Figure 4.
Matrix metalloproteinases are required for COL4A2 incorporation, basement membrane expansion and hair follicle morphogenesis. (A) Schematic of the experimental procedure for MMP inhibition. (B) Snapshot images of eGFP-COL4A2 FRAP experiments in follicles from eGFP-Col4a2 explants, with or without MMP inhibitor. eGFP-COL4A2 (green) recovery after photobleaching is shown at the tip (blue arrowheads), lower stalk (orange arrowheads), and junction (gray arrowheads) regions in control and MMP-inhibited follicles (open arrowheads). Scale bar: 20 μm. (C) Normalized mean intensity values of eGFP-COL4A2 in bleached regions (n = 6 follicles, each from a separate explant in an independent experiment for the control, and n = 6 follicles from four explants, each from an independent experiment for the inhibitor-treated condition). Values normalized to 0 h. MMPi = MMP inhibitor treatment. Values: means ± SD. The control data of this experiment are shown in Fig. 3 B. (D) Quantification of the mean intensity at 3 h 30 min in C. Values: means ± SD. Two-tailed unpaired t tests were used. (E) BM length changes during the early stage of MMP inhibition (0–7 h) (n = 8 follicles from four explants, each from an independent experiment for the control, and n = 12 follicles from four explants, each from an independent experiment for the inhibitor-treated). Re-photobleaching of the targeted BM was performed every 2–3 h. Values: means ± SD. Two-way ANOVA with Tukey’s post-hoc tests was used. (F) BM length changes during the later stage of MMP inhibition (16–23 h) (n = 9 follicles from six explants, each from an independent experiment for the control, and n = 5 follicles from three explants, each from an independent experiment for the inhibitor-treated). Re-photobleaching of the targeted BM was performed. Values: means ± SD. Two-tailed unpaired t tests were used. The control data of this experiment are shown in Fig. 2 C. (G–O) Morphological changes in follicles during the early stage of MMP inhibition (0–16 h). (G) Confocal images. (H) Shape factor: S = follicle length (LHF)/bulb width (WHF). Changes in the length of follicles (I), the normalized length (J), width (K), normalized width (L), shape factor changes (M), shape factor at 0 h (N), and at 16 h (O) (n = 10 follicles from five explants, each from an independent experiment for the control, and n = 14 follicles from four explants, each from an independent experiment for the inhibitor-treated). The values were normalized to 0 h (J and L). Values: means ± SD. Two-tailed unpaired t tests were used. Two-way ANOVA with Tukey’s post-hoc tests was used for J and L. Scale bars: 20 μm. (P–U) Morphological changes during the later stage of MMP inhibition (16–36 h). (P) Confocal images. Changes in follicle length (Q), width (R), width at 16 h (S), shape factor changes (T), and shape factor at 36 h (U). (n = 8 follicles from seven explants, each from an independent experiment for the control, and n = 8 follicles from five explants, each from an independent experiment for the inhibitor-treated). Values: means ± SD. Two-tailed unpaired t tests were used. Scale bars: 20 μm. All statistical tests in this figure assumed normal data distribution, but this was not formally tested.

Matrix metalloproteinases are required for COL4A2 incorporation, basement membrane expansion and hair follicle morphogenesis. (A) Schematic of the experimental procedure for MMP inhibition. (B) Snapshot images of eGFP-COL4A2 FRAP experiments in follicles from eGFP-Col4a2 explants, with or without MMP inhibitor. eGFP-COL4A2 (green) recovery after photobleaching is shown at the tip (blue arrowheads), lower stalk (orange arrowheads), and junction (gray arrowheads) regions in control and MMP-inhibited follicles (open arrowheads). Scale bar: 20 μm. (C) Normalized mean intensity values of eGFP-COL4A2 in bleached regions (n = 6 follicles, each from a separate explant in an independent experiment for the control, and n = 6 follicles from four explants, each from an independent experiment for the inhibitor-treated condition). Values normalized to 0 h. MMPi = MMP inhibitor treatment. Values: means ± SD. The control data of this experiment are shown in Fig. 3 B. (D) Quantification of the mean intensity at 3 h 30 min in C. Values: means ± SD. Two-tailed unpaired t tests were used. (E) BM length changes during the early stage of MMP inhibition (0–7 h) (n = 8 follicles from four explants, each from an independent experiment for the control, and n = 12 follicles from four explants, each from an independent experiment for the inhibitor-treated). Re-photobleaching of the targeted BM was performed every 2–3 h. Values: means ± SD. Two-way ANOVA with Tukey’s post-hoc tests was used. (F) BM length changes during the later stage of MMP inhibition (16–23 h) (n = 9 follicles from six explants, each from an independent experiment for the control, and n = 5 follicles from three explants, each from an independent experiment for the inhibitor-treated). Re-photobleaching of the targeted BM was performed. Values: means ± SD. Two-tailed unpaired t tests were used. The control data of this experiment are shown in Fig. 2 C. (G–O) Morphological changes in follicles during the early stage of MMP inhibition (0–16 h). (G) Confocal images. (H) Shape factor: S = follicle length (LHF)/bulb width (WHF). Changes in the length of follicles (I), the normalized length (J), width (K), normalized width (L), shape factor changes (M), shape factor at 0 h (N), and at 16 h (O) (n = 10 follicles from five explants, each from an independent experiment for the control, and n = 14 follicles from four explants, each from an independent experiment for the inhibitor-treated). The values were normalized to 0 h (J and L). Values: means ± SD. Two-tailed unpaired t tests were used. Two-way ANOVA with Tukey’s post-hoc tests was used for J and L. Scale bars: 20 μm. (P–U) Morphological changes during the later stage of MMP inhibition (16–36 h). (P) Confocal images. Changes in follicle length (Q), width (R), width at 16 h (S), shape factor changes (T), and shape factor at 36 h (U). (n = 8 follicles from seven explants, each from an independent experiment for the control, and n = 8 follicles from five explants, each from an independent experiment for the inhibitor-treated). Values: means ± SD. Two-tailed unpaired t tests were used. Scale bars: 20 μm. All statistical tests in this figure assumed normal data distribution, but this was not formally tested.

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