Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: PCR primers used in this study

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques:

Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: Cordyceps cicadae polysaccharides administration attenuated cell apoptosis and epithelial–mesenchymal transition in the kidneys of diabetic nephropathy mice. (a, b) At the end of 12 weeks of treatment, the expression of miR ‐30a‐3p and TRIM16 was determined in the kidney tissues of mice in each group by RT‐PCR , n = 8. (c) The protein expression level of TRIM16 in kidney tissues of mice was analyzed by western blot, n = 5. (d, e) Protein expression of TRIM16 in the kidney tissues using IHC staining, scale bar = 50 μm, n = 5. (f) TUNEL staining was used to detect cell apoptosis in the kidney tissues, scale bar, 20 μm, n = 5. (g) Protein expression of Bcl‐2 and Bax in the kidney tissues were detected by IHC staining, scale bar = 50 μm, n = 5. (h) Representative images of immunofluorescence staining for P‐cadherin (red), N‐cadherin (green), and DAPI (blue), scale bar, 50 μm, n = 5, scale bar = 20 μm. Data are expressed as mean ± SD . * P < 0.05, ** P < 0.01.

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, TUNEL Assay, Staining, Immunofluorescence

Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: Expression of miR‐30a‐3p and TRIM16 in MPC5 podocytes treated with high glucose. The MPC5 podocytes were treated with normal glucose, mannitol, and high glucose conditions for 6, 12, and 24 h. (a) The expression level of miR‐30a‐3p in mouse podocyte was detected by RT‐qPCR. (b, c) The mRNA and protein expression of TRIM16 was determined by RT‐PCR and western blotting. * P < 0.05, ** P < 0.01.

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: miR‐30a‐3p regulates TRIM16 in MPC5 podocytes. (a) TRIM16 is a predicted target of miR‐30a‐3p by TargetScan. (b) A luciferase reporter plasmid containing wildtype or mutant TRIM16 was cotransfected into MPC5 podocytes with miR‐30a‐3p or miR‐NC. Luciferase activity was determined at 48 h after transfection using the dual‐luciferase assay and was normalized to Renilla activity. (c–e) RT‐PCR and western blot analysis of TRIM16 in podocytes transfected with miR‐30a‐3p or miR‐NC or miR‐30a‐3p inhibitor or inhibitor of negative control. Data are presented as mean ± SD, n = 3. ** P < 0.01, *** P < 0.001. ## P < 0.001.

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control

Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: Cordyceps cicadae polysaccharides protect mouse podocyte from high glucose induced apoptosis and epithelial–mesenchymal transition by controlling miR‐30a‐3p expression. (a) Relative miR‐30a‐3p and TRIM16 expression were detected by RT‐PCR and western blotting analysis. (b) Cell viability was determined by MTT assay. (c, d) The apoptosis of cells as detected by flow cytometry. (e) Representative images of TUNEL staining to illustrate apoptotic MPC5 podocytes in each group, cell nuclei were labeled in blue by DAPI, scale bar, 50 μm. (f) Representative immunofluorescence images of E‐cadherin (E‐cad, red) and N‐cadherin (N‐cad, green), cell nuclei were labeled in blue by DAPI, scale bar, 20 μm. Data are presented as mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, MTT Assay, Flow Cytometry, TUNEL Assay, Staining, Labeling, Immunofluorescence

Journal: Scientific Reports

Article Title: Withaferin A activates TRIM16 for its anti-cancer activity in melanoma

doi: 10.1038/s41598-020-76722-x

Figure Lengend Snippet: Knock-down of TRIM16 protects melanoma cells against WFA. ( A ), Western blot analysis of TRIM16 expression in MelJD and MelCV cells following TRIM16 knock-down. GAPDH was used as internal control. The full-length blots are presented in the Supplementary Fig. B. MelJD and MelCV cells were transfected with control siRNA (siControl) or TRIM16 siRNAs (siTRIM16) for 24 h, then treated with WFA for additional 48 h followed by ( B ), Alamar Blue cell viability and ( C ), and BrdU cell proliferation measurements. Results are mean ± SEM, differences in cell viability and proliferation were compared to the vehicle (DMSO) treated siRNA control cells. ( D ), MelJD and MelCV cells expressing TRIM16 siRNAs (siTRIM16) or control siRNA (siControl) were seeded for colony formation assay and treated with indictaed concentrations of WFA for 72 h then allowed for colonies to form for 10 days, followed by crystal violet staining. ( E ), Quantification of colony formation assay based on crystal violet absorbance (590 nm). Differences in colony formation were compared to the vehicle treated control siRNA. MelJD and MelCV cells were transfected with control siRNA (siControl) or TRIM16 siRNAs (siTRIM16) for 24 h, then treated with WFA for additional 48 h followed by ( F, ) MITOPROBE DILC 1 (5) and ( G ), SYTOX Green measurements. Results are mean ± SEM, differences in cell viability and proliferation were compared to the vehicle control siRNA control cells.

Article Snippet: Nitrocellulose membranes (GE HEALTHCARE, Rydalmere, NSW, Australia) were blocked with 5% (wt/vol) nonfat dry milk in Tris-buffered saline with Tween-20 (20 mM Tris–HCl (pH 7.6), 137 mM NaCl, 0.1% Tween-20), then incubated overnight at 4 °C with the following primary antibodies: TRIM16 (1:1000; BETHYL LABORATORIES, TX, USA) and GAPDH antibody (1:1000; SANTA CRUZ BIOTECHNOLOGIES, Texas, USA).

Techniques: Knockdown, Western Blot, Expressing, Control, Transfection, Colony Assay, Staining