Journal: Autophagy

Article Title: Pycard deficiency inhibits microRNA maturation and prevents neointima formation by promoting chaperone-mediated autophagic degradation of AGO2/argonaute 2 in adipose tissue.

doi: 10.1080/15548627.2023.2277610

Figure Lengend Snippet: Figure 3. AGO2 is degraded through chaperone-mediated autophagy pathway. Stromal vascular fraction isolated from WT mice were differentiated into adipocytes and used for the following experiments. (A-B) immunoblotting analysis of AGO2 expression in the cells treated with 10 µM lactacystin for indicated time. (C-D) cells were treated with NH4Cl combined with leupeptin for indicated time, and AGO2 level was measured. (E-G) cells were transfected with control siRNA (siCtrl) or cathepsin B siRNA (siCtsb) or cathepsin D siRNA (siCtsd), and AGO2 level was analyzed. (H-I) immunoblotting analysis of AGO2 expression in the cells transfected with control siRNA (siCtrl) or Ulk1 siRNA (siUlk1). (J-K) cells were treated with 6-AN or starvation for indicated time, and AGO2 level was measured. (L-N) cells were transfected with control siRNA (siCtrl) or Hspa8 siRNA (siHspa8) or Lamp2 siRNA (siLamp2), and AGO2 expression was analyzed. (O) interaction of AGO2 and HSPA8 in adipose tissue were detected by immunoprecipitation and immunoblotting analysis. (P) co-staining of AGO2 and lysosomal HSPA8 in abdominal adipose tissue. Red: AGO2; green: HSPA8; blue: DAPI. (Q) the interaction of AGO2 and LAMP2 in adipose tissue was detected by immunoprecipitation and immunoblotting analysis. (R) co-staining of AGO2 and LAMP2 in abdominal adipose tissue. Red: AGO2; green: LAMP2; blue: DAPI. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, and n. S. non-significant, derived from Student’s t tests.

Article Snippet: For transfections of Cathepsin B siRNA (Santa Cruz Biotechnology, sc -29,933), Cathepsin D siRNA (Santa Cruz Biotechnology, sc -29,934), Hspa8 siRNA (Santa Cruz Biotechnology, sc -35,593), Lamp2 siRNA (Santa Cruz Biotechnology, sc -35,791), Ulk1 siRNA (Santa Cruz Biotechnology, sc -44,849), Prmt8 siRNA (Santa Cruz Biotechnology, sc -152,473), and Mir106b mimic in adipocytes, the cells that had been differentiated for 6 days were electroporated using SE Primary Cell 4D-Nucleofector X Kit (Lonza, V4XC– 1012) according to the manufacturer’s instructions.

Techniques: Isolation, Western Blot, Expressing, Transfection, Control, Immunoprecipitation, Staining, Derivative Assay

Journal: Autophagy

Article Title: Pycard deficiency inhibits microRNA maturation and prevents neointima formation by promoting chaperone-mediated autophagic degradation of AGO2/argonaute 2 in adipose tissue.

doi: 10.1080/15548627.2023.2277610

Figure Lengend Snippet: Figure 4. Pycard deficiency promotes CMA-mediated AGO2 degradation in adipocytes. Stromal vascular fraction isolated from WT and pycard−/− mice were differentiated into adipocytes and used for the following experiments. (A-B) cells were treated with NH4Cl (N) combined with leupeptin (L) for 24 h. AGO2 level was detected by immunoblotting analysis. The ratio of N/L to veh treatment after normalization by endogenous control. (C-D) cells were transfected with control siRNA (siCtrl) or Hspa8 siRNA (siHspa8), AGO2 protein level was measured by immunoblotting analysis. (E-F) cells were transfected with control siRNA (siCtrl) or Lamp2 siRNA (siLamp2), and western blot was performed to measure AGO2 protein expression. (G-H) cells were transfected with control siRNA (siCtrl) or Ulk1 siRNA (siUlk1), andAGO2 protein expression was analyzed by immunoblotting analysis. (I) the interaction of AGO2 and HSPA8 in differentiated adipocytes was determined by immunoprecipitation and immunoblotting analysis. (J) the interaction of AGO2 and LAMP2 was analyzed in differentiated adipocytes. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, and n.S. non-significant, derived from Student’s t tests.

Article Snippet: For transfections of Cathepsin B siRNA (Santa Cruz Biotechnology, sc -29,933), Cathepsin D siRNA (Santa Cruz Biotechnology, sc -29,934), Hspa8 siRNA (Santa Cruz Biotechnology, sc -35,593), Lamp2 siRNA (Santa Cruz Biotechnology, sc -35,791), Ulk1 siRNA (Santa Cruz Biotechnology, sc -44,849), Prmt8 siRNA (Santa Cruz Biotechnology, sc -152,473), and Mir106b mimic in adipocytes, the cells that had been differentiated for 6 days were electroporated using SE Primary Cell 4D-Nucleofector X Kit (Lonza, V4XC– 1012) according to the manufacturer’s instructions.

Techniques: Isolation, Western Blot, Control, Transfection, Expressing, Immunoprecipitation, Derivative Assay

Journal: Autophagy

Article Title: Pycard deficiency inhibits microRNA maturation and prevents neointima formation by promoting chaperone-mediated autophagic degradation of AGO2/argonaute 2 in adipose tissue.

doi: 10.1080/15548627.2023.2277610

Figure Lengend Snippet: Figure 5. Pycard deficiency promotes PRMT8-mediated AGO2 methylation and increases AGO2 binding to HSPA8. (A) the differentiated WT and pycard−/− adipocytes were treated with NH4Cl combined with leupeptin for 24 h. Acetylated AGO2 level was determined by immunoprecipitation (IP) of acetylate lysin (ac-lysin) and followed by immunoblotting analysis of AGO2. (B) the differentiated WT and pycard−/− adipocytes were treated with NH4Cl combined with leupeptin for 24 h. Methylated AGO2 level was determined by IP of AGO2 and followed by immunoblotting analysis of either SYM10 or ASYM24. (C) Messenger RNA levels of PRMTs in the differentiated WT and pycard−/− adipocytes were analyzed by qRT-PCR. ** p < 0.01. (D) immunoblotting analysis of PRMT8 in epididymal adipose tissue (EpiAdi) or perivascular adipose tissue (PVAT) collected from WT or pycard−/− mice. (E) the differentiated WT or pycard−/− adipocytes were transfected with control siRNA (siCtrl) or Prmt8 siRNA (siPrmt8), protein levels of AGO2 and PRMT8 were detected by immunoblotting analysis. (F) primary mature adipocytes were treated with PRMT8 inhibitor (PRMT8i, GSK3368715, 2 nM) or vehicle (veh) for 48 h. AGO2 methylation level was determined by IP and immunoblotting analysis. (G) primary mature adipocytes were treated with PRMT8 inhibitor (PRMT8i, GSK3368715, 2 nM) or vehicle (veh) for 48 h. The interaction between AGO2 and HSPA8 was determined by IP and immunoblotting analysis. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, and n.S. non-significant, derived from Student’s t tests.

Article Snippet: For transfections of Cathepsin B siRNA (Santa Cruz Biotechnology, sc -29,933), Cathepsin D siRNA (Santa Cruz Biotechnology, sc -29,934), Hspa8 siRNA (Santa Cruz Biotechnology, sc -35,593), Lamp2 siRNA (Santa Cruz Biotechnology, sc -35,791), Ulk1 siRNA (Santa Cruz Biotechnology, sc -44,849), Prmt8 siRNA (Santa Cruz Biotechnology, sc -152,473), and Mir106b mimic in adipocytes, the cells that had been differentiated for 6 days were electroporated using SE Primary Cell 4D-Nucleofector X Kit (Lonza, V4XC– 1012) according to the manufacturer’s instructions.

Techniques: Methylation, Binding Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Transfection, Control, Derivative Assay

Journal: Clinical Cancer Research

Article Title: A Novel Topical Fluorescent Probe for Detection of Glioblastoma

doi: 10.1158/1078-0432.ccr-20-4518

Figure Lengend Snippet: Figure 4. siRNA-based knockdown of CAPN1 and CTSD in U87 cells. A, The RNA expression levels of CAPN1 and CTSD are normalized to those of the housekeeping genes, GAPDH and HPRT1. The boxplot shows the genomic fold change SEM. Note: “” indicates significance (P < 0.05) and “” indicates significance (P < 0.01) compared with respective controls. B, In confocal fluorescence evaluation of U87 cells, knock- down of CAPN1, calpain-2 (CAPN2), and CTSD sig- nificantly reduced the fluorescence intensity of U87 cells. Cells have been incubated with 1 mmol/L con- centration of proline-arginine-hydroxymethyl rhoda- mine green for 30 minutes. DIC, differential interfer- ence contrast.

Article Snippet: U87 cells (4 104 cells) were seeded in 8-well chamber (Ibidi) and transfected with 10 nmol/L concentration of siRNAs targeting CAPN1 (CAPN1 siRNA, Santa Cruz Biotechnology, SC-29885, CA), CTSD (cathepsin D siRNA, Santa Cruz Biotechnology, SC-29239), or control siRNA (Santa Cruz Biotechnology, SC-37007) using the Lipofectamine RNAiMAX transfection reagent (#13778030, Invitrogen).

Techniques: Knockdown, RNA Expression, Incubation

Journal: Clinical Cancer Research

Article Title: A Novel Topical Fluorescent Probe for Detection of Glioblastoma

doi: 10.1158/1078-0432.ccr-20-4518

Figure Lengend Snippet: Figure 6. Immunoblotting analysis and IHC staining of CAPN1 and CTSD in human surgical specimens. A, Protein expression of CAPN1 and CTSD assessed by immuno- blotting in the positive (n ¼ 5) and negative (n ¼ 2) groups. B, The boxplot represents protein expression fold change for CAPN1 and CTSD with respect to that of b-actin. The analysis indicates statistically signifi- cant difference in the CAPN1 expression level between the two groups (Welch’s two sample t-test, P ¼ 0.007). Note: “n.s.” indicates not significant, whereas “” indicates significant difference (P < 0.01). C, IHC- based expression of CAPN1 and CTSD in the tumor and peritumoral tissues in the positive and negative groups. Scale bar, 100 mm. D, Fluorescent immunos- taining for CAPN1 and CTSD. Each subpanel indicates expression/staining for: (a) TOTO-3, cell nuclei; (b) MIB-1; (c) CAPN1; (d) merged fluorescent image; (e) TOTO-3, cell nuclei; (f) MIB-1; (g) CTSD; and (h) merged fluorescent image. The expression of MIB-1 (b, blue arrow) and CAPN1 (c, yellow arrow) show overlap in the merged image (d, green arrow) but not for MIB-1 (f, blue arrow) and CTSD (g, yellow arrow). CAPN1 can be observed in the cytoplasm (c, yellow arrow), while CTSD in the lysosome (g, yellow arrow). Scale bar, 20 mm.

Article Snippet: U87 cells (4 104 cells) were seeded in 8-well chamber (Ibidi) and transfected with 10 nmol/L concentration of siRNAs targeting CAPN1 (CAPN1 siRNA, Santa Cruz Biotechnology, SC-29885, CA), CTSD (cathepsin D siRNA, Santa Cruz Biotechnology, SC-29239), or control siRNA (Santa Cruz Biotechnology, SC-37007) using the Lipofectamine RNAiMAX transfection reagent (#13778030, Invitrogen).

Techniques: Western Blot, Immunohistochemistry, Expressing, Staining