Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Oligonucleotide primer sets for RT-qPCR.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Sequencing

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Exosomal transfer of NEAT1 to EC cells leads to increased expression of STAT3 and YKL-40. (A) RT-qPCR analysis of NEAT1 expression level in NFs, CAFs, normal endometrial epithelial cells, and EC cells. (B) HEC-1A and RL95-2 cells were cultured in control CM, CAFs-CM, or exosome-depleted CAFs-CM for 48 h, then NEAT1 level was assessed by RT-qPCR. HEC-1A and RL95-2 cells were maintained in control CM, CAFs-CM, or 20 μM GW4869-treated CAFs-CM for 48 h. (C) NEAT1 expression was determined by RT-qPCR. (D) The NEAT1 level was assessed by RT-qPCR. (E) STAT3, p-STAT3 and YKL-40 levels were assessed by Western blot. HEC-1A and RL95-2 cells were co-cultured with CAFs or GW4869-treated CAFs for 48 h. (F) Western blot analysis of STAT3, p-STAT3, and YKL-40 levels. (G) Western blot analysis of Rab27a protein level in CAFs infected with lentivirus expressing si-NC or si-Rab27a. (H) HEC-1A and RL95-2 cells were cultured in control CM, CAFs-CM, or Rab27a-silenced CAFs-CM for 48 h, then NEAT1 level was detected by RT-qPCR. (I) HEC-1A cells and RL95-2 were co-cultured with CAFs or Rab27a-silenced CAFs for 48 h, and NEAT1 level was detected by RT-qPCR. (J)&(K) Protein levels of STAT3, p-STAT3, and YKL-40 were assessed by Western blot. (L) RT-qPCR analysis of NEAT1 level in exosomes isolated from CAFs infected with lentivirus expressing sh-NEAT1 or NEAT1 gene. (M) HEC-1A and RL95-2 cells were incubated with exosomes derived from CAFs, NEAT1-overexpressed CAFs, or NEAT1-silenced CAFs for 48 h. RT-qPCR was performed to assess NEAT1 level. (N)&(O) STAT3 and YKL-40 expression was detected by RT-qPCR and Western blot assays. (P) The proliferation of HEC-1A and RL95-2 cells was detected by MTT assay. (Q) The growth of HEC-1A and RL95-2 cells was evaluated by colony formation assay. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Western Blot, Infection, Isolation, Incubation, Derivative Assay, MTT Assay, Colony Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Exosomal NEAT1 sponges miR-26a/b-5p to facilitate STAT3 and YKL-40 expression in EC cells. (A) RT-qPCR for miR-26a/b-5p level in exosomes derived from CAFs, NEAT1-overexpressed CAFs, or NEAT1-silenced CAFs. (B) HEC-1A and RL95-2 cells were co-cultured with NEAT1-overexpressed or silenced CAFs, and miR-26a/b-5p level was detected by RT-qPCR. (C) Expression of miR-26a/b-5p in HEC-1A and RL95-2 cells infected with lentivirus containing miR-26a/b-5p mimics or inhibitor was detected by RT-qPCR. (D) Expression of YKL-40 in HEC-1A and RL95-2 cells after overexpression or silencing of miR-26a/b-5p was assessed by Western blot. (E) The binding sites between NEAT1 and miR-26a/b-5p. Luciferase analysis (F) and RIP assay (G) was performed to determine interaction between NEAT1 and miR-26a/b-5p. The expression of STAT3 and YKL-40 in HEC-1A and RL95-2 cells received various treatments was assessed by RT-qPCR (H) and Western blot (I). All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001, ns = no significant.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Cell Culture, Infection, Over Expression, Western Blot, Binding Assay, Luciferase

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: MiR-26a/b-5p suppresses YKL-40 expression via targeting STAT3 in EC cells. (A) Two binding sites of miR-26a/b-5p to the 3’-UTR of STAT3. The interaction between STAT3 and miR-26a/b-5p was detected by luciferase analysis (B) and RIP assay (C). (D) The protein level of STAT3 in HEC-1A and RL95-2 cells after the transfection of STAT3 expression plasmid was assessed by Western blot. RT-qPCR (E) and Western blot (F) were used for determining YKL-40 and STAT3 expression in HEC-1A cells from various groups. (G) Three p-STAT3-binding sites (BS1, BS2, and BS3) at YKL-40 promoter were shown. (H) ChIP assay using anti-p-STAT3 antibody was used to verify the binding between p-STAT3 and the promoter of YKL-40 under exosome treatment. (I) The interaction between p-STAT3 and YKL-40 was assessed by the dual luciferase reporter assay. All data from three independent experiments were shown as mean ± SD (n = 6). **, P < 0.01; ***, P < 0.001, ns = no significant.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Reporter Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: CAFs-derived exosomal NEAT1 accelerates tumor growth in vivo via up-regulating YKL-40. Nude mice were subcutaneously injected with HEC-1A cells with or without CAFs. GW4869 (2 mg/kg) was intraperitoneally injected into the mice every other day. (A) The tumor growth curve was shown. (B) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (C) Immunohistochemical staining for Ki-67 expression in tumor tissues. (D) Western blot analysis of YKL-40 and STAT3 protein level in tumor tissues. Nude mice were subcutaneously injected with HEC-1A cells with or without CAFs that were stably transfected with sh-NC or sh-Rab27a. (E) The tumor growth curve was shown. (F) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (G) Immunohistochemical staining for Ki-67 expression in tumor tissues. (H) Western blot analysis of YKL-40 and STAT3 protein level in tumor tissues. HEC-1A cells with or without CAFs stably transfected with sh-NEAT1 or NEAT1 gene were subcutaneously injected into the nude mice. (I) The tumor growth curve was shown. (J) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (K) Immunohistochemical staining for Ki-67 expression in tumor tissues. (L) Western blot analysis of YKL-40 and STAT3 protein levels in tumor tissues. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Derivative Assay, In Vivo, Injection, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining, Western Blot, Stable Transfection, Transfection

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Overexpression of miR-26a/b-5p reverses exosomal NEAT1-mediated protumorigenic effect in vivo . HEC-1A cells stably transfected with miR-26a/b-5p mimics or NC mimics were mixed with or without CAFs stably expressing NC vector or NEAT1, which were subcutaneously injected into the nude mice. (A) The tumor growth curve was shown. (B) Immunohistochemical staining for Ki-67 expression in tumor tissues. (C) Western blot analysis of YKL-40 and STAT3 protein levels in tumor tissues. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Over Expression, In Vivo, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Injection, Immunohistochemical staining, Staining, Western Blot

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: Reagents and resources used in this study.

Article Snippet: pmRFP C1-CHMP4C , Sino Biological , HG14241-ANR.

Techniques: Protease Inhibitor, Electron Microscopy, Silver Staining, Plasmid Preparation, Negative Control, Subcloning, Recombinant, Expressing, shRNA

Journal: Journal of Biomedical Science

Article Title: Beyond the vaccines: a glance at the small molecule and peptide-based anti-COVID19 arsenal

doi: 10.1186/s12929-022-00847-6

Figure Lengend Snippet: FDA approvals (monoclonal antibodies) [ 20 ]

Article Snippet: Resultantly, the biotinylated peptide sequence derived from human ACE2 demonstrated substantial selective binding affinity to Sino Biological insect-derived SARS-CoV-2 spike protein RBD [ ].

Techniques: Virus, Suspension, Activity Assay, Inhibition, Binding Assay, Comparison, Variant Assay, Neutralization

Journal: iScience

Article Title: Prestimulation of CD2 confers resistance to HIV-1 latent infection in blood resting CD4 T cells

doi: 10.1016/j.isci.2021.103305

Figure Lengend Snippet: CD2 prestimulation inhibits HIV-1 latent infection of resting CD4 T cells. (A–C) Resting CD4 T cells were prestimulated with α-CD2 or α-CXCR4 beads and then infected with HIV-1(NL4-3) for 2 h. Cells were washed, cultured for 5 days, and then activated by α-CD3/CD28 beads to induce viral replication, which was measured by p24 release. The experiment was repeated in another two donors, and levels of p24 were quantified in triplicate in each donor. (D–F) Resting memory CD4 T cells were prestimulated with α-CD2 or α-CXCR4 beads and then infected with HIV-1(AD8) for 2 h. Cells were washed, cultured for 5 days, and then activated by α-CD3/CD28 beads to induce viral replication. Levels of p24 were quantified in triplicate. (G and H) Resting CD4 T cells were cultured in IL-2 or IL-7 for 3 days, prestimulated with α-CD2 beads, and then infected with HIV-1(NL4-3). Cells were washed, cultured for 5 days, and then activated by α-CD3/CD28 beads to induce viral replication. Levels of p24 were quantified in triplicate. (I) Resting CD4 T cells were prestimulated with soluble α-CD2 antibody (α-CD2s, 2 μg/mL) or recombinant human LFA-3 (2 μg/mL) for 1 h and then infected with HIV-1(NL4-3). Cells were similarly washed, cultured, and then activated at day 5. The experiment was repeated in two additional donors. Levels of p24 were quantified in triplicate. (J) Resting CD4 T cells were prestimulated with α-CD2 beads or soluble α-CD2 antibody (α-CD2s, 200 ng/mL) and then similarly infected and activated. The experiment was repeated in two additional donors. Levels of p24 were quantified in triplicate, and data are represented as mean ± SEM. Statistical significance was determined using two-tailed t test in Prism 7 (Graph Pad). Significance p values are indicated. See also .

Article Snippet: pCMV3-CXCR4-C-OFPSpark , Sino Biological , Cat#HG11325-ACR.

Techniques: Infection, Cell Culture, Recombinant, Two Tailed Test

Journal: iScience

Article Title: Prestimulation of CD2 confers resistance to HIV-1 latent infection in blood resting CD4 T cells

doi: 10.1016/j.isci.2021.103305

Figure Lengend Snippet: Lack of interaction and co-localization between CD2 and CXCR4 on CD4 T cells (A) Surface expression of CD2, CXCR4, and CD3ε. Jurkat CD4 T cells were stained by anti-CD2, -CXCR4, or CD3ε antibody and then analyzed by flow cytometry. (B) Cells were also stimulated with HIV-1(NL4-3). FRET signal between CD2 and CXCR4 was quantified by flow cytometry, using FRET between CXCR4 and CD3ε as a control. (C) Cells were also electroporated with a CD2-GFPSpark vector (green) and a CXCR4-OFPSpark vector (red) and then stimulated with HIV-1(gp160). CD2 and CXC4 colocalization before (top panel) and during HIV-1 infection (middle panel), and following α-CD2 bead prestimulation and HIV-1 infection (bottle panel), was analyzed by confocal microscopy. See also Figure S7 .

Article Snippet: pCMV3-CXCR4-C-OFPSpark , Sino Biological , Cat#HG11325-ACR.

Techniques: Expressing, Staining, Flow Cytometry, Plasmid Preparation, Infection, Confocal Microscopy

Journal: iScience

Article Title: Prestimulation of CD2 confers resistance to HIV-1 latent infection in blood resting CD4 T cells

doi: 10.1016/j.isci.2021.103305

Figure Lengend Snippet: CD2 signaling inhibits HIV-1 nuclear entry (A) Resting CD4 T cells were prestimulated with α-CD2 beads and infected with HIV-1(gp160). Actin cytoskeleton-associated viral DNA was quantified (left), and viral nuclear 2-LTR circles were also quantified by real-time PCR. (B) Model of CD2 signaling in blocking HIV-1 latent infection of CD4 T cells. CD2 signaling spatially inhibits viral entry and nuclear migration through triggering cofilin activation and actin polymerization around CD2, which does not spatially co-localize with CXCR4. CD2 signaling may competitively inhibit HIV-1-mediated CXCR4 signaling needed for viral entry and nuclear migration. Samples were analyzed in triplicate, and data are represented as mean ± SEM. Statistical significance was determined using two-tailed t test in Prism 7 (Graph Pad). Significance p values are indicated. See also Figure S8 .

Article Snippet: pCMV3-CXCR4-C-OFPSpark , Sino Biological , Cat#HG11325-ACR.

Techniques: Infection, Real-time Polymerase Chain Reaction, Blocking Assay, Migration, Activation Assay, Two Tailed Test

Journal: iScience

Article Title: Prestimulation of CD2 confers resistance to HIV-1 latent infection in blood resting CD4 T cells

doi: 10.1016/j.isci.2021.103305

Figure Lengend Snippet:

Article Snippet: pCMV3-CXCR4-C-OFPSpark , Sino Biological , Cat#HG11325-ACR.

Techniques: Purification, Recombinant, Transfection, Software