Journal: Clinical Science (London, England : 1979)

Article Title: YTHDF2 regulates ACSL4-dependent ferroptosis of keratinocytes in diabetic wound healing

doi: 10.1042/CS20255877

Figure Lengend Snippet: ( A ) MDA contents were detected in the wound exudates from control and patients with diabetes mellitus ( n = 6). ( B ) MDA contents were detected in the skin tissues of control and diabetic rats ( n = 4). ( C ) Histological analysis to detect 4HNE levels in skin tissues from patients with diabetes mellitus ( n = 6) and diabetic rats ( n = 4). Original magnification × 400. ( D )TEM analysis of skin tissues from diabetic rats and control. Arrowheads indicate mitochondria. ( E and F ) The skin tissues from diabetic rats and controls were subjected to qPCR of ACSL4 ( E ) and immunoblotting ( F ) ( n = 4). ( G ) Skin tissues from patients with diabetes mellitus ( n = 3) and diabetic rats ( n = 4) were subjected to Histological analysis of ACSL4. Original magnification × 400. Bars represent the mean ± SD; * P < 0.05; ** P < 0.01; *** P < 0.001. ACSL4, acyl-CoA synthetase long-chain family member 4; MDA, malondialdehyde.

Article Snippet: Biotinylated random and ACSL4 RNA probes were added to the pretreated streptavidin magnetic beads (#HY-K0202, MedChemExpress, U.S.A.).

Techniques: Control, Western Blot

Journal: Clinical Science (London, England : 1979)

Article Title: YTHDF2 regulates ACSL4-dependent ferroptosis of keratinocytes in diabetic wound healing

doi: 10.1042/CS20255877

Figure Lengend Snippet: ( A ) Representative images of the wounds of control rats treated with control solvents and diabetic rats treated with Fer-1 or control solvents ( n = 4). ( B and C ) Masson’s staining of the wounds of control and diabetic rats treated with the formulas, n = 4; original magnification 20 × 100. ( B and D ) Histological analysis of 4HNE levels in skin tissues from control and diabetic rats treated with the formulations ( n = 4). Original magnification × 200. ( E ) MDA content in the wound tissues of control and diabetic rats treated with the formulations ( n = 4). ( F and G ) Skin tissues from control and diabetic rats treated with the formulas were subjected to real-time PCR for ACSL4 ( F ) and immunoblotting ( G ), n = 4. The significance of CON versus DM is shown as * and that of DM versus DM+Fer-1 as # . Bars represent the mean ± SD; * , # P <0.05; ** , ## P <0.01; *** P <0.001.

Article Snippet: Biotinylated random and ACSL4 RNA probes were added to the pretreated streptavidin magnetic beads (#HY-K0202, MedChemExpress, U.S.A.).

Techniques: Control, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Clinical Science (London, England : 1979)

Article Title: YTHDF2 regulates ACSL4-dependent ferroptosis of keratinocytes in diabetic wound healing

doi: 10.1042/CS20255877

Figure Lengend Snippet: ( A ) Cell viability of human primary keratinocytes treated with NG (normal glucose: 5.6 mM of glucose), G (mid-high glucose: 16.5 mM of glucose (HG16.5); high glucose: 33 mM of glucose (HG33)), or M (16.5 mM: 5.6 mM of glucose + 10.9 mM of Mannitol (HM16.5); 33 mM: 5.6 mM of glucose + 27.4 mM of Mannitol (HM33) for 72 h, n = 3. ( B and C ) Rate of cell death in human primary keratinocytes treated with HM33 and HG33 for 72 h, n = 3. ( D ) Rate of lipid peroxidation in human primary keratinocytes treated with HM33 or HG33 for 72 h, n = 3. ( E ) ACSL4 mRNA levels in HaCaT cells treated with NG, HM16.5, HG16.5, HM33, and HG33 for 72 hours were measured by qPCR ( n = 3). ( F ) ACSL4 protein levels in HaCaT cells treated with NG, HM16.5, HG16.5, HM33, and HG33 for 72 hours were measured by immunoblotting ( n = 3). NG: normal glucose, HM16.5:5.6 mM of glucose + 10.9 mM of Mannitol, HG16.5:16.5 mM of glucose, HM33:5.6 mM of glucose + 27.4 mM of Mannitol, HG33:33 mM of glucose. Bars represent the mean ± SD; * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Biotinylated random and ACSL4 RNA probes were added to the pretreated streptavidin magnetic beads (#HY-K0202, MedChemExpress, U.S.A.).

Techniques: Western Blot

Journal: Clinical Science (London, England : 1979)

Article Title: YTHDF2 regulates ACSL4-dependent ferroptosis of keratinocytes in diabetic wound healing

doi: 10.1042/CS20255877

Figure Lengend Snippet: ( A ) qPCR analysis of ACSL4 mRNA expression in control and ACSL4 overexpression HaCaT cells, n = 3. ( B ) Immunoblotting analysis of ACSL4 protein expression in control and ACSL4 overexpression HaCaT cells. ( C ) Cell viability of HaCaT cells transfected with control and ACSL4 overexpression plasmids, n = 4. ( D ) Rate of cell death of HaCaT cells transfected with control and ACSL4 overexpression plasmids, n = 3. ( E ) Rate of lipid peroxidation of HaCaT cells transfected with control and ACSL4 overexpression plasmids, n = 3. ( F and G ) Representative images of in vitro wound-healing assays in HaCaT cells transfected with control and ACSL4 overexpression plasmids. HaCaT cells were scratched 72 hours after plasmids transfection, n = 3. Bars represent the mean ± SD; * P <0.05; ** P <0.01. ACSL4, acyl-CoA synthetase long-chain family member 4.

Article Snippet: Biotinylated random and ACSL4 RNA probes were added to the pretreated streptavidin magnetic beads (#HY-K0202, MedChemExpress, U.S.A.).

Techniques: Expressing, Control, Over Expression, Western Blot, Transfection, In Vitro

Journal: Clinical Science (London, England : 1979)

Article Title: YTHDF2 regulates ACSL4-dependent ferroptosis of keratinocytes in diabetic wound healing

doi: 10.1042/CS20255877

Figure Lengend Snippet: ( A ）High (33 mM) glucose and mannitol-stimulated HaCaT cells were treated with actinomycin D (5 μg/mL) for 0, 3, 6, and 9 hours. The expression of ACSL4 mRNA was analyzed using qPCR ( n = 3). ( B ) Pull-down and silver staining analysis of ACSL4 mRNA-binding proteins in HaCaT cells. Arrowheads indicate YTHDF2 protein. ( C ) Immunoblot analysis of YTHDF2 protein expression in HaCaT cells treated with NG, HM16.5, HG16.5, HM33, or HG33 for 72 hours ( n = 3). ( D ) Skin tissues from patients with diabetes ( n = 3) and diabetic rats ( n = 4) were subjected to histological analysis for YTHDF2 expression. Original magnification × 200. ( E ) Pull-down and Immunoblotting analyses of ACSL4 mRNA binding to YTHDF2 protein from HaCaT lysates. Arrowheads indicate YTHDF2 protein. ( F ) MeRIP-qPCR analysis of N6-methyladenosine levels in ACSL4 mRNA in HaCaT cells treated with normal glucose ( n = 3). ( G ) RIP-qPCR and RNA affinity isolation analysis of the interaction between the YTHDF2 protein and ACSL4 mRNA in HaCaT cells ( n = 3). ( H ) The N6-methyladenosine modification of ACSL4 WT plasmid and mutation (MUT) plasmid were transfected in the 293 T cells. RIP-qPCR and RNA affinity isolation analysis of the interaction between YTHDF2 protein and ACSL4 mRNA in 293 T cells. ( n = 3). NG: normal glucose, HM16.5: 5.6 mM of glucose + 10.9 mM of mannitol, HG16.5: 16.5 mM of glucose, HM33: 5.6 mM of glucose + 27.4 mM of mannitol, HG33: 33 mM of glucose. Bars represent the mean ± SD; * P <0.05; ** P <0.01; *** P <0.001. ACSL4, acyl-CoA synthetase long-chain family member 4; YTHDF2, YTH N6-methyladenosine RNA binding protein 2.

Article Snippet: Biotinylated random and ACSL4 RNA probes were added to the pretreated streptavidin magnetic beads (#HY-K0202, MedChemExpress, U.S.A.).

Techniques: Expressing, Silver Staining, Binding Assay, Western Blot, Isolation, Modification, Plasmid Preparation, Mutagenesis, Transfection, RNA Binding Assay

Journal: Clinical Science (London, England : 1979)

Article Title: YTHDF2 regulates ACSL4-dependent ferroptosis of keratinocytes in diabetic wound healing

doi: 10.1042/CS20255877

Figure Lengend Snippet: ( A ) qPCR analysis of YTHDF2 mRNA expression in control and YTHDF2 knockdown HaCaT cells, n = 3. ( B ) qPCR analysis of ACSL4 mRNA expression in control and YTHDF2 knockdown HaCaT cells, n = 3. ( C ) Control and YTHDF2 knockdown HaCaT cells were subjected to immunoblotting of YTHDF2 and ACSL4 protein expression, n = 3. ( D ) Control and YTHDF2 knock down in HaCaT cells were treated with actinomycin D (5 μg/mL) for 0, 3, 6, and 9 hours, and cells were subjected to qPCR of ACSL4 mRNA. ( E, F ) Representative images of in vitro wound-healing assays in HaCaT cells transfected with control, siYTHDF2 and stimulated with ACSL4 inhibitor PRGL493. HaCaT cells were scratched at 48 hours after siRNAs transfection, n = 3. Bars represent the mean ± SD; * P <0.05; ** P <0.01; *** P <0.001. ACSL4, acyl-CoA synthetase long-chain family member 4; YTHDF2, YTH N6-methyladenosine RNA binding protein 2.

Article Snippet: Biotinylated random and ACSL4 RNA probes were added to the pretreated streptavidin magnetic beads (#HY-K0202, MedChemExpress, U.S.A.).

Techniques: Expressing, Control, Knockdown, Western Blot, In Vitro, Transfection, RNA Binding Assay

Journal: Clinical Science (London, England : 1979)

Article Title: YTHDF2 regulates ACSL4-dependent ferroptosis of keratinocytes in diabetic wound healing

doi: 10.1042/CS20255877

Figure Lengend Snippet: ( A ) Representative images of the wounds of SD rats treated with YTHDF2-knockdown adenovirus and control adenovirus ( n = 5). ( B ) Skin tissues from SD rats treated with YTHDF2-knockdown adenovirus and control adenovirus were subjected to immunoblotting of YTHDF2, control adenovirus rats n = 3, YTHDF2-knockdown adenovirus n = 4. ( C ) MTC staining of the wounds of SD rats treated with YTHDF2-knockdown adenovirus and control adenovirus, n = 5. Original magnification × 20, × 100.( D ) Histological analysis to detect 4HNE levels in skin tissues from SD rats treated with YTHDF2-knockdown adenovirus and control adenovirus, n = 5. ( E ) Skin tissues from SD rats treated with YTHDF2-knockdown adenovirus and control adenovirus were subjected to immunoblotting of ACSL4, n = 3. Bars represent the mean ± SD; * P <0.05; ** P <0.01; *** P <0.001. SD, Sprague-Dawley; YTHDF2, YTH N6-methyladenosine RNA binding protein 2

Article Snippet: Biotinylated random and ACSL4 RNA probes were added to the pretreated streptavidin magnetic beads (#HY-K0202, MedChemExpress, U.S.A.).

Techniques: Knockdown, Control, Western Blot, Staining, RNA Binding Assay

Journal: Clinical Science (London, England : 1979)

Article Title: YTHDF2 regulates ACSL4-dependent ferroptosis of keratinocytes in diabetic wound healing

doi: 10.1042/CS20255877

Figure Lengend Snippet: YTHDF2 interacts with and decays ACSL4 mRNA in an N6-methyladenosine-dependent manner in normal skin keratinocytes. In diabetic keratinocytes, YTHDF2 down-regulation induces ACSL4 mRNA up-regulation by decreasing ACSL4 mRNA decay, which causes lipid ROS in keratinocytes, ultimately leading to ferroptosis and delayed diabetic wound healing. ACSL4, acyl-CoA synthetase long-chain family member 4; YTHDF2, YTH N6-methyladenosine RNA binding protein 2.

Article Snippet: Biotinylated random and ACSL4 RNA probes were added to the pretreated streptavidin magnetic beads (#HY-K0202, MedChemExpress, U.S.A.).

Techniques: RNA Binding Assay