activation plasmid m  (Santa Cruz Biotechnology)

Journal: Advanced Science

Article Title: Tumor‐Activatable Nanoparticles Target Low‐Density Lipoprotein Receptor to Enhance Drug Delivery and Antitumor Efficacy

doi: 10.1002/advs.202201614

Figure Lengend Snippet: LDLR‐mediated endocytosis determines NCP particle uptake by tumor cells. Uptake of a) Chol‐pyro NCP and b) Ce6‐NCP by MC38 cells after LDLR blockade with 1 or 10 µg mL −1 α ‐LDLR. The Dil‐LDL uptake served as control. c) Cellular uptake of Chol‐pyro‐NCP and Ce6‐NCP on WT and LDLR KO MC38 cells. CLSM d) statistical analysis and e) images of Chol‐SN38 uptaken by MC38 cells 24 h after treatment with 10 µg mL −1 IgG or α ‐LDLR. Scale bar: 20 µm. f) Lysosome was stained with LysoTracker and the colocalization of lysosome and Chol‐SN38 was evaluated based on Pearson's R value. g) Immunofluorescence analysis showing the in vivo tumor uptake of Chol‐pyro NCP at 24 and 48 h with 1 µg of IgG or α ‐LDLR. Scale bar: 20 µm. h) Flow cytometry results and i) mean fluorescent intensity of Chol‐pyro NCP tumor uptake at 24 and 48 h post i.v. injection with 1 µg of IgG or α ‐LDLR. Time‐dependent j) Pt and k) SN38 accumulation after i.v. injection of OxPt (3.5 mg kg −1 ) plus irinotecan (6.2 mg SN38/kg equivalent), OxPt/SN38 (3.5 mg OxPt/kg equivalent, 6.2 mg SN38/kg equivalent) to MC38‐bearing mice with and without 1 µg of intratumorally injected α ‐LDLR. Data are expressed as means ± SD. The data were analyzed by one‐way analysis of variance (ANOVA; a,b) with Tukey's multiple comparison test, or Student's two‐tailed t‐test (c,d,i).

Article Snippet: LDLR KO MC38 cells were obtained following the protocol of LDLR CRISPR Plasmids (m) (Santa Cruz Biotechnology).

Techniques: Control, Staining, Immunofluorescence, In Vivo, Flow Cytometry, Injection, Comparison, Two Tailed Test

Journal: Advanced Science

Article Title: Tumor‐Activatable Nanoparticles Target Low‐Density Lipoprotein Receptor to Enhance Drug Delivery and Antitumor Efficacy

doi: 10.1002/advs.202201614

Figure Lengend Snippet: LDLR‐mediated endocytosis determines the anticancer efficacy of OxPt/SN38. a) Anticancer efficacy of OxPt/SN38 with intratumorally injected 1 µg IgG or α ‐LDLR on MC38 tumor‐bearing C57BL/6 mice at a dose of 3.5 mg OxPt/kg equivalent. n = 6. b) Anticancer efficacy of OxPt/SN38 on WT and LDLR KO MC38 tumor‐bearing C57BL/6 mice at a dose of 3.5 mg OxPt/kg equivalent. n = 6. c) H&E staining (top), TUNEL (middle), and Caspase 3 (bottom) showing differences in apoptosis and necrosis of MC38 tumors after OxPt/SN38 treatments with intratumorally injected IgG or α ‐LDLR. Scale bar: 100 µm. d) Scheme showing OxPt/SN38 delivery by hitchhiking LDL and anticancer mechanism. Once injected intravenously, OxPt/SN38 binds to LDL and transfers Chol‐SN38 to LDL. LDL‐bound OxPt/SN38 is taken up by cancer cells through LDLR‐mediated endocytosis. Acidic endo/lysosomal environment triggers the release of OxPt and SN38 from OxPt/SN38. The released OxPt and SN38 synergistically inhibit DNA replication by crosslinking DNA bases and binding to topoisomerase I, respectively. The DNA damage causes MMP disruption, resulting in the early apoptosis of cancer cells. Data are expressed as means ± SD. The data were analyzed by one‐way ANOVA with Tukey's multiple comparison test. (d) was created with BioRender.com.

Article Snippet: LDLR KO MC38 cells were obtained following the protocol of LDLR CRISPR Plasmids (m) (Santa Cruz Biotechnology).

Techniques: Injection, Staining, TUNEL Assay, Binding Assay, Disruption, Comparison

Journal: Bioscience Reports

Article Title: Ubiquitin fold modifier 1 activates NF-κB pathway by down-regulating LZAP expression in the macrophage of diabetic mouse model

doi: 10.1042/BSR20191672

Figure Lengend Snippet: ( A ) The mRNA levels of TNF-α, IL-6, IL-1β, and Ufm1 in MPMs of db/m and db/db mice were tested by real-time PCR. ( B ) The secreted proteins levels of TNF-α, IL-6, and IL-1β in serum of db/m and db/db mice were tested by ELISA. ( C ) The protein expression of Ufm1 in MPMs of db/m and db/db mice were tested by Western blot. ( D ) Raw264.7 cells were treated with LPS (10, 100, and 1000 ng/ml), and the mRNA levels of TNF-α, IL-6, and Ufm1 were tested by real-time PCR. ( E ) Raw264.7 cells were treated with LPS (6, 12, and 24 h), and the mRNA levels of TNF-α, IL-6, and Ufm1 were tested by real-time PCR. Similar results were obtained in three independent experiments, and one of three representative experiments was shown. The values presented were the mean ± SEM; * P < 0.05, ** P < 0.01 versus the control group.

Article Snippet: CDK5RAP3 CRISPR activation plasmid (m) (sc-429782-ACT), UFM1 CRISPR activation plasmid (m) (sc-426807-ACT), and Control CRISPR activation plasmid (sc-437275) (Santa Cruz Biotechnology) were also transfected to cells to overexpress LZAP and Ufm1 separately following the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control

Journal: Bioscience Reports

Article Title: Ubiquitin fold modifier 1 activates NF-κB pathway by down-regulating LZAP expression in the macrophage of diabetic mouse model

doi: 10.1042/BSR20191672

Figure Lengend Snippet: ( A ) GFP-LZAP, Flag-Ufm1, Myc-Uba5 (E1), Myc-Ufc1 (E2), and Myc-Ufl1 (E3) vectors were transfected in HEK293 cells. The cell lysates were immunoprecipitated (IP) with anti-Flag antibody, followed by immunoblot analysis with anti-GFP antibodies. The arrowheads indicated GFP-LZAP and Flag-Ufm1conjugate. ( B ) GFP-LZAP and Flag-Ufm1 vectors were transfected in Raw264.7 cells. The cell lysates were IP with anti-GFP antibody, followed by immunoblot analysis with anti-Flag or anti-GFP antibodies. ( C ) Raw264.7 cells were treated with sh-Ufm1 plasmid and LPS, and the proteins levels of Ufm1 and LZAP were tested by Western blot. β-Actin acted as a loading control. ( D ) Raw264.7 cells were treated with Ufm1 overexpressed plasmid and LPS, and the proteins levels of Ufm1 and LZAP were tested by Western blot. β-Actin acted as a loading control. Similar results were obtained in three independent experiments, and one of three representative experiments was shown.

Article Snippet: CDK5RAP3 CRISPR activation plasmid (m) (sc-429782-ACT), UFM1 CRISPR activation plasmid (m) (sc-426807-ACT), and Control CRISPR activation plasmid (sc-437275) (Santa Cruz Biotechnology) were also transfected to cells to overexpress LZAP and Ufm1 separately following the manufacturer’s instructions.

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

Journal: Bioscience Reports

Article Title: Ubiquitin fold modifier 1 activates NF-κB pathway by down-regulating LZAP expression in the macrophage of diabetic mouse model

doi: 10.1042/BSR20191672

Figure Lengend Snippet: ( A ) SP600125 and LPS were treated to Raw264.7 cells correspondingly the proteins levels of Ufm1, p-ATF2, ATF2, p-c-Jun and c-Jun were tested by Western blot. β-Actin acted as a loading control. ( B ) SP600125 and LPS were treated to Raw264.7 cells, and the Ufm1 mRNA level was detected by real-time PCR. ( C ) SP600125 and LPS were treated to Raw264.7 cells, and the Ufm1 promoter activity was tested by luciferase reporter assay. Similar results were obtained in three independent experiments, and one of three representative experiments was shown. The values presented were the mean ± SEM. * P < 0.05 versus the control group.

Article Snippet: CDK5RAP3 CRISPR activation plasmid (m) (sc-429782-ACT), UFM1 CRISPR activation plasmid (m) (sc-426807-ACT), and Control CRISPR activation plasmid (sc-437275) (Santa Cruz Biotechnology) were also transfected to cells to overexpress LZAP and Ufm1 separately following the manufacturer’s instructions.

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Reporter Assay