Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: Immunohistochemical stainings for HSP60 A. and p27 Kip1 B. in pancreatic sections from db/db and db/m + mice were shown. Original magnification, ×400, scale bar: 100 μm; x1000, scale bar: 50 μm. The protein expressions of HSP60 C. and p27 Kip1 D. in pancreatic islets isolated from db/db and db/m + were determined by Western blotting. Moreover, the nuclear p27 Kip1 protein expression was also determined E. . Protein levels were quantified by densitometry and normalized by β-actin levels. Data are presented as means ± SEM ( n = 4). * P < 0.05, versus db/m + mice.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Immunohistochemical staining, Isolation, Western Blot, Expressing

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: Effects of AGEs on the cell viability A. , p27 Kip1 protein expression B. , and HSP60 protein expression C. in RINm5f cells were shown. Cells were treated with AGE-BSA (5-100 μg/ml in A or 0.02-1 μg/ml in B and C) for 24 hours. Cell viability was determined by WST-8 assay. The protein expression was determined by Western blotting. Protein levels were quantified by densitometry and normalized by GAPDH levels. Moreover, effects of AGEs on the cell number (D-a), cell hypertrophy index (D-b), and cell area (D-c) of RINm5f cells were investigated. Cells were treated with AGE-BSA (0.1-1 μg/ml) for 24 hours. The viable cell number was determined by trypan blue exclusion assay. The cell hypertrophy index and cell diameter were measured as described under “Materials and Methods”. Data are presented as means ± SEM (n ≥ 5). * P < 0.05, versus BSA.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Expressing, Western Blot, Trypan Blue Exclusion Assay

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: A. The effect of AGEs on RAGE protein expression in RINm5f cells. Cells were treated with AGE-BSA or non-glycated BSA (0.02-0.5 μg/ml) for 24 hours. The protein expression was determined by Western blotting. Protein levels were quantified by densitometry and normalized by GAPDH levels. Data are presented as means ± SEM (n ≥ 5). * P < 0.05, versus BSA. C: control, B: BSA, A: AGE-BSA. (B-E) After the pretreatment of RAGE neutralizing antibody (10 μg/ml) for 1 hour, RINm5f cells were treated with AGE-BSA or non-glycated BSA (0.5 μg/ml) for 24 hours. The protein expression of HSP60 B. , cell hypertrophy index C. , ATP content D. , and insulin production E. were detected as described under “Materials and Methods”. Data are presented as means ± SEM ( n = 4). * P < 0.05, versus BSA, # P < 0.05, versus AGE-BSA.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Expressing, Western Blot, Control

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: RINm5f cells were transfected with pM51-HSP60 (0-4 μg/ml) for 48 hours. The pM51 empty vector was as a negative control. Transfection of pM51-HSP60 or pM51 vector control (1 μg/ml) for 48 hours, and then cells were treated with AGE-BSA and non-glycated BSA (0.5 μg/ml) for 24 hours. The protein expressions of HSP60 and p27 Kip1 A. , cell hypertrophy index B. , cell diameter C. , insulin secretion D. , and ATP production E. were detected as described under “Materials and Methods”. Data are presented as means ± SEM ( n ≥ 5). * P < 0.01, versus BSA, # P < 0.01, versus pM51/AGE-BSA. NS: non-significant.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Transfection, Plasmid Preparation, Negative Control, Control

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: RINm5f cells were treated with AGE-BSA (0.5-50 μg/ml) and non-glycated BSA (50 μg/ml) for 24 hours. A. The levels of cellular H 2 O 2 were detected by ELISA. Data are presented as means ± SEM ( n ≥ 5). * P < 0.05, versus BSA. B. ROS production was also determined by flow cytometric assay. NAC, N-acetyl-L-cysteine. C. RINm5f cells were treated with AGE-BSA and non-glycated BSA (10 μg/ml) for 24 hours in the presence or absence of RAGE neutralizing antibody. The levels of cellular H 2 O 2 were detected by ELISA. D. Effect of antioxidant N-acetyl-L-cysteine (NAC) on HSP60 protein expression in AGE-BSA-treated RINm5f cells. After pretreatment with NAC (2 mM) for 1 hour, the cells were treated with AGE-BSA or non-glycated BSA (0.5 μg/ml) for 24 hours. The protein expression of HSP60 was determined by Western blotting. Protein levels were quantified by densitometry and normalized by GAPDH levels. Data are presented as means ± SEM ( n = 4). * P < 0.05, versus BSA, # P < 0.05, versus AGE-BSA. In some experiments, the H 2 O 2 productions in islets of db/db and db/m+ mice were measured E. . Data are presented as means ± SEM ( n ≥ 10). **P < 0.01, versus db/m+ mice.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Western Blot

Journal: Oncotarget

Article Title: The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction

doi: 10.18632/oncotarget.8604

Figure Lengend Snippet: The immunohistochemical staining for insulin (A-a), AGEs (A-b), and HSP60 (A-c) were performed on the pancreatic sections (islet areas) of normal subject and diabetic patient. Original magnification, ×400, scale bar: 100 μm; x1000, scale bar: 50 μm. Moreover, the islet area B. and β-cell area C. in islets of normal subject and diabetic patient with 6 random areas per section was determined by ImageJ software. Data are presented as mean ± SEM. * P < 0.05, diabetic patient versus normal subject.

Article Snippet: For immunohistochemistry, the primary antibodies for p27 Kip1 , AGEs (ab23722; Immunogen: AGE-BSA and AGE-human serum albumin (HSA); Cross-reacts with BSA and HSA alone < 1%; Abcam, Cambridge, MA, USA), HSP60, RAGE, and insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Techniques: Immunohistochemical staining, Staining, Software

Journal: Scientific reports

Article Title: IFITM proteins inhibit HIV-1 protein synthesis.

doi: 10.1038/s41598-018-32785-5

Figure Lengend Snippet: Figure 1. IFITMs inhibit HIV viral output and viral protein production in transfected cells. HEK293T cells were transfected with a titration of expression vectors for FLAG tagged IFITMs, with the total quantity of 0.5 μg, plus 0.5 μg of HIV-1 NL4-3 proviral DNA. (A) Levels of virus production were measured by p24 ELISA 48 h post-transfection, and (B) intracellular viral proteins and IFITMs were analyzed by immunoblotting. HEK293T cells were transfected with 0.5 μg of FLAG-tagged IFITM expression vectors and 0.5 μg of HIV-1 NL4-3 proviral DNA. Levels of virus production from indicated HIV-1 proviral DNAs were measured by (C) p24 ELISA and (D) reverse transcriptase (RT) activity assay and (E) immunoblotting of intracellular proteins 48 h post-transfection. (F) Mean fluorescence intensity (MFI) of GFP expression in HEK293T cells co-transfected with 0.5 μg CMV-driven GFP vector and 0.5 μg IFITM-expression vectors or empty vectors measured by flow cytometry 48 hours post-transfection. (G) HIV-1 NL4-3 and 89.6 viruses were produced from HEK293T cells transfected with vector or the indicated IFITMs in TZM-bl cells for 48 hours. Infectivity in TZM-bl cells was measured by luciferase activity assay 48 hours after infection with virus stock with equivalent p24 concentration. Data show mean + S.E.M. of more than 3 independent experiments. All differences were assessed with Student’s t-test and *indicates p < 0.05.

Article Snippet: ELISA plates (Nunc) were pre-coated with 5 μg/ml sheep anti-HIV-1 p24 antibody (Aalto Bio Reagents) at 4 C overnight.

Techniques: Transfection, Titration, Expressing, Virus, Enzyme-linked Immunosorbent Assay, Western Blot, Reverse Transcription, RT Activity Assay, Fluorescence, Plasmid Preparation, Flow Cytometry, Produced, Infection, Luciferase, Activity Assay, Concentration Assay

Journal: Scientific reports

Article Title: IFITM proteins inhibit HIV-1 protein synthesis.

doi: 10.1038/s41598-018-32785-5

Figure Lengend Snippet: Figure 2. Inducible expression of IFITMs after viral entry inhibits HIV viral output and viral protein production in infected SupT1 cells. (A) SupT1 cells were infected with the indicated dilutions of wild type HIV-1 NL4-3 inoculum and then treated with 1 μg/ml doxycycline to induce IFITMs post-entry and 5 μM AMD3100 to limit infection to a single cycle. At 72 h post-infection levels of virus production were measured by p24 ELISA and differences assessed by Two-way ANOVA with Bonferroni’s multiple comparison test. (B) Cellular viral proteins and IFITMs were analyzed by immunoblotting and (C) densitometry. Data show mean + S.E.M. of 3 independent experiments. Differences were assessed with Student’s t-test and *indicates p < 0.05.

Article Snippet: ELISA plates (Nunc) were pre-coated with 5 μg/ml sheep anti-HIV-1 p24 antibody (Aalto Bio Reagents) at 4 C overnight.

Techniques: Expressing, Infection, Virus, Enzyme-linked Immunosorbent Assay, Comparison, Western Blot

Journal: Scientific reports

Article Title: IFITM proteins inhibit HIV-1 protein synthesis.

doi: 10.1038/s41598-018-32785-5

Figure Lengend Snippet: Figure 3. Knockdown of IFITMs rescues HIV-1 output and viral protein production in TZM-bl cells. TZM-bl cells transduced with shRNAs against the indicated IFITMs or scrambled control (sc) were treated with 100IU/ml IFNβ for 72 hours. (A) Levels of IFITM expression were analyzed by immunoblotting. (B) Scrambled control TZM-bl cells were transfected with HIV-1 NL4-3 proviral DNA and treated with AMD3100 and 100IU/ml IFNβ for 4 h post-transfection. Level of virus production was measured by p24 ELISA 72 h post-transfection. TZM-bl cells transduced with shRNAs against the indicated IFITMs or scrambled control were transfected with HIV-1 NL4-3 proviral DNA and then treated with AMD3100 and IFNβ 4 h post-transfection. Level of virus output and viral proteins was measured by (C) p24 ELISA and (D) immunoblotting, respectively, at 72 hours post-transfection. (E) Immunoblotting was further analyzed by densitometry. Data show mean + S.E.M. of 3 independent experiments. Differences were assessed with Student’s t-test and *indicates p < 0.05.

Article Snippet: ELISA plates (Nunc) were pre-coated with 5 μg/ml sheep anti-HIV-1 p24 antibody (Aalto Bio Reagents) at 4 C overnight.

Techniques: Knockdown, Transduction, Control, Expressing, Western Blot, Transfection, Virus, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: IFITM proteins inhibit HIV-1 protein synthesis.

doi: 10.1038/s41598-018-32785-5

Figure Lengend Snippet: Figure 4. Knockdown of IFITMs rescues HIV-1 output and viral protein production in primary human T cells. Activated human CD4+ T cells were transduced with lentivirus expressing the indicated shRNAs against IFITMs or scrambled sequence (sc) for 48 hours. (A) Level of IFITM expression was analyzed by immunoblotting and (B) densitometry; data was normalized to GAPDH and scrambled control. Cells were then infected with HIV-1 89.6 with equivalent p24 concentrations. Cells were pre-treated with AMD3100 for 2 hours prior to infection and throughout. Viral proteins in infected cells was analyzed by immunoblotting. Immediately after infection (3 h post-infection), cells were treated with trypsin and washed with PBS before intracellular staining of p24 and flow cytometry to measure virus uptake. Intracellular levels of p24 and median fluorescence intensity are shown in (D) representative histograms and (E) summary bar chart. (F) Level of virus production was measured by p24 ELISA 72 hours post-infection and normalized. Levels of viral proteins in infected cells from one of the blood donors were shown by (G) immunoblotting and (H) densitometry. Data show mean + S.E.M. of 2 blood donors. Differences were assessed with Student’s t-test and *indicates p < 0.05.

Article Snippet: ELISA plates (Nunc) were pre-coated with 5 μg/ml sheep anti-HIV-1 p24 antibody (Aalto Bio Reagents) at 4 C overnight.

Techniques: Knockdown, Transduction, Expressing, Sequencing, Western Blot, Control, Infection, Staining, Flow Cytometry, Virus, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: IFITM proteins inhibit HIV-1 protein synthesis.

doi: 10.1038/s41598-018-32785-5

Figure Lengend Snippet: Figure 6. HIV-1 RNA is a determinant of IFITM-mediated inhibition of protein synthesis. (A) Normalized levels of unspliced viral transcripts (measured by qPCR) in the polysome fractions of HEK293T cells transfected with codon-optimized HIV-1 NL4-3 Gag DNA and IFITM expression vectors at 48 h post-transfection. (B) Levels of extracellular p24 in HEK293T cells transfected with codon-optimized HIV-1 Gag DNA measured by p24 ELISA 48 h post-transfection and normalized. Cellular levels of HIV-1 Gag (p55 and p24) were analyzed by (C) immunoblotting. (D) Levels of virus production in HEK293T cells transfected with 0.5 μg wild-type HIV-1 NL4-3 proviral DNA and 0.5 μg IFITM-expression plasmids or empty vector was measured by p24 ELISA 48 hours post-transfection and normalized. Cellular levels of HIV-1 Gag (p55 and p24) in cells transfected were analyzed by (E) immunoblotting and (F) densitometry. Data show mean + SEM of 3 independent experiments and differences were assessed by Student’s t-test, *denotes p < 0.05 compared to vector control.

Article Snippet: ELISA plates (Nunc) were pre-coated with 5 μg/ml sheep anti-HIV-1 p24 antibody (Aalto Bio Reagents) at 4 C overnight.

Techniques: Inhibition, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Virus, Plasmid Preparation, Control

Journal: Scientific reports

Article Title: IFITM proteins inhibit HIV-1 protein synthesis.

doi: 10.1038/s41598-018-32785-5

Figure Lengend Snippet: Figure 7. HIV Nef can help overcome IFITM-mediated restriction of protein synthesis. (A) Level of virus production in HEK293T cells transfected with 0.5 μg expression vectors for IFITMs and 0.5 μg HIV-1 NL4-3 proviral DNA with a deletion (ΔNef) was measured by p24 ELISA 48 hours post-transfection and the data normalized, fold change of virus production compared to vector control is indicated. Differences were assessed with Student’s t tests. (B) Intracellular level of p55/p24 and IFITMs was measured by immunoblotting. (C) SupT1 cells were infected with the indicated dilutions of wild type or Nef-deleted (ΔNef) HIV-1 NL4-3 inoculum and then treated with 1 μg/ml doxycycline to induce IFITM expression post-entry and 5 μM AMD3100 to limit infections to a single round. Level of virus production was measured by p24 ELISA 72 hours post-infection. Differences were assessed with Two-way ANOVA and Bonferroni post-tests. (D) C8166 cells constitutively expressing either vector control or IFITM1 were infected with either wild type or Nef-deleted (ΔNef) HIV-1 NL4-3. Levels of virus production were measured by p24 ELISA at the indicated time-points post-infection and were normalized to the levels of virus produced from vector controls. Differences were assessed with Two-way ANOVA. (E) HEK293T cells were transfected with 0.5 μg expression vectors for IFITMs and 0.5 μg HIV-1 NL4-3 proviral DNA with either wildtype NL4-3 nef or the indicated lentiviral nef alleles. Virus production was measured by p24 ELISA 48 hours post-transfection and the data normalized. Differences were assessed with Student’s t tests. (F) HEK293T cells were transfected with ΔNef HIV-1 NL4-3 proviral DNA, expression vectors for IFITMs and an increasing proportion of HIV-1 Nef-encoding vector versus empty vector in a fixed total quantity of 1 μg. Level of virus production was measured by p24 ELISA 48 hours post- transfection, while levels of viral proteins and IFITM-FLAG expression was analyzed by (G) immunoblotting. Differences were assessed by Student’s t-test. All data show mean + SEM from 3 independent experiments and *denotes p < 0.05.

Article Snippet: ELISA plates (Nunc) were pre-coated with 5 μg/ml sheep anti-HIV-1 p24 antibody (Aalto Bio Reagents) at 4 C overnight.

Techniques: Virus, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Control, Western Blot, Infection, Produced