Journal: Bioengineering & Translational Medicine

Article Title: E3 ligase activity of Carboxyl terminus of Hsc70 interacting protein ( CHIP) in Wharton's jelly derived mesenchymal stem cells improves their persistence under hyperglycemic stress and promotes the prophylactic effects against diabetic cardiac damages

doi: 10.1002/btm2.10234

Figure Lengend Snippet: CHIP targets HG induced‐PTEN for ubiquitin‐mediated proteasomal degradation cooperated by HSP70 under HG conditions. (a–c) Cells transfected with HA‐vector or HA‐CHIP in the presence and absence of MG‐132 for 6 h were subjected to HG challenge for 24 h. Whole cell lysate was immunoprecipitated with the anti‐HA, anti‐CHIP, and anti‐PTEN antibody, and subsequently immunoblotted with the primary antibodies, including anti‐HA, anti‐PTEN, and anti‐ubiquitin. (d, e) Cells transfected with HA‐vector, HA‐CHIP, and CHIP mutants (K30A, an H260Q) were treated with or without MG‐132 for 6 h in the presence of HG for 24 h. Whole cell lysate was immunoprecipitated with the anti‐HA and anti‐PTEN antibody followed by immunoblotting with the anti‐HA, anti‐PTEN, and anti‐ubiquitin antibody. (f) Cells were transfected with increasing concentrations of siHSP70 (10, 20, 30 nM) after challenged with HG for 24 h, and the expression level of PTEN and HSP70 was measured employing Western blot analysis. (g) Following cotransfection of GFP‐vector or GFP‐CHIP with increasing concentration of siHSP70 in WJMSCs were challenged with HG for 24 h, and the protein expression was measured via immunoblotting. (h) WJMSCs transfected with sicontrol, CHIP siRNA, or siHSP70 were subjected to HG challenge for 24 h, and the total cell extract was immunoblotted with CHIP, PTEN, and HSP70. β‐actin served as a loading control. (i) Docking studies demonstrating the molecular interaction of HSP70 with PTEN forming a heteromer complex (HSP70 and PTEN shown in quaternary structure with helices and sheets in complex). Values shown are mean ± SD. Quantification of the results are shown ( n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 indicates the significant difference. CHIP, carboxyl terminus of Hsc70 interacting protein; HG, high glucose; PTEN, phosphatase and tensin homolog; WJMSCs, Wharton's jelly derived mesenchymal stem cells

Article Snippet: Lentiviral GFP‐CHIP was purchased from the Sino biological (RG83573‐ACGLN), while the lentiviral expressing small hairpin RNAs (shRNAs), including shcontrol (pLAS.Void), shCHIP (TRCN0000007528 NM_005861), shPTEN (TRCN0000002746 NM_000314), and lentiviral packaging plasmids (pCMVΔR8.91 and pMD.G), were obtained from the national RNAi core facility (Academia Sinica).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Cotransfection, Concentration Assay, Derivative Assay

Journal: Bioengineering & Translational Medicine

Article Title: E3 ligase activity of Carboxyl terminus of Hsc70 interacting protein ( CHIP) in Wharton's jelly derived mesenchymal stem cells improves their persistence under hyperglycemic stress and promotes the prophylactic effects against diabetic cardiac damages

doi: 10.1002/btm2.10234

Figure Lengend Snippet: CHIP overexpressed WJMSCs rescued hyperglycemic effects under diabetic conditions. (a) Schematic illustration of STZ‐induced diabetes, and WJMSCs administration expressing different plasmids, including GFP‐CHIP, shCHIP, and shPTEN. (b) The oral glucose tolerance test (OGTT) was performed after 6 weeks treatment for the indicated time points (0, 30, 60, 90, and 120 min) in various experimental groups, including control, STZ‐induced diabetes (STZ), STZ‐induced diabetes administered with WJMSCs alone (STZ + WJMSCs), STZ‐induced diabetes injected with CHIP overexpressed WJMSCs (STZ + CHIP‐WJMSCs), STZ‐induced diabetes transplanted with CHIP knockdown WJMSCs (STZ + shCHIP‐WJMSCs), and STZ‐induced diabetic rats infused with PTEN knockdown WJMSCs (STZ + shPTEN‐WJMSCs) after the rats were fasted for 14 h. (c) Morphological assessment of cardiac tissues in different experimental groups. (d) Echocardiographic evaluation of cardiac function in different experimental groups (control, STZ, STZ + WJMSCs, STZ + CHIP‐WJMSCs, STZ + shCHIP‐WJMSCs, and STZ + shPTEN‐WJMSCs). (e) Total cell lysate from the left ventricle was quantified and measured using Western blot. Protein expression levels of the apoptosis (p‐AKT, Bax, and Cyt‐c) and oxidative stress markers (catalase, SOD2, and gp91 PHOX ) were assessed. GAPDH act as a loading control. Values shown are mean ± SD. Quantification of the results are shown ( n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 shows the significance. CHIP, carboxyl terminus of Hsc70 interacting protein; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; PTEN, phosphatase and tensin homolog; STZ, streptozotocin; WJMSCs, Wharton's jelly derived mesenchymal stem cells

Article Snippet: Lentiviral GFP‐CHIP was purchased from the Sino biological (RG83573‐ACGLN), while the lentiviral expressing small hairpin RNAs (shRNAs), including shcontrol (pLAS.Void), shCHIP (TRCN0000007528 NM_005861), shPTEN (TRCN0000002746 NM_000314), and lentiviral packaging plasmids (pCMVΔR8.91 and pMD.G), were obtained from the national RNAi core facility (Academia Sinica).

Techniques: Expressing, Injection, Western Blot, Derivative Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Differential HspBP1 expression accounts for the greater vulnerability of neurons than astrocytes to misfolded proteins

doi: 10.1073/pnas.1710549114

Figure Lengend Snippet: Hsp70-dependent association of CHIP with mHtt. (A) PU-H71 induction promoted the association of transfected Htt-73Q with CHIP (Upper) and also increased the expression of Hsp70 (Lower). The ratios of immunoprecipitated CHIP to Htt-73Q in transfected HEK293 cells untreated (control) or treated with PU-H71 from three independent experiments are also shown. (B) Knocking down Hsp70 via siRNA abolished the coimmunoprecipitation of Htt-73Q with CHIP (Upper) and diminished the level of Hsp70 in transfected HEK293 cells (Lower). Right shows the ratios of CHIP to Htt-73Q in coimmunoprecipitation from three independent experiments. *P < 0.05, ****P < 0.0001 (unpaired two-tailed Student’s t test). Data are represented as mean ± SEM. Error bars indicate SEM.

Article Snippet: The shRNA of CHIP and negative control shRNA were products of Origene (TL503190).

Techniques: Transfection, Expressing, Immunoprecipitation, Control, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Differential HspBP1 expression accounts for the greater vulnerability of neurons than astrocytes to misfolded proteins

doi: 10.1073/pnas.1710549114

Figure Lengend Snippet: High activity of CHIP contributes to astrocytic resistance to hyperthermia. (A) Apparent demise of cultured cortical neurons at DIV 10–12 after heat-shock stress (45 °C, 30 min). The same heat-shock stress (45 °C, 30 min) did not affect viability of cultured cortical astrocytes at DIV 40–45. Neurons and astrocytes were fixed and immunostained at 24 h after heat shock. (B) CHIP knockdown caused death of a portion of CHIP shRNA-transfected astrocytes, which was characterized by shrunken nucleus, at 24 h after heat shock (45 °C, 30 min). Arrows indicate corpse of astrocytes. (C) Quantitative results of Fig. 5 A and B. At least 10 visual fields were randomly picked up for each group, and the live cells in each visual field were quantified. (D and E) TUNEL assay showing increased susceptibility of CHIP knockdown astrocytes to heat-shock stress (45 °C, 30 min). Arrows indicate apoptotic astrocytes (TUNEL-positive cells indicated by green fluorescence). ***P < 0.001, ****P < 0.0001 (unpaired two-tailed Student’s t test); ns, not significant. Data are represented as mean ± SEM. Error bars indicate SEM. (Scale bar, 10 μm.)

Article Snippet: The shRNA of CHIP and negative control shRNA were products of Origene (TL503190).

Techniques: Activity Assay, Cell Culture, Knockdown, shRNA, Transfection, TUNEL Assay, Fluorescence, Two Tailed Test