5) or 1,000 IU/ml (lanes 3 & 6) of IFN-α. Cells were harvested 5 days after 2nd transfection. Viral total RNA (top panel), encapsidated pgRNA (upper middle panel), and core DNA (lower middle panel) were subjected to Northern and Southern analyses, respectively. ISG20 protein expression was revealed by Western blot, and β-actin served as loading control (bottom panels). The relative levels of viral nucleic acids and ISG20 expression in the siISG20 transfected or IFN-α treated samples (lanes 2–6) are expressed as the percentage of the control sample (lane 1). The data presented here are representative of two independent experiments.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2-NTCP12 cells stably transduced by control lentiviral shRNA (shcontrol) or ISG20 lentiviral shRNA (shISG20) were spinoculated with HBV at 100 vge/cell. 16 h later, the infected cells were mock treated or treated with 1,000 IU/ml of IFN-α for 6 days, and the cells were subjected to the following analyses: (A) The expression of ISG20 was analyzed by Western blot. (B) HBV infectivity was assessed by HBcAg immunofluorescence, and the percentage of HBcAg-positive cells were calculated from multiple microscopic field of view (mean±SD, n = 5). Nuclei were stained with DAPI. (C) HBV total RNA were quantified by qPCR and the relative expression levels to β-actin mRNA were plotted as fold change to control samples (HBV infected shcontrol cells without IFN-α treatment) (mean±SD, n = 3).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Stable Transfection, Control, shRNA, Infection, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were transfected with pHBV1.3 and equal amount of control vector (lanes 1 & 2) or F-ISG20 (lanes 3 & 4) or F-ISG20 D94G (lanes 5 & 6). Cells were harvested 5 days post-transfection and levels of HBV RNA (1st panel from the top) and encapsidated pgRNA (4th panel from the top) were determined by Northern blot hybridization. The assembled HBV capsid was revealed by native capsid gel EIA assay (3rd panel from the top) and the viral DNA in capsid was detected in situ by hybridization (5th panel from the top). HBV core DNA replicative intermediates were extracted and analyzed by Southern blot (6th panel from the top). Expression of FLAG-tagged ISG20 proteins was revealed by Western blot and β-actin served as loading control (bottom two panels). Results from duplicate experiments are presented.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Hybridization, Enzyme Immunoassay, In Situ, Southern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were co-transfected with plasmid pCMVHBVΔCΔP and either control vector (lane 1) or FLAG-Pol (lanes 2&3), or pCMVHBV with either control vector (lane 4) or F-ISG20 D94G (lanes 5&6). Cells were harvested 4 days post-transfection. Input HBV RNA was determined by Northern blot (top panels). Input FLAG-Pol and F-ISG20 D94G proteins were determined by Western blot using FLAG Ab (top panels). Cell lysates were immunoprecipitated with beads coated with FLAG Ab, the immunoprecipitated Pol and ISG20 D94G were revealed by Western blot using FLAG Ab (lanes 3&6, bottom panel), and the bound RNA was extracted by Trizol and analyzed by Northern blot (lanes 3&6, bottom panel). HA Ab pull-down served as negative controls (lanes 2 & 5, bottom panel). See for more experimental details.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Control, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells in 6-well-plate were co-transfected with 1 μg of pCMVHBVΔCΔP and 5 μg control vector (lane 1) or 1 μg of FLAG-Pol in the absence of HA-ISG20 D94G (lane 2) or increased amount of HA-ISG20 D94G (1 μg, 2 μg, 4 μg; lanes 3–5). The total amount of transfected DNA was kept constant (6 μg/well) by adding control vector plasmid (lanes 2–4). 5 days later, total cellular HBV RNA and protein (FLAG-Pol and HA-ISG20 D94G ) were determined by Northern and Western blot, respectively, as input controls (top panels). Immunoprecipitation was performed by using antibodies against HA or FLAG epitopes, followed by Northern blot analysis of HBV RNA (bottom panels).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of HBV pgRNA deletion clones. Plasmid pHBV1.3 contains a 1.3 overlength HBV genome (Genbank Accession Number U95551), starting at nt 1000. The HBV nucleotide positions are according to Galibert et al . Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5’ cap of mRNA; and the sawtooth line represents the polyA tail at the 3’ terminus of mRNA. The internal deletion clones (pg-IDs) are described in details in . The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 5’ or 3’ terminus of pgRNA coding sequences (pg-Δ5TR and pg-Δ3TR, respectively.), or both (pg-Δ5/3TR). The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector. (B) Sensitivity of HBV RNA with TR deletion to ISG20-mediated RNA reduction. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or F-ISG20 plasmid. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization. ( C) HBV TR insertion renders Luc gene to be sensitive to ISG20. The schematic illustration indicates the reporter construct EnII/Cp-Luc with HBV TR insertion at the flanking non-translational region of luciferase ORF. HepG2 cells were transfected with each indicated reporter plasmid and control vector or plasmid expressing ISG20. Cells were lysed at day 3 post transfection and luciferase activity was measured. The plotted relative luciferase activity (RLA) represents the mean ± SD (n = 3) of the percentage of absorbance obtained from wells transfected with ISG20 over control vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Clone Assay, Plasmid Preparation, Transfection, Control, Northern Blot, Hybridization, Construct, Luciferase, Expressing, Activity Assay

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic stem-loop structure of HBV ε RNA. Ribonucleotide sequences (nt 1847–1991, genotype D, subtype ayw) are presented with base paring indicated by dotted line. (B) Verification of the purified recombinant 6×His-tagged ISG20 by SDS-PAGE Coomassie staining. (C) EMSA assay of ISG20-ε binding. The indicated amount of ISG20 proteins were incubated with 100 ng 32 P-end-labeled ε RNA in binding buffer to form nucleoprotein complexes. Monoclonal anti-His antibody was used for supershifting of the His-ISG20/ HBV ε complex. Excessive amount of cold unlabeled HBV ε RNA (10×, 20×, 40×) were used to compete with the binding of ISG20 to 100 ng radiolabeled HBV ε. The nucleoprotein complexes were separated by native PAGE and the shifted bands were detected by autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Purification, Recombinant, SDS Page, Staining, Binding Assay, Incubation, Labeling, Clear Native PAGE, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustrations of the wildtype HBV ε RNA and mutants. The substructural domains of ε, including the lower stem, bulge, upper stem, and apical loop, are marked on the full-length form. Shorter versions of ε include the upper stem loop (US+L), lower stem with wildtype or mutant bulge sequence as loop (LS+B, LS+Bm), and LS+B with bottom 4 base-pairs removed from the lower stem (LSΔ4+B). These RNA fragments were chemically synthesized and 5’ end radiolabeled for ISG20 EMSA. (B) EMSA of ISG20 binding with full-length (FL) ε, US+L, and LS+B. (C) EMSA of ISG20 binding with LS+B, LS+Bm, and LS+B. (D) HepG2 cells were transfected with plasmid pMS transcribing the 2.1kb HBV RNA, or pMSΔ4bp transcribing the 2.1kb HBV with 4 nucleotides removed from the bottom right arm of the lower stem of ε, in the absence or presence of F-ISG20. HBV RNA and FLAG-tagged ISG20 were analyzed by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Mutagenesis, Sequencing, Synthesized, Binding Assay, Transfection, Plasmid Preparation, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of ISG20. The amino acid (a.a) positions are labeled with numbers. The gray boxes indicate the predicted Exo motifs. The enzymatic mutant site (D94G) is marked with an asterisk. (B) Bacterially expressed His-tagged ISG20 and mutants were purified and examined by SDS-PAGE Coomassie staining. The asterisk indicates a nonspecific protein band co-purified with the recombinant ΔExoII mutant. (C) EMSA of ε binding by wildtype ISG20 and the indicated mutants. (D) HepG2 cells were co-transfected with pHBV1.3 and control vector or indicated FLAG-ISG20 constructs. HBV RNA and ISG20 proteins were detected by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Labeling, Mutagenesis, Purification, SDS Page, Staining, Recombinant, Binding Assay, Transfection, Control, Plasmid Preparation, Construct, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: 0.1 µg of 5’-radiolabeled synthetic RNA substrates, specifically (A) the intact ε, upper stem-loop region (US+L), and lower stem with bulge serving as loop (LS+B); and (B) the intact ε, single-stranded left arm portion of ε, and 30-mer poly(rA), were incubated with the indicated amount of RNase A or purified His-ISG20 in nuclease reaction buffer for 15 min, then the reactions were terminated and the mixtures were fractionated through 10% TBE-Urea denaturing polyacrylamide gel, and the dried gel was subjected to autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Incubation, Purification, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: The major viral intermediates and products at each major HBV replication steps are illustrated. cccDNA (or other HBV transcription template)-derived 3.5kb RNA (including precore mRNA and pgRNA) and other shorter subgenomic RNA species (2.4/2.1kb surface mRNA and 0.7kb X mRNA) are aligned to show the location of ε on different RNA species. The black circle dots indicate the 5’ cap of mRNA, the zigzag lines represent the polyA tails. ISG20 is shown as a rectangle box and its targeting sites on HBV RNA are indicated by arrowheads. As a consequence of ISG20-mediated HBV RNA degradation, the illustrations and labels of viral proteins/antigens, pgRNA encapsidation and DNA replication are shown in gray color schemes. The solid gray triangles indicate capsid proteins, viral polymerase is shown in an oval shape before ε binding and then a gray circle dot in the nucleocapsids after pgRNA encapsidation.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Derivative Assay, Binding Assay

Journal: Oncotarget

Article Title: Down regulation of human positive coactivator 4 suppress tumorigenesis and lung metastasis of osteosarcoma

doi: 10.18632/oncotarget.18290

Figure Lengend Snippet: (A) Western blotting analysis of PC4 knockdown efficiency in 143B cells. (B) Cell proliferation assays show slight inhibition by lv-shRNA-PC4 (* p<0.01 ). (C) Cell cycle distribution of 143B cells with lv-shRNA transfection (* p<0.05 ). (D) Cell attachment assay. 2*10 4 cells were seeded into 96-well culture plates, and incubated at 37°C for 30 min or 60 min or 120min or 24 hours. After incubation, unattached cells were washed with PBS, and adherent cells were counted with CCK-8 kits, (* p <0.01). (E, F) Spheroid development in semisolid soft agar medium after 7 days. 143B showed decreased efficiency of sphere-forming in 143B PC4− group. Spheres were counted in five random fields of vision. (G) Invasion assay. 143B cells seeded on the upper chamber with pre-coated matrigel for 24 hours. Cells on the underside of the membrane were fixed, stained with crystal violet. (H, I) Effect of lv-shRNA-PC4 on 143B cell migration by wound-healing assay. The 143B cells were seeded in 6-well plates for 24h, after cell reached 100% density wounds were created. Cell migration was observed at 0h, 24h, 48h, and 72h after wounding. The migration distance was calculated as the width at indicated time (* p <0.05 versus control).

Article Snippet: The Sp1 siRNA (Cat. # sc-90024, Santa Cruz) and PC4 siRNA (Cat.# sc-38583, Santa Cruz).

Techniques: Western Blot, Knockdown, Inhibition, shRNA, Transfection, Cell Attachment Assay, Incubation, CCK-8 Assay, Invasion Assay, Membrane, Staining, Migration, Wound Healing Assay, Control

Journal: Oncotarget

Article Title: Down regulation of human positive coactivator 4 suppress tumorigenesis and lung metastasis of osteosarcoma

doi: 10.18632/oncotarget.18290

Figure Lengend Snippet: (A) The mRNA expression levels of PC4, MMP9, MMP2, and FN after lv-shRNA-PC4 transfected in seven osteosarcoma cells. All the cell lines were compared to parental cells respectively (* P <0.05). (B) 143B treated with 35ug/ml FN protein for 4h or FN siRNA, the mRNA expression levels of MMP9, MMP2 (* P <0.05). (C) MG63 treated with FN protein or FN siRNA, the mRNA expression levels of MMP9, MMP2 (* P <0.05). (D) Luciferase reporter. PMA was used as an inducer for MMP9 luciferase activity. Both PC4 siRNA and SP1 siRNA decrease the luciferase activity of MMP9 promoter region, and PC4 siRNA and SP1 siRNA have the combined effect (*P<0.05, **P<0.05). (E) Quantitative ChIP assay of endogenous PC4 interact with MMP9 promoter region in 143B cells. MMP9 cDNA was detectable in the immunoprecipitated chromatin samples of 143B cells using a PC4 antibody, suggesting PC4 binds to the MMP9 promoter region. The same amount of isotype antibody was used as control (IgG), as well as no antibody controls (Negative control). RT-PCR results are expressed as percentages of the total input DNA (* P <0.05). (F) PC4 binds to SP1 in 143B. 143B cell extracts protein was used. Co-Immunoprecipitation was performed using PC4 antibody and SP1 antibody, IgG as control.

Article Snippet: The Sp1 siRNA (Cat. # sc-90024, Santa Cruz) and PC4 siRNA (Cat.# sc-38583, Santa Cruz).

Techniques: Expressing, shRNA, Transfection, Luciferase, Activity Assay, Immunoprecipitation, Control, Negative Control, Reverse Transcription Polymerase Chain Reaction