Figure 8.
Dynamic regulation of Mi-2β plays a key role for the induction of stress response and the recovery to homeostasis.(A) A transient decrease in Chd4 (Mi-2β) expression was detected by RNA-seq during the time course of TS and was validated by qPCR. The means and SEM of expression data in each group of qPCR samples are shown (n = 3 for each group). *, P < 0.01; **, P < 0.005 (one-way ANOVA followed by Dunnett’s test). (B) Immunohistochemistry for Mi-2β protein expression in WT and tape-stripped (6 and 24 h) epidermis. Scale bar = 10 µm. Arrows point to nuclei with reduced Mi-2β expression. (C) ChIP-qPCR for Mi-2β at select target sites associated with stress response genes (e.g., Aloxe3, Mapk6, Ppid, Jun, and JunB) at 6 h after TS. Krt14 was used as a positive control bound by Mi-2β but not affected by its deletion, and Myod1 was used as a negative control not bound by Mi-2β. Relative fold enrichment with mean and SEM is shown (n = 5 for each gene target tested by ChIP-qPCR). Significance in enrichment (*, P < 0.05) was calculated by a two-tailed unpaired t test. (D) Experimental design for testing the induction and recovery phase after TS in WT and Mi2Δ mice. For evaluating the induction phase, depletion of Mi2Δ in the epidermis was performed by two i.p. injections of 4-OHT (days −7 and −4). For evaluating the recovery phase, mosaic depletion of Mi-2β in the epidermis was achieved by one i.p. injection of 4-OHT (day −7). WT and Mi2Δ mice were then tape-stripped (day 0), and skin was harvested and evaluated by histochemistry and immunofluorescence (day 5). (E) TEWL was measured before (day 0; left) and up to 6 h after TS of WT and Mi2Δ mice (right). The change in TEWL at the indicated time point (1, 3, and 6 h) after TS was calculated as a percentage of the change immediately after TS. Means and SEM for data points in each group are shown (n = 8 for each group). (F) Representative phenotypes (upper panel) and H&E staining (lower panel) of WT and Mi2Δ mosaic skin at 5 d after barrier disruption are shown (left). Not-injured (NI) areas in WT and Mi2Δ mosaically depleted skin are shown as controls. Epidermal thickness measurements are shown in a bar graph (right) with mean and SEM (n = 5 for each group). P values were calculated by one-way ANOVA followed by Dunnett’s test. Scale bar = 25 µm. (G) Immunofluorescence detection for Ki67 (top) and K6 (bottom) expression in regions with or without barrier disruption in WT and Mi2Δ mice (left). The percentage of Ki67-positive cells in each group is shown as a bar graph with mean and SEM (right; n = 4 for each group). P values were calculated by one-way ANOVA followed by Dunnett’s test. Scale bar = 10 µm (Ki67) and 25 µm (K6). qPCR data of three experiments with a total of three mice per group are shown in A; immunohistochemistry shown in B is from one representative of three independent experiments performed with a total of three mice per group; ChIP-q-PCR shown in C is from two experiments with a total of five WT mice and five mice from 6 h after TS; TEWL data shown in E are from four independent experiments with a total of four to six mice per group. Histological studies shown in F and G are representative of two independent experiments with a total of five mice per group.

Dynamic regulation of Mi-2 β plays a key role for the induction of stress response and the recovery to homeostasis. (A) A transient decrease in Chd4 (Mi-2β) expression was detected by RNA-seq during the time course of TS and was validated by qPCR. The means and SEM of expression data in each group of qPCR samples are shown (n = 3 for each group). *, P < 0.01; **, P < 0.005 (one-way ANOVA followed by Dunnett’s test). (B) Immunohistochemistry for Mi-2β protein expression in WT and tape-stripped (6 and 24 h) epidermis. Scale bar = 10 µm. Arrows point to nuclei with reduced Mi-2β expression. (C) ChIP-qPCR for Mi-2β at select target sites associated with stress response genes (e.g., Aloxe3, Mapk6, Ppid, Jun, and JunB) at 6 h after TS. Krt14 was used as a positive control bound by Mi-2β but not affected by its deletion, and Myod1 was used as a negative control not bound by Mi-2β. Relative fold enrichment with mean and SEM is shown (n = 5 for each gene target tested by ChIP-qPCR). Significance in enrichment (*, P < 0.05) was calculated by a two-tailed unpaired t test. (D) Experimental design for testing the induction and recovery phase after TS in WT and Mi2Δ mice. For evaluating the induction phase, depletion of Mi2Δ in the epidermis was performed by two i.p. injections of 4-OHT (days −7 and −4). For evaluating the recovery phase, mosaic depletion of Mi-2β in the epidermis was achieved by one i.p. injection of 4-OHT (day −7). WT and Mi2Δ mice were then tape-stripped (day 0), and skin was harvested and evaluated by histochemistry and immunofluorescence (day 5). (E) TEWL was measured before (day 0; left) and up to 6 h after TS of WT and Mi2Δ mice (right). The change in TEWL at the indicated time point (1, 3, and 6 h) after TS was calculated as a percentage of the change immediately after TS. Means and SEM for data points in each group are shown (n = 8 for each group). (F) Representative phenotypes (upper panel) and H&E staining (lower panel) of WT and Mi2Δ mosaic skin at 5 d after barrier disruption are shown (left). Not-injured (NI) areas in WT and Mi2Δ mosaically depleted skin are shown as controls. Epidermal thickness measurements are shown in a bar graph (right) with mean and SEM (n = 5 for each group). P values were calculated by one-way ANOVA followed by Dunnett’s test. Scale bar = 25 µm. (G) Immunofluorescence detection for Ki67 (top) and K6 (bottom) expression in regions with or without barrier disruption in WT and Mi2Δ mice (left). The percentage of Ki67-positive cells in each group is shown as a bar graph with mean and SEM (right; n = 4 for each group). P values were calculated by one-way ANOVA followed by Dunnett’s test. Scale bar = 10 µm (Ki67) and 25 µm (K6). qPCR data of three experiments with a total of three mice per group are shown in A; immunohistochemistry shown in B is from one representative of three independent experiments performed with a total of three mice per group; ChIP-q-PCR shown in C is from two experiments with a total of five WT mice and five mice from 6 h after TS; TEWL data shown in E are from four independent experiments with a total of four to six mice per group. Histological studies shown in F and G are representative of two independent experiments with a total of five mice per group.

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