Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

doi: 10.1186/s12967-024-05691-w

Figure Lengend Snippet: Elevated m6A methylation levels in HG induced HLEB3 and LECs of STZ mice. ( A - B ) m6A dot blot assay with anti-m6A antibody ( A ) and m6A ELISA kit ( B ), showing the level of m6A in HLEB3 cells induced by HG over different periods ( n = 3). ( C ) RT-qPCR analysis of the relative mRNA levels of m6A-related methylase and demethylase after 72 h of HG induction in HLEB3 cells ( n = 3). ( D - E ) Relative METTL3 levels in HLEB3 cells under NG and HG environment ( n = 3). ( F - G ) m6A dot blot assay ( F ) with anti-m6A antibody and m6A ELISA kit ( G ), showing the level of m6A in the lens capsule of STZ mice after induction for different times ( n = 3). ( H ) Representative images of the lens morphology in control mice (left) and 16-week STZ mice (right). ( I ) Relative m6A-related methylase and demethylase mRNA levels in lens capsules of STZ mice induced for 16 weeks ( n = 3). ( J-K ) Relative METTL3 levels in the lens capsule of control mice and 16-week STZ mice ( n = 3). ( L ) Representative images of HE staining and IHC staining of METTL3 in lens tissues of control mice (left) and STZ mice (right). Scale bar = 100 μm. ( M ) IHC score of METTL3 in lens tissues ( n = 5). Statistical significance was determined by a two-sided Student’s t-test as appropriate

Article Snippet: The human LEC line HLEB3 (ATCC, RRID: CVCL_6367) was cultured in Eagle’s medium (MEM, HyClone) that contained 20% FBS (Gibco) and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO 2 (normal glucose, NG).

Techniques: Methylation, Dot Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Capsules, Staining, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

doi: 10.1186/s12967-024-05691-w

Figure Lengend Snippet: HG-induced impaired autophagic flux and accelerated senescence in HLEB3 cells, and knockdown of METTL3 restored autophagic flux and delayed senescence. ( A - C ) Assessment of METTL3 silencing effectiveness in siMETTL3-transfected HLEB3 cells at mRNA level using RT-qPCR and protein level using western blot analysis ( n = 3). ( D ) m6A level in siMETTL3-transfected HLEB3 cells with an anti-m6A antibody. ( E - F ) HLEB3 cells were cultured in HG for 72 h post-transfection. Autophagy expression following various treatments using western blot ( n = 3). ( G-H ) Ad-mCherry-GFP-LC3B transfection into HLEB3 cells post-various treatments. Representative confocal microscopy images and quantification of fluorescent puncta per cell are shown. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 10 μm. ( I ) Representative images of autophagosomes visualized via the MDC method; Scale bar = 50 μm. ( J-K ) Senescence biomarkers levels in HLEB3 cells subjected to different treatments using western blot ( n = 3). ( L-M ) SA-β-Gal staining was used to detect the senescence levels in differently treated HLEB3 cells. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 100 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Article Snippet: The human LEC line HLEB3 (ATCC, RRID: CVCL_6367) was cultured in Eagle’s medium (MEM, HyClone) that contained 20% FBS (Gibco) and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO 2 (normal glucose, NG).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Western Blot, Cell Culture, Expressing, Confocal Microscopy, Staining

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

doi: 10.1186/s12967-024-05691-w

Figure Lengend Snippet: Overexpression of METTL3 further impairs HG-induced autophagic flux in HLEB3 cells and accelerates senescence. ( A - C ) Assessment of METTL3 overexpressing effectiveness in OE-METTL3-transfected HLEB3 cells at mRNA level using RT-qPCR and protein level using western blot analysis ( n = 3). ( D ) m6A level in OE-METTL3-transfected HLEB3 cells with an anti-m6A antibody. (E - F ) Autophagy-related biomarker expression in HLEB3 cells under different treatments using western blot ( n = 3). ( G - H ) Ad-mCherry-GFP-LC3B was transfected into HLEB3 cells, followed by various treatments. Representative confocal microscopy images and quantification of fluorescent puncta per cell are shown. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments ( n = 3). Scale bar = 10 μm. ( I ) Representative images of autophagosomes visualized via the MDC method. Scale bar = 50 μm. ( J - K ) Senescence biomarkers levels in HLEB3 cells subjected to different treatments using western blot ( n = 3). ( L - M ) SA-β-Gal staining was used to detect the senescence levels in differently treated HLEB3 cells. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 100 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Article Snippet: The human LEC line HLEB3 (ATCC, RRID: CVCL_6367) was cultured in Eagle’s medium (MEM, HyClone) that contained 20% FBS (Gibco) and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO 2 (normal glucose, NG).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Biomarker Discovery, Expressing, Confocal Microscopy, Staining

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

doi: 10.1186/s12967-024-05691-w

Figure Lengend Snippet: Knocking down METTL3 in vivo increases the autophagic flux of HLEB3 cells and decelerates senescence. ( A ) Fluorescent representative images of GFP expression in the anterior capsule membrane of mice lens transfected with AAV2. Scale bar = 100 μm. ( B - C ) METTL3 expression changes following AAV2-shMETTL3 transfection using western blot analysis ( n = 3). ( D - E ) Autophagy-related biomarker expression, and ( F - G ) senescence-related biomarker expression in LECs of control, STZ-AAV2-sh-NC, and STZ-AAV2-sh-METTL3 mice ( n = 3). ( H-I ) HE staining, IHC images and IHC score in lens tissues of mice: Control, STZ-AAV2 sh-NC, and STZ-AAV2 sh-METTL3. Data were quantitatively analyzed using randomized fields of view from 5 independent experiments. Scale bar = 50 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Article Snippet: The human LEC line HLEB3 (ATCC, RRID: CVCL_6367) was cultured in Eagle’s medium (MEM, HyClone) that contained 20% FBS (Gibco) and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO 2 (normal glucose, NG).

Techniques: In Vivo, Expressing, Membrane, Transfection, Western Blot, Biomarker Discovery, Control, Staining

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

doi: 10.1186/s12967-024-05691-w

Figure Lengend Snippet: SIRT1, a downstream target, negatively governed by METTL3-mediated m6A modification in vitro and in vivo. ( A - B ) SRAMP bioinformatics ( https://www.cuilab.cn/sramp/ ) analysis was used to predict m6A methylation sites on SIRT1 mRNA in humans and mice. ( C-D ) Representative images of IHC staining and IHC score of SIRT1 in control mice, STZ-AAV2 sh-NC mice, and STZ-AAV2 sh-METTL3 mice ( n = 5). ( E - F ) METTL3 and SIRT1 protein levels in HLEB3 cells when subjected to NG, HG, HG + vector, and HG + OE-METTL3 using western blot ( n = 3). ( G ) Detecting the SIRT1 enrichment level in HLEB3 cells after METTL3 knockdown using RIP-qPCR ( n = 3). Scale bar = 50 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Article Snippet: The human LEC line HLEB3 (ATCC, RRID: CVCL_6367) was cultured in Eagle’s medium (MEM, HyClone) that contained 20% FBS (Gibco) and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO 2 (normal glucose, NG).

Techniques: Modification, In Vitro, In Vivo, Methylation, Immunohistochemistry, Control, Plasmid Preparation, Western Blot, Knockdown

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

doi: 10.1186/s12967-024-05691-w

Figure Lengend Snippet: The activation of SIRT1 increases autophagy and delays the progression of senescence. ( A - B ) Detecting the SIRT1 and autophagy-related biomarker expression levels following various treatments using western blot ( n = 3). ( C - D ) Ad-mCherry-GFP-LC3B transfection into HLEB3 cells, followed by various treatments. Representative confocal microscopy images and quantification of fluorescent puncta per cell are shown. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 10 μm. ( E - F ) Detecting the senescence biomarkers in HLEB3 cells under various treatments using western blot ( n = 3). ( G - H ) SA-β-Gal staining was used to detect the senescence levels in differently treated HLEB3 cells. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 100 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Article Snippet: The human LEC line HLEB3 (ATCC, RRID: CVCL_6367) was cultured in Eagle’s medium (MEM, HyClone) that contained 20% FBS (Gibco) and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO 2 (normal glucose, NG).

Techniques: Activation Assay, Biomarker Discovery, Expressing, Western Blot, Transfection, Confocal Microscopy, Staining

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

doi: 10.1186/s12967-024-05691-w

Figure Lengend Snippet: METTL3 reduces SIRT1 mRNA stability in a YTHDF2-dependent manner. ( A ) Relative SIRT1 mRNA levels following the knockdown of the YTHDF family ( n = 3). YTHDF2 mRNA expression in OE-YTHDF2 ( B ) and siYTHDF2 ( C ), both in transfected HLEB3 cells ( n = 3). ( D ) Detecting the SIRT1 enrichment level in HLEB3 cells after YTHDF2 knockdown using RIP-qPCR ( n = 3). ( E - F ) YTHDF2 and SIRT1 protein levels in HG-induced HLEB3 cells transfected with si-NC + vector, siMETTL3, OE-YTHDF2, siMETTL3 + OE-YTHDF2 ( n = 3). ( G ) SIRT1 mRNA stability at different time points after Act D (10 μg/ml) treatment ( n = 3). ( H-I ) YTHDF2 and SIRT1 protein levels in HG-induced HLEB3 cells transfected with si-NC + vector, OE-METTL3, siYTHDF2 and OE-METTL3 + siYTHDF2 ( n = 3). ( J ) Schematic representation of METTL3-mediated regulation of SIRT1 in accelerating the progression of diabetes-associated cataracts via an m6A-dependent mechanism. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Article Snippet: The human LEC line HLEB3 (ATCC, RRID: CVCL_6367) was cultured in Eagle’s medium (MEM, HyClone) that contained 20% FBS (Gibco) and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO 2 (normal glucose, NG).

Techniques: Knockdown, Expressing, Transfection, Plasmid Preparation

Journal: Retrovirology

Article Title: Intestinal endothelial cells increase HIV infection and latency in resting and activated CD4 + T cells, particularly affecting CCR6 + CD4 + T cells

doi: 10.1186/s12977-023-00621-y

Figure Lengend Snippet: Fig. 1 IEC stimulation increases HIV infection of resting CD4 + T cells. Here and throughout the paper, boxplots show the median (horizontal line), and the central box extends to the 25th and 75th percentiles. The whiskers then extend 1.5 inter-quartile ranges or to the minimum or maximum observed value, whichever is closer to the median. A Infection rates in T cells stimulated with endothelial cells. Resting T cells were cultured alone (Resting), or co-cultured with human umbilical vein endothelial cells (EC), human lymphatic endothelial cells (LEC), or human intestinal endothelial cells (IEC). + and − indicate treatment with or without IFN-γ respectively in EC, LEC, or IEC. All T cells were infected with an HIV reporter virus expressing GFP 1 day after co-culture, and the %GFP+ cells were measured on day 6 post-infection. Samples were taken in triplicate for each donor, and different donors are represented by different symbols (n = 5). P-values are from post-hoc pairwise comparisons of marginal means based on beta generalized linear models (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). B Expression of activation markers versus GFP expression in IEC + stimulated resting CD4 + T cells. Similar to A, but on day 6 post-infection, T cells were stained for three activation markers: CD25, CD69, and HLA-DR. C Expression of activation markers and infection rates in T cells with and without IEC stimulation (n = 3), similar to ( B ). D Infection rates in resting memory and naïve T cells stimulated with IEC. Memory and naïve T cells were isolated, and the rest of the experimental procedures are the same as ( A ). Samples were taken in triplicate for each donor, and different donors are represented by different symbols (n = 3). P-values are from post-hoc pairwise comparisons of marginal means based on beta generalized linear models (****p<0.0001).

Article Snippet: LEC were obtained from ScienCell Research Laboratories (isolated from human lymph nodes) and cultured in basal endothelial cell medium supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution (P/S) (Invitrogen).

Techniques: Infection, Cell Culture, Virus, Expressing, Co-Culture Assay, Activation Assay, Staining, Isolation