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The 4E-BP1 depletion slightly rescues the skin barrier function in Rap EKO mice. ( a ) Schematic representation of the floxed Rptor locus and complete Eif4ebp1 knockout, showing PCR fragment lengths before and after recombination. Genomic DNA PCR analysis from mouse tails confirms successful recombination of the floxed Rptor region in the presence of K14-driven Cre and complete Eif4ebp1 knockout. ( b ) Western blot analysis of 4E-BP1 protein expression in back skin from indicated genotypes. ( c ) Macroscopic appearance and body weight measurements of control, Rap EKO , and dKO newborn mice at P0. ( d ) Representative toluidine blue dye penetration assay with newborn mice and the quantification of the blue stained area (n = 5). ( e ) Representative H&E-stained sections of back skin and tongue from control, Rap EKO , and dKO newborns at P0. Dashed lines indicate the basement membrane. Bar = 100 μm. Right panels show quantification of epidermal thickness and HF density (n = 5). ( f ) Left: immunohistochemical staining for <t>p-S6</t> and p-4E-BP1 in back skin sections at P0. Bar = 50 μm. Right: quantitative analysis of p-S6 and p-4E-BP1 immunoreactivity (n = 5). Dashed lines indicate the basement membrane. Data are presented as mean ± SEM; each dot represents an individual mouse. Statistical significance was determined by 2-way ANOVA with multiple comparisons. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .001. dKO denotes Rap EKO / Eif4ebp1 −/− , e denotes epidermis, and d denotes dermis. dKO, double-knockout; HF, hair follicle; K14, keratin 14; ns, not significant; p-4E-BP1, <t>phosphorylated</t> 4E-BP1; P0, postnatal day 0; P2, P0, postnatal day 2; p-S6, phosphorylated S6; WT, wild-type.
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The 4E-BP1 depletion slightly rescues the skin barrier function in Rap EKO mice. ( a ) Schematic representation of the floxed Rptor locus and complete Eif4ebp1 knockout, showing PCR fragment lengths before and after recombination. Genomic DNA PCR analysis from mouse tails confirms successful recombination of the floxed Rptor region in the presence of K14-driven Cre and complete Eif4ebp1 knockout. ( b ) Western blot analysis of 4E-BP1 protein expression in back skin from indicated genotypes. ( c ) Macroscopic appearance and body weight measurements of control, Rap EKO , and dKO newborn mice at P0. ( d ) Representative toluidine blue dye penetration assay with newborn mice and the quantification of the blue stained area (n = 5). ( e ) Representative H&E-stained sections of back skin and tongue from control, Rap EKO , and dKO newborns at P0. Dashed lines indicate the basement membrane. Bar = 100 μm. Right panels show quantification of epidermal thickness and HF density (n = 5). ( f ) Left: immunohistochemical staining for <t>p-S6</t> and p-4E-BP1 in back skin sections at P0. Bar = 50 μm. Right: quantitative analysis of p-S6 and p-4E-BP1 immunoreactivity (n = 5). Dashed lines indicate the basement membrane. Data are presented as mean ± SEM; each dot represents an individual mouse. Statistical significance was determined by 2-way ANOVA with multiple comparisons. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .001. dKO denotes Rap EKO / Eif4ebp1 −/− , e denotes epidermis, and d denotes dermis. dKO, double-knockout; HF, hair follicle; K14, keratin 14; ns, not significant; p-4E-BP1, <t>phosphorylated</t> 4E-BP1; P0, postnatal day 0; P2, P0, postnatal day 2; p-S6, phosphorylated S6; WT, wild-type.
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The 4E-BP1 depletion slightly rescues the skin barrier function in Rap EKO mice. ( a ) Schematic representation of the floxed Rptor locus and complete Eif4ebp1 knockout, showing PCR fragment lengths before and after recombination. Genomic DNA PCR analysis from mouse tails confirms successful recombination of the floxed Rptor region in the presence of K14-driven Cre and complete Eif4ebp1 knockout. ( b ) Western blot analysis of 4E-BP1 protein expression in back skin from indicated genotypes. ( c ) Macroscopic appearance and body weight measurements of control, Rap EKO , and dKO newborn mice at P0. ( d ) Representative toluidine blue dye penetration assay with newborn mice and the quantification of the blue stained area (n = 5). ( e ) Representative H&E-stained sections of back skin and tongue from control, Rap EKO , and dKO newborns at P0. Dashed lines indicate the basement membrane. Bar = 100 μm. Right panels show quantification of epidermal thickness and HF density (n = 5). ( f ) Left: immunohistochemical staining for <t>p-S6</t> and p-4E-BP1 in back skin sections at P0. Bar = 50 μm. Right: quantitative analysis of p-S6 and p-4E-BP1 immunoreactivity (n = 5). Dashed lines indicate the basement membrane. Data are presented as mean ± SEM; each dot represents an individual mouse. Statistical significance was determined by 2-way ANOVA with multiple comparisons. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .001. dKO denotes Rap EKO / Eif4ebp1 −/− , e denotes epidermis, and d denotes dermis. dKO, double-knockout; HF, hair follicle; K14, keratin 14; ns, not significant; p-4E-BP1, <t>phosphorylated</t> 4E-BP1; P0, postnatal day 0; P2, P0, postnatal day 2; p-S6, phosphorylated S6; WT, wild-type.
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The 4E-BP1 depletion slightly rescues the skin barrier function in Rap EKO mice. ( a ) Schematic representation of the floxed Rptor locus and complete Eif4ebp1 knockout, showing PCR fragment lengths before and after recombination. Genomic DNA PCR analysis from mouse tails confirms successful recombination of the floxed Rptor region in the presence of K14-driven Cre and complete Eif4ebp1 knockout. ( b ) Western blot analysis of 4E-BP1 protein expression in back skin from indicated genotypes. ( c ) Macroscopic appearance and body weight measurements of control, Rap EKO , and dKO newborn mice at P0. ( d ) Representative toluidine blue dye penetration assay with newborn mice and the quantification of the blue stained area (n = 5). ( e ) Representative H&E-stained sections of back skin and tongue from control, Rap EKO , and dKO newborns at P0. Dashed lines indicate the basement membrane. Bar = 100 μm. Right panels show quantification of epidermal thickness and HF density (n = 5). ( f ) Left: immunohistochemical staining for <t>p-S6</t> and p-4E-BP1 in back skin sections at P0. Bar = 50 μm. Right: quantitative analysis of p-S6 and p-4E-BP1 immunoreactivity (n = 5). Dashed lines indicate the basement membrane. Data are presented as mean ± SEM; each dot represents an individual mouse. Statistical significance was determined by 2-way ANOVA with multiple comparisons. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .001. dKO denotes Rap EKO / Eif4ebp1 −/− , e denotes epidermis, and d denotes dermis. dKO, double-knockout; HF, hair follicle; K14, keratin 14; ns, not significant; p-4E-BP1, <t>phosphorylated</t> 4E-BP1; P0, postnatal day 0; P2, P0, postnatal day 2; p-S6, phosphorylated S6; WT, wild-type.
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The 4E-BP1 depletion slightly rescues the skin barrier function in Rap EKO mice. ( a ) Schematic representation of the floxed Rptor locus and complete Eif4ebp1 knockout, showing PCR fragment lengths before and after recombination. Genomic DNA PCR analysis from mouse tails confirms successful recombination of the floxed Rptor region in the presence of K14-driven Cre and complete Eif4ebp1 knockout. ( b ) Western blot analysis of 4E-BP1 protein expression in back skin from indicated genotypes. ( c ) Macroscopic appearance and body weight measurements of control, Rap EKO , and dKO newborn mice at P0. ( d ) Representative toluidine blue dye penetration assay with newborn mice and the quantification of the blue stained area (n = 5). ( e ) Representative H&E-stained sections of back skin and tongue from control, Rap EKO , and dKO newborns at P0. Dashed lines indicate the basement membrane. Bar = 100 μm. Right panels show quantification of epidermal thickness and HF density (n = 5). ( f ) Left: immunohistochemical staining for <t>p-S6</t> and p-4E-BP1 in back skin sections at P0. Bar = 50 μm. Right: quantitative analysis of p-S6 and p-4E-BP1 immunoreactivity (n = 5). Dashed lines indicate the basement membrane. Data are presented as mean ± SEM; each dot represents an individual mouse. Statistical significance was determined by 2-way ANOVA with multiple comparisons. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .001. dKO denotes Rap EKO / Eif4ebp1 −/− , e denotes epidermis, and d denotes dermis. dKO, double-knockout; HF, hair follicle; K14, keratin 14; ns, not significant; p-4E-BP1, <t>phosphorylated</t> 4E-BP1; P0, postnatal day 0; P2, P0, postnatal day 2; p-S6, phosphorylated S6; WT, wild-type.
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The 4E-BP1 depletion slightly rescues the skin barrier function in Rap EKO mice. ( a ) Schematic representation of the floxed Rptor locus and complete Eif4ebp1 knockout, showing PCR fragment lengths before and after recombination. Genomic DNA PCR analysis from mouse tails confirms successful recombination of the floxed Rptor region in the presence of K14-driven Cre and complete Eif4ebp1 knockout. ( b ) Western blot analysis of 4E-BP1 protein expression in back skin from indicated genotypes. ( c ) Macroscopic appearance and body weight measurements of control, Rap EKO , and dKO newborn mice at P0. ( d ) Representative toluidine blue dye penetration assay with newborn mice and the quantification of the blue stained area (n = 5). ( e ) Representative H&E-stained sections of back skin and tongue from control, Rap EKO , and dKO newborns at P0. Dashed lines indicate the basement membrane. Bar = 100 μm. Right panels show quantification of epidermal thickness and HF density (n = 5). ( f ) Left: immunohistochemical staining for <t>p-S6</t> and p-4E-BP1 in back skin sections at P0. Bar = 50 μm. Right: quantitative analysis of p-S6 and p-4E-BP1 immunoreactivity (n = 5). Dashed lines indicate the basement membrane. Data are presented as mean ± SEM; each dot represents an individual mouse. Statistical significance was determined by 2-way ANOVA with multiple comparisons. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .001. dKO denotes Rap EKO / Eif4ebp1 −/− , e denotes epidermis, and d denotes dermis. dKO, double-knockout; HF, hair follicle; K14, keratin 14; ns, not significant; p-4E-BP1, <t>phosphorylated</t> 4E-BP1; P0, postnatal day 0; P2, P0, postnatal day 2; p-S6, phosphorylated S6; WT, wild-type.
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The 4E-BP1 depletion slightly rescues the skin barrier function in Rap EKO mice. ( a ) Schematic representation of the floxed Rptor locus and complete Eif4ebp1 knockout, showing PCR fragment lengths before and after recombination. Genomic DNA PCR analysis from mouse tails confirms successful recombination of the floxed Rptor region in the presence of K14-driven Cre and complete Eif4ebp1 knockout. ( b ) Western blot analysis of 4E-BP1 protein expression in back skin from indicated genotypes. ( c ) Macroscopic appearance and body weight measurements of control, Rap EKO , and dKO newborn mice at P0. ( d ) Representative toluidine blue dye penetration assay with newborn mice and the quantification of the blue stained area (n = 5). ( e ) Representative H&E-stained sections of back skin and tongue from control, Rap EKO , and dKO newborns at P0. Dashed lines indicate the basement membrane. Bar = 100 μm. Right panels show quantification of epidermal thickness and HF density (n = 5). ( f ) Left: immunohistochemical staining for p-S6 and p-4E-BP1 in back skin sections at P0. Bar = 50 μm. Right: quantitative analysis of p-S6 and p-4E-BP1 immunoreactivity (n = 5). Dashed lines indicate the basement membrane. Data are presented as mean ± SEM; each dot represents an individual mouse. Statistical significance was determined by 2-way ANOVA with multiple comparisons. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .001. dKO denotes Rap EKO / Eif4ebp1 −/− , e denotes epidermis, and d denotes dermis. dKO, double-knockout; HF, hair follicle; K14, keratin 14; ns, not significant; p-4E-BP1, phosphorylated 4E-BP1; P0, postnatal day 0; P2, P0, postnatal day 2; p-S6, phosphorylated S6; WT, wild-type.

Journal: JID Innovations

Article Title: The 4E-BP1 deletion modestly mitigates mTORC1-deficient epidermal barrier defects

doi: 10.1016/j.xjidi.2026.100453

Figure Lengend Snippet: The 4E-BP1 depletion slightly rescues the skin barrier function in Rap EKO mice. ( a ) Schematic representation of the floxed Rptor locus and complete Eif4ebp1 knockout, showing PCR fragment lengths before and after recombination. Genomic DNA PCR analysis from mouse tails confirms successful recombination of the floxed Rptor region in the presence of K14-driven Cre and complete Eif4ebp1 knockout. ( b ) Western blot analysis of 4E-BP1 protein expression in back skin from indicated genotypes. ( c ) Macroscopic appearance and body weight measurements of control, Rap EKO , and dKO newborn mice at P0. ( d ) Representative toluidine blue dye penetration assay with newborn mice and the quantification of the blue stained area (n = 5). ( e ) Representative H&E-stained sections of back skin and tongue from control, Rap EKO , and dKO newborns at P0. Dashed lines indicate the basement membrane. Bar = 100 μm. Right panels show quantification of epidermal thickness and HF density (n = 5). ( f ) Left: immunohistochemical staining for p-S6 and p-4E-BP1 in back skin sections at P0. Bar = 50 μm. Right: quantitative analysis of p-S6 and p-4E-BP1 immunoreactivity (n = 5). Dashed lines indicate the basement membrane. Data are presented as mean ± SEM; each dot represents an individual mouse. Statistical significance was determined by 2-way ANOVA with multiple comparisons. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .001. dKO denotes Rap EKO / Eif4ebp1 −/− , e denotes epidermis, and d denotes dermis. dKO, double-knockout; HF, hair follicle; K14, keratin 14; ns, not significant; p-4E-BP1, phosphorylated 4E-BP1; P0, postnatal day 0; P2, P0, postnatal day 2; p-S6, phosphorylated S6; WT, wild-type.

Article Snippet: Primary antibodies and dilutions included Ki-67 (1:200, Abcam, ab15580), K14 (1:500, Abcam, ab181595), loricrin (1:200, Invitrogen, PA5-30583), keratin 10 (1:200, Santa Cruz, SC-23877), FLG (1:250, BioLegend, #905804), phosphorylated S6 (1:200, Cell Signaling Technology, #5364s), and phosphorylated 4EBP1 (1:200, Cell Signaling Technology, #2855).

Techniques: Knock-Out, Western Blot, Expressing, Control, Staining, Membrane, Immunohistochemical staining, Double Knockout