Journal: Molecular Medicine Reports

Article Title: Critical role of SIRT1 upregulation on the protective effect of lncRNA ANRIL against hypoxia/reoxygenation injury in H9c2 cardiomyocytes

doi: 10.3892/mmr.2021.12186

Figure Lengend Snippet: ANRIL upregulates SIRT1 expression following H/R injury. (A-C) H9c2 cardiomyocytes were cultured under a normoxic condition (normoxia) or exposed to hypoxia for 4 h followed by reoxygenation (H/R) for 2 to 24 h, as indicated. (A) ANRIL and (B) SIRT1 mRNA expression levels were determined by reverse transcription-quantitative PCR analysis. β-actin served as an internal control. Results are expressed as relative to the normoxia group. (C) SIRT1 protein expression was detected by an immunoblotting assay. β-actin served as a loading control. Results are representative of three independent experiments. (D-G) H9c2 cardiomyocytes were transiently transfected with pcDNA3.1-vector or pcDNA3.1-ANRIL plasmids (1, 2 or 4 µg per well). At 2 days after transfection, cells were cultured under normoxia or exposed to H/R (4 h hypoxia followed by 8 h reoxygenation) as indicated. The expression of (D) ANRIL following transfection was detection. (E) ANRIL and (F) SIRT1 mRNA expression levels, and (G) SIRT1 protein expression were determined. Data are presented as the mean ± SD. n=3. **P<0.01 vs. control group. NS, not significant; ANRIL, antisense non-coding RNA in the INK4 locus; SIRT1, sirtuin 1; H/R, hypoxia/reoxygenation.

Article Snippet: H9c2 cardiomyocytes (cat. no. CRL-1446) were purchased from the American Type Culture Collection and then cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in an incubator at 37°C with 5% CO 2 .

Techniques: Expressing, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Western Blot, Transfection, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: Critical role of SIRT1 upregulation on the protective effect of lncRNA ANRIL against hypoxia/reoxygenation injury in H9c2 cardiomyocytes

doi: 10.3892/mmr.2021.12186

Figure Lengend Snippet: ANRIL upregulates SIRT1 expression by sponging miR-181a. (A) Putative binding sites (red) between miR-181a and the 3′-UTR of ANRIL and SIRT1. Mutant sequences (blue) of ANRIL were depicted at the top. (B) RNA-binding protein immunoprecipitation assay was conducted to detect the enrichment of miR-181a and ANRIL by using Ago2 antibody to precipitate the lysates of H9c2 cells. Isotype IgG antibody was used as a control. Results are expressed as relative to IgG control (n=3). (C) 293T cells were co-transfected with NC-mimic or miR-181a mimic along with wt or mut ANRIL luciferase reporter plasmids. Luciferase activity was measured at 2 days after transfection. Results are expressed as relative to NC transfection (n=5). (D) H9c2 cells were transfected with pcDNA3.1-vector or pcDNA3.1-ANRIL plasmids, or si-NC or siANRIL as indicated. ANRIL (left) and miR-181a (right) expression levels were determined at 2 days after transfection. (E) 293T cells were co-transfected with NC-mimic or miR-181a mimic along with wt or mut SIRT1 luciferase reporter plasmids. Luciferase activity was measured at 2 days after transfection. Results are expressed as relative to NC transfection (n=5). (F and G) H9c2 cells were transfected with NC-mimic or miR-181a mimic, or antagomir-NC or antagomir-181a. At 2 days after transfection, the (F) mRNA expression levels of miR-181a and SIRT1, and the (G) protein expression levels of SIRT1 were determined (n=3). (H) H9c2 cells were transfected with pcDNA3.1-vector or pcDNA3.1-ANRIL plasmids (4 µg per well) along with or without 50 or 150 nM miR-181a mimic. At 2 days after transfection, cells were cultured under normoxia or exposed to H/R (4 h hypoxia followed by 8 h reoxygenation). SIRT1 protein expression was determined by an immunoblotting assay (n=3). Data are presented as the mean ± SD. **P<0.01. NS, not significant; ANRIL, antisense non-coding RNA in the INK4 locus; SIRT1, sirtuin 1; miR, microRNA; UTR, untranslated region; NC, negative control; wt, wild-type; mut, mutant; si/siRNA, small interfering RNA; H/R, hypoxia/reoxygenation.

Article Snippet: H9c2 cardiomyocytes (cat. no. CRL-1446) were purchased from the American Type Culture Collection and then cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in an incubator at 37°C with 5% CO 2 .

Techniques: Expressing, Binding Assay, Mutagenesis, RNA Binding Assay, Immunoprecipitation, Control, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Cell Culture, Western Blot, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Critical role of SIRT1 upregulation on the protective effect of lncRNA ANRIL against hypoxia/reoxygenation injury in H9c2 cardiomyocytes

doi: 10.3892/mmr.2021.12186

Figure Lengend Snippet: ANRIL protects against H/R injury. (A-E) H9c2 cells were transiently transfected with pcDNA3.1-vector or pcDNA3.1-ANRIL plasmids (2 or 4 µg per well). At 2 days after transfection, cells were cultured under normoxia or exposed to H/R (4 h hypoxia followed by 8 h reoxygenation). (A) ANRIL expression was determined via reverse transcription-quantitative PCR analysis. β-actin served as an internal control. Results are expressed as relative to normoxia group. (B) The release of LDH from cells was measured and expressed as a percentage of the total LDH activity (n=5). (C and D) Apoptosis was detected using a TUNEL assay and defined as a percentage of TUNEL positive cells (green) of the total number of cells (DAPI, blue) (n=12). Scale bar, 100 µm. (E) The expression levels of Bax, Bcl-2 and cleaved caspase-3 were determined using an immunoblotting assay (n=3). Data are presented as the mean ± SD. **P<0.01. NS, not significant; LDH, lactate dehydrogenase; ANRIL, antisense non-coding RNA in the INK4 locus; H/R, hypoxia/reoxygenation.

Article Snippet: H9c2 cardiomyocytes (cat. no. CRL-1446) were purchased from the American Type Culture Collection and then cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in an incubator at 37°C with 5% CO 2 .

Techniques: Transfection, Plasmid Preparation, Cell Culture, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Activity Assay, TUNEL Assay, Western Blot

Journal: Molecular Medicine Reports

Article Title: Critical role of SIRT1 upregulation on the protective effect of lncRNA ANRIL against hypoxia/reoxygenation injury in H9c2 cardiomyocytes

doi: 10.3892/mmr.2021.12186

Figure Lengend Snippet: ANRIL protects against H/R injury via the miR-181a/SIRT1 axis. (A-C) H9c2 cells were transiently transfected with pcDNA3.1-vector or pcDNA3.1-ANRIL plasmids (4 µg per well) along with or without 50 or 150 nM miR-181a mimic. At 2 days after transfection, cells were cultured under normoxia or exposed to H/R (4 h hypoxia followed by 8 h reoxygenation). (A) The release of LDH from cells was measured and expressed as a percentage of the total LDH activity (n=5). (B) Apoptosis was detected using a TUNEL assay and defined as a percentage of TUNEL positive cells (green) of the total number of cells (DAPI, blue) (n=12). Scale bar, 100 µm. (C) The expression levels of Bax, Bcl-2 and cleaved caspase-3 were analyzed. (D) The efficiency of siRNA targeting SIRT1 was confirmed in H9c2 cells under normoxia. (E-G) H9c2 cells were transiently transfected with pcDNA3.1-vector or pcDNA3.1-ANRIL plasmids (4 µg per well) along with or without 10 or 20 nM siSIRT1. At 2 days after transfection, cells were cultured under normoxia or exposed to H/R (4 h hypoxia followed by 8 h reoxygenation). (E) LDH release, (F) cell apoptosis (scale bar, 100 µm) and (G) expression of Bax, Bcl-2 and cleaved caspase-3 were analyzed as described in A-C. Data are presented as the mean ± SD. **P<0.01. NS, not significant; ANRIL, antisense non-coding RNA in the INK4 locus; H/R, hypoxia/reoxygenation; SIRT1, sirtuin 1; miR, microRNA; LDH, lactate dehydrogenase; si/siRNA, small interfering RNA; NC, negative control.

Article Snippet: H9c2 cardiomyocytes (cat. no. CRL-1446) were purchased from the American Type Culture Collection and then cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in an incubator at 37°C with 5% CO 2 .

Techniques: Transfection, Plasmid Preparation, Cell Culture, Activity Assay, TUNEL Assay, Expressing, Small Interfering RNA, Negative Control

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: H&E staining of dorsal skin of wild-type (WT) mice. TLR2 dynamic during the second telogen. ( A ) Confocal images of dorsal skin hair follicles co-immunostained for TLR2 and GFP. Scale bars are 10 µm. ( B ) A scatter graph shows a high level of correlation between TLR2 and GFP intensity. N=3. ( C ) Representative H&E staining of the dorsal skin of WT mice at indicated time points demonstrates the typical changes in hair follicle morphology. Scale bars are 100 µm. ( D ) Confocal images of dorsal skin hair follicles from p46 and p69 mice immunostained for TLR2. Scale bars are 10 µm. ( E ) Bar graph showing an elevated level of TLR2 in hair follicle stem cell (HFSC) of p69 mice (late second telogen) compared to p46 (early second telogen). N=4. Correlation analysis and the Mann-Whitney test were used to determine the statistical significance. All data are mean ± s.e.m. A p-value ≤ 0.05 was considered to be statistically significant.

Article Snippet: After 24 hr cells were washed with 1× D-PBS and incubated in HFDPC Basal Medium contains no growth supplement (Cell Applications, Inc cat.# 610-500) for the next 24 hr.

Techniques: Staining, MANN-WHITNEY

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: Representative confocal images of hair follicles immunostained for P-cad showing changes in the size of sHG during the progression of the hair cycle. At anagen onset, sHG cells proliferate and grow downward to envelop the dermal papilla. The proliferation of sHG cells results in a larger sHG compared to telogen. Dashed lines outline the sHG. Scale bars are 20 μm.

Article Snippet: After 24 hr cells were washed with 1× D-PBS and incubated in HFDPC Basal Medium contains no growth supplement (Cell Applications, Inc cat.# 610-500) for the next 24 hr.

Techniques:

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: ( A ) Representative confocal images of telogen hair follicles from Tlr2 lox/lox and TLR2 HFSC-KO mice immunostained for Ker15. Stars label the hair shaft. Scale bars are 10 μm. ( B ) Bar graph showing no difference in numbers of Ker15 + cells in bulge area in Tlr2 lox/lox compared with TLR2 HFSC-KO telogen hair follicles from images in A. N=4 and 5 mice for Tlr2 lox/lox and TLR2 HFSC-KO respectively. ( C ) Telogen hair follicles from Tlr2 lox/lox and TLR2 HFSC-KO mice immunostained for CD34. Scale bars are 10 μm. ( D ) Quantification of CD34 + cell numbers in bulge area in images from C showing no difference in Tlr2 lox/lox compared with TLR2 HFSC-KO follicles. N=5 per group. ( E ) Representative confocal images of Tlr2 lox/lox and TLR2 HFSC-KO telogen hair follicles immunostained for Sox9. Scale bars are 10 μm. ( F ) Bar graph showing no difference in Sox9 + cell numbers in bulge area in Tlr2 lox/lox and TLR2 HFSC-KO follicles from images in E. N=4. Mann-Whitney test was used to determine the statistical significance. All data are mean ± s.e.m. A p-value ≤ 0.05 was considered to be statistically significant.

Article Snippet: After 24 hr cells were washed with 1× D-PBS and incubated in HFDPC Basal Medium contains no growth supplement (Cell Applications, Inc cat.# 610-500) for the next 24 hr.

Techniques: MANN-WHITNEY

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: BMP signaling in the hair bulge of wild-type (WT) and TLR2 HFSC-KO mice. ( A ) GEO2R analysis of published RNA data from sorted follicle populations in the second telogen to anagen transition demonstrates the declined expression of Bmp7 mRNA and key transcriptional target of the BMP signaling pathway in the skin, Id1 , Id2 , and Id3 mRNA. ( B ) Western blot analysis of pNFkB, NFkB, pSMAD1/5/9, SMAD1, ikBα protein level in human epidermal keratinocytes treated with/without 10 µg/ml Pam3SCK4 and 10 ng/ml BMP4. ( C ) Bar graphs show the activation effect of BMP4 treatment on the BMP signaling which was abolished in the presence of TLR2 ligand Pam3SCK4. N=3 independent experiments. ( D ) Representative microphotographs of human hair follicle stem cells (HFSCs) pre-treated with 10 µg/ml MAb-mTLR2 or DMSO and co-cultured with/without 5 µg/ml Pam3SCK4. Representative images from at least three independent assays are shown. Scale bar 50 µm. ( E ) Bar graphs show increased proliferation of HFSC in the presence of TLR2 ligand Pam3SCK4 with no effect in MAb-mTLR2 pre-treated cells compared to control. N=6 independent experiments. ( F ) Dysregulated genes were confirmed by RNA sequencing or quantitative polymerase chain reaction (qPCR) of FACS-sorted HFSCs from the first telogen dorsal skin of Tlr2 lox/lox and TLR2 HFSC-KO mice. HFSCs from four mice in each group were sorted and sequenced. Dysregulated genes in RNA sequencing were those with an adjusted p-value <0.05. ( G ) Dysregulated pathways in HFSCs lacking TLR2. ( H ) Representative confocal images of competent telogen follicles from WT or TLR2 KO mice immunostained for CD34 and pSmad1/5/9. Stars label hair shaft. Scale bars are 10 μm. ( I ) Quantification of pSmad1/5/9 + bulge stem cell number in images from H shows significantly more pSmad1/5/9 + cells in WT hair follicle bulge region compared with TLR2 KO hair follicles. N=4 for each group. ( J ) Representative confocal images of Tlr2 lox/lox and TLR2 HFSC-KO telogen hair follicles immunostained for β-catenin showed no difference between the two groups. Stars label the hair shaft. Scale bars are 20 μm. All bar graphs are mean ± s.e.m. Non-parametric Mann-Whitney test ( I ), or Kruskal-Wallis test with Dunn’s multiple comparisons test ( C ), or one-way ANOVA with Tukey’s multiple comparisons test ( E ) was used to determine statistical differences. A p-value ≤ 0.05 was considered to be statistically significant. For the western blot (WB) quantification with experiments with n=3, the minimum achievable p-value for the non-parametric tests is 0.1000. Figure 4—figure supplement 1—source data 1. Uncropped WB gels.

Article Snippet: After 24 hr cells were washed with 1× D-PBS and incubated in HFDPC Basal Medium contains no growth supplement (Cell Applications, Inc cat.# 610-500) for the next 24 hr.

Techniques: Expressing, Western Blot, Activation Assay, Cell Culture, Control, RNA Sequencing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: The promotion of hair follicle regeneration after wound healing is dependent on TLR2. ( A ) Representative confocal images of Nile red-labeled (sebaceous gland) wild-type (WT) telogen hair follicles co-immunostained for MPO showing complete co-localization of MPO to the sebaceous gland. The isotype control panel shows the images of hair follicles stained with MPO isotype control antibody. Scale bars are 20 μm. ( B ) Representative confocal images of hair follicles from old vs young mice stained for MPO. Scale bars are 10 μm. ( C ) Quantification of MPO fluorescent intensity in B showing significantly less MPO in hair follicles from older mice. N=6 for each group. ( D ) Representative microphotographs of human hair follicle stem cells (HFSCs) pre-treated with 10 µg/ml MAb-mTLR2 or DMSO and co-cultured with/without 2.5 µM of CEP. Representative images from at least three independent assays are shown. Scale bar 50 µm. ( E ) Bar graphs show increased proliferation of HFSC in the presence of TLR2 endogenous ligand CEP compared to control, which was abolished in the presence of TLR2 blocking antibody. N=6 independent experiments. ( F ) Bar graphs show increased proliferation of human hair follicle dermal papilla cells incubated with 5 µM of CEP compared to the control. N=9 independent experiments. ( G ) Representative confocal images of Ki67 immunostaining of dorsal skin adjacent to wound of CEP-treated WT bone marrow transplanted WT and TLR2 KO mice. Scale bars are 50 μm. ( H ) Quantitative results showed increased Ki67 intensity in hair follicles around wounds of CEP-treated WT bone marrow transplanted WT mice with no differences in TLR2 KO with WT bone marrow. N=4 per group. ( I ) Representative photographs of dorsal skin (upper panels), inner skin flaps (middle panels), and representative confocal images of Ki67 immunostaining (lower panels) of vehicle- or CEP-treated WT or TLR2 KO skin. Scale bars are 1 mm for dorsal skin, 500 μm for skin flaps, and 50 μm for confocal images. ( J ) Bar graph showing quantification of hair follicle numbers of vehicle or CEP-treated skin from I. N=5 per group. ( K ) Bar graph showing quantification of Ki67 fluorescent intensity of Ki67 staining of vehicle or CEP-treated skin from I. N=4 per group. Unpaired two-tailed t-test ( C ), or non-parametric Mann-Whitney test ( H, J, K ), or Kruskal-Wallis test with Dunn’s multiple comparisons test ( F ), or one-way ANOVA with Tukey’s multiple comparisons test ( E ) was used to determine statistical differences. All bar graphs are mean ± s.e.m. A p-value ≤ 0.05 was considered to be statistically significant.

Article Snippet: After 24 hr cells were washed with 1× D-PBS and incubated in HFDPC Basal Medium contains no growth supplement (Cell Applications, Inc cat.# 610-500) for the next 24 hr.

Techniques: Labeling, Control, Staining, Cell Culture, Blocking Assay, Incubation, Immunostaining, Two Tailed Test, MANN-WHITNEY