Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Development of a 4D imaging method for visualizing basement membrane dynamics. (A) Schematic model of BM molecular structure and fluorescent protein insertion site in the collagen IV α2 chain. (B) Genotypes of pups from eGFP-Col4a2 het × eGFP-Col4a2 het crosses. Sample sizes in . (C) Appearance of 8-wk-old wild-type and eGFP-Col4a2 homo mice. (D) Immunofluorescence image of E15.5 embryos from eGFP-Col4a2 mice stained for eGFP. Scale bar: 3 mm. (E–H) Immunofluorescence images of adult tissues from eGFP-Col4a2 mice. (E) In P56 adult telogen dorsal skin from het mice, eGFP (green) colocalized with COL4A2 (magenta) in BMs of epidermis (white arrowheads), follicle epithelium (yellow arrowheads), and vessel-like tissues (open arrowheads). (F) In homo mice, eGFP (green) colocalizes similarly with collagen IV (magenta) and was also found in arrector pili muscles (arrows). The same field of view is presented in . (G) In adult kidneys, eGFP (green) was detected in Bouman’s capsules (closed arrowheads), the mesangial matrix (arrows), and collecting ducts (open arrowheads). (H) In adult brain, eGFP (green) localized to capillaries (closed arrowheads) and fractone-like structures (open arrowheads). DAPI (blue) = nuclear counterstaining. Scale bars: 50 μm. (I) Fluorescence images of cultured dorsal skin explants from E12.5 embryos of eGFP-Col4a2 mice. Magnified areas show primary (closed arrowheads) and secondary (open arrowheads) hair follicles. Scale bars: 200 μm. (J) Snapshot images of the 3D maximum projection of cultured E12.5 skin explants from eGFP-Col4a2 mice. Closed arrowheads indicate ring-like accumulations of the eGFP-COL4A2 signal at the neck regions of hair follicles. Hair follicles, indicated by closed arrowheads in the left panel, grow vertically from the plane of observation. The dashed rectangle indicates a follicle growing horizontally. The snapshot images in the right panels were obtained from a separate scan of the follicle outlined by the dashed rectangle in the 3D time-lapse maximum projection shown in the main image. The open arrowheads indicate vessel-like structures. Scale bars: 50 μm.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Imaging, Membrane, Immunofluorescence, Staining, Muscles, Capsules, Fluorescence, Cell Culture

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Generation of eGFP-Col4a2 knock-in mice. (A) Targeting strategy for generating eGFP-Col4a2 knock-in mice. An eGFP protein was inserted into the N-terminus region of the 7S domain of the COL4A2 protein (between A 28 and Q 29 ). To minimize the structural interference of the inserted fluorescent protein with COL4A2 protein structure and functions, a glycine- and serine-rich flexible linker, GGSGGSGGSGGS, was added to both ends of the EGFP protein. L, linker; LF, left forward primer; LR, left reverse primer; RF, right forward primer; RR, right reverse primer. (B) Image of PCR screening used to distinguish wild-type, heterozygous, and homozygous eGFP-Col4a2 mice. Fabpi-200: An internal control primer pair for detecting the Fabpi gene. A 50 bp DNA ladder RTU (GeneDirex) was used. (C) Genome sequences of boundaries between the Col4a2 gene and linkers. (D) Immunofluorescence images of E13.5, E15.5, and E17.5 embryonic tissues from eGFP-Col4a2 mice stained for eGFP. Scale bars: 3 mm. (E–G) Immunofluorescence images of tissues from eGFP-Col4a2 mouse embryos. (E) At E15.5 (left panel), the eGFP (green) signal is detected at the epidermal BM (white closed arrowheads) below the keratin 14-positive (magenta) basal epidermis, the BMs of the hair placode (yellow closed arrowheads), the dermal vessel-like structures (open arrowheads), and the panniculus carnosus muscle-like layers (arrows). At E17.5 (middle and right panels), eGFP (green) is detected in perlecan-positive (magenta) epidermal and vessel-like BMs. (F) In the E15.5 kidney, eGFP (green) appeared around the tubular epithelial structures (closed arrowheads). (G) In the E15.5 brain, eGFP (green) appears in pial BM zones (closed arrowheads) and vessel capillaries (arrows). DAPI (blue) was used for nuclear counterstaining. Scale bars: (E and F) 20 μm, (G) 200 μm. (H–K) Immunofluorescence images of adult tissues from eGFP-Col4a2 mice. (H) In P56 adult telogen dorsal skin tissues from het mice, eGFP (green) colocalizes with collagen IV (magenta) and perlecan (magenta) in the BMs of the epidermis (closed white arrowheads), hair follicle epithelium (closed yellow arrowheads), arrector pili muscle (arrows), and vessel-like tissues (open arrowheads). (I) In P56 adult telogen dorsal skin tissues from homo mice, eGFP (green) is distributed as it is in het mice and colocalizes with perlecan (magenta). (J) In adult kidneys, eGFP (green) is detected in Bouman’s capsules (closed arrowheads), the mesangial matrix (arrows), and collecting ducts (open arrowheads). The same field of view is presented in . (K) In the subventricular zone, eGFP (green) colocalizes with perlecan (magenta) in capillaries (closed arrowheads) and fractone-like structures (open arrowheads) around GFAP+ (magenta) astrocytes. DAPI (blue) was used for nuclear counterstaining. Scale bars: 50 μm. Source data are available for this figure: .

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Knock-In, Control, Immunofluorescence, Staining, Capsules

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Genotypes of pups from eGFP-Col4a2 het × eGFP-Col4a2 het crosses

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques:

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Representative time-lapse video capturing eGFP-COL4A2 signals in mouse embryonic dorsal skin explant cultures. Representative time-lapse video of the 3D maximum projection of cultured skin explants from E12.5 embryos of eGFP-Col4a2 mice, corresponding to . The BM was visualized with eGFP-COL4A2 (green). The BMs of hair follicles, interfollicular epidermis, and vessels are visualized. Live imaging was performed under a TCS SP8X (Leica) confocal microscope. Frames were collected approximately every 29 min and 24 s over the 19-h and 6-min time-lapse. The video is 13 s in total and displayed at ∼3.08 frames per second.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Cell Culture, Imaging, Microscopy

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Spatially distinct basement membrane expansion rates synchronize with directional cell movement. (A) Schematic of region definitions in the developing hair follicle. Tip: Interface region between the prematrix and dermal condensate, defined by two bending points at the follicle tip. Lower stalk: The lower half of the follicle extends to the tip. Upper stalk: The upper half extends to the junction region. Junction: Bending neck region where follicular epidermis meets interfollicular epidermis. BM dynamics were measured in these regions. (B) Confocal images of hair follicles in dorsal skin explants from eGFP-Col4a2 mice show that BM length changes over 7 h. ROIs (dashed rectangles) indicate photobleached areas. Re-photobleaching of the targeted BM was performed every 2–3 h to maintain clear photobleached edges. See for details. BM length changes were measured at the tip (blue lines), lower stalk (orange lines), and upper stalk (gray lines). Scale bar: 20 μm. (C) Percent BM length changes over a 7-h ( n = 9 follicles from seven explants, each from an independent experiment). Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. (D) Measurement of BM thickness in hair follicles of dorsal embryonic skin explants from eGFP-Col4a2 mice. BMs at the tip (blue lines), lower stalk (orange lines), and upper stalk (gray lines) were measured. Scale bars: 20 μm (left two images), 2 μm (right three images). (E) Quantification of BM thickness ( n = 13 follicles from three explants, each from an independent experiment). Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. (F) EdU incorporation assays. Dividing cells were labelled with EdU (magenta) with Hoechst counterstaining (blue). EdU-positive nuclei in the basal layer of the tip (blue line), lower stalk (orange line), and upper stalk (gray line) were measured across z-planes (−10, 0, +10, and +15 μm). Scale bar: 20 μm. (G) Percentage of EdU-positive nuclei in the tip, lower stalk, and upper stalk regions ( n = 18 follicles from seven explants, each from an independent experiment). Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. (H) Snapshot images of 3D time-lapse videos of developing hair follicles in eGFP-Col4a2; mem-tdTomato explants. The relative locations of the BM (lower bleached edge) and epithelial basal progenitors (white arrowheads) from reference points on the BM (upper bleached edge, yellow arrowheads) in lower (orange lines) and upper stalk regions (gray lines) were measured at 0 and 9 h 30 min. Re-photobleaching of the targeted BM was performed. See for detailed tracking procedures. Scale bar: 20 μm. (I) Normalized length changes in cell displacement and BM expansion ( n = 7 cells and BM regions from five follicles, each from a separate explant in an independent experiment). Values: means ± SD. Two-tailed unpaired t tests were used. (J) Normalized length changes (relative to cell displacement) of BM expansion and the value subtracted BM expansion from the cell displacement (data from ). Values: means ± SD. Two-tailed unpaired t tests were used. (K) A schematic of the composite cell and BM movement. All statistical tests in this figure assumed normal data distribution, but this was not formally tested.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Membrane, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Re-photobleaching procedures for the measurement of basement membrane expansion. (A) Images of re-photobleaching procedures (see Materials and methods for detailed procedures and explanations). A z-plane exhibiting sharp and narrow signals from the BM within the targeted area was selected for photobleaching (panels a, b, and b′). The ROIs (indicated by rectangular boxes) were set on the targeted z-plane. The ROI was photobleached across 10 ± 3 continuous z-planes (with 1 µm intervals), centered on the target z-plane. For example, the BMs in the tip and lower stalk regions presented sharp and narrow signals; thus, the purple, green, and yellow ROIs were set around the main z-plane (panel b). As the upper stalk BM showed sharper signals in the 1 µm lower z-plane (additional z-plane: panel b′), the cyan ROI was set around the additional z-plane. Additional z-planes are displayed only if some target areas in the main z-plane do not show sharp eGFP-COL4A2 signals. The same photobleaching procedure was applied to each ROI. At intervals of 2–2 h and 30 min of culture following each photobleaching session, the edges of the photobleached areas were identified (distinguishable edges are indicated by arrowheads: panels c, d, f, g, i, j, and j′), and a new ROI was drawn along the identified photobleached edges (panels e, e′, h, and h′). This procedure was repeated twice until the end of the imaging session (7 h after initial photobleaching: panels j and j′). Scale bar: 20 µm. (B) Images of BM length changes. These images show the procedures used to quantify BM length changes in A. Measurements were conducted in different z-planes to determine the length of the BM at the upper and lower stalk regions (upper panels) and the tip region (upper and lower panels). Scale bar: 20 µm. (C) Maximum projection images of eGFP-COL4A2 in hair follicles in A are presented. Side views of the dotted cuboidal areas are shown. As no significant shape changes were observed at the boundaries of the 3D cuboidal bleached area during the imaging period, we can conclude that the BM maintains a consistent shape and expansion rate, even with shifts in the circumferential location of the hair follicle. This suggests that any potential displacement around the circumference does not significantly affect the accuracy of the measurements. Scale bars: 20 µm for maximum projection images; 5 µm for magnified side view images.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Membrane, Imaging

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Analysis of the 3D basement membrane structure. (A) Confocal images of the hair follicles enclosed by a continuous line-like BM structure. Images show z-stack maximum projection images and single z-plane images of developing hair follicles with a continuous line-like BM structure at the tip (observed in 9 out of 13 follicles examined), lower stalk (observed in 12 out of 13 follicles examined), and upper stalk regions (observed in all 13 follicles examined). These hair follicles were developed in cultured skin explants from E12.5 embryos of eGFP-Col4a2 mice ( n = 13 follicles from five explants, each from an independent experiment). Scale bars: 10 μm. (B) Confocal z-stack maximum projection and single z-plane images of the hair follicles exhibiting pores (arrowheads) in its lower part. The BM with pores (a dotted rectangular area) is magnified. Scale bars: 10 μm. (C) Confocal z-stack maximum projection and single z-plane images of the hair follicles treated with an MMP inhibitor (batimastat). No clear pore structures were observed in the 13 MMP inhibitor-treated hair follicles examined. ( n = 13 follicles from five explants, each from an independent experiment for the inhibitor-treated condition). Scale bars: 10 μm.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Membrane, Cell Culture

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Snapshot images of 3D time-lapse videos of developing hair follicles from eGFP-Col4a2; mem-tdTomato mice. (A and B) Snapshot single z-plane images of cells and BMs tracking at 30-min intervals from 0 to 9 h 30 min. Cells and the BMs in the lower (A) and upper stalk regions (B) were tracked. The BM was re-photobleached several times to maintain a clear photobleached edge. The lower photobleached edge of the BM (indicated by white arrowheads) and epithelial basal progenitors initially located at this photobleached edge (indicated by yellow dots) were tracked. A white dot marks the other daughter cell following the division of the cell marked with a yellow dot. Scale bar: 20 μm.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques:

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Spatially distinct collagen IV turnover. (A) Time-lapse images of eGFP-COL4A2 FRAP experiments. Confocal single-color images (upper) and pseudocolor images (lower) show the BM of a developing hair follicle in explants that were photobleached in the tip (blue arrowheads), lower stalk (orange arrowheads), and follicular–interfollicular junction (gray arrowheads) regions before and after photobleaching at selected recovery times. Scale bar: 20 μm. (B) Line graph of the normalized fluorescence recovery of eGFP-COL4A2 over 4 h ( n = 6 hair follicles, each from a separate explant in an independent experiment). The cyan lines are simple linear regressions for the data derived from each region. Values: means ± SD. (C) Comparison of the recovery rates of eGFP-COL4A2 signals between BMs in different regions. The normalized mean intensity values of eGFP-COL4A2 fluorescence in the bleached regions from the FRAP experiments at 3 h and 30 min, as shown in , were used. Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. Data distribution was assumed to be normal, but this was not formally tested. (D) Maximum projection images of mKikGR-COL4A2 in developing hair follicles in embryonic skin explants from mKikGR-Col4a2 mice at the indicated times after photoconversion. Scale bar: 20 μm.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Fluorescence, Derivative Assay, Comparison

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Representative time-lapse video of FRAP experiments with eGFP-COL4A2 in developing hair follicles. eGFP-COL4A2 signals (green) were photobleached in designated regions and signal recovery was monitored. Cultured skin explants from E12.5 embryos of eGFP-Col4a2 mice were used, corresponding to . Live imaging was performed under a TCS SP8X (Leica) confocal microscope. Frames were collected approximately every 10 min and 7 s over the 5-h and 43-min time-lapse. The video is 37 s in total, with the first and last frames each held for 2 s to indicate regions of interest. The remaining frames are displayed at ∼0.95 frames per second.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Cell Culture, Imaging, Microscopy

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Representative time-lapse video of pulse-chase experiments in the developing hair follicles of skin explants from mKikGR-Col4a2 mouse embryos. The green mKikGR-COL4A2 signals were photoconverted to red (magenta), and the changes in these mKikGR-COL4A2 signals were chased over ∼19 h at 30 min intervals, corresponding to . Live imaging was performed under a TCS SP8X (Leica) confocal microscope. Frames were collected approximately every 28 min and 27 s during the 18-h and 57-min time-lapse. The video is 41 s in total and displayed at one frame per second.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Pulse Chase, Imaging, Microscopy

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Imaging of mKikGR-COL4A2 in developing hair follicles. Snapshot images of z-stack maximum projections of mKikGR-COL4A2 in developing hair follicles in embryonic skin explants from mKikGR-Col4a2 mice at the indicated time points after photoconversion. Scale bar: 20 μm.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Imaging

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: 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 . (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 . (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 (L HF )/bulb width (W HF ). 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.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Membrane, Inhibition, Control, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Representative time-lapse video of FRAP experiments with eGFP-COL4A2 in developing hair follicles under MMP inhibition. EGFP-COL4A2 signals (green) were photobleached in designated regions and signal recovery was monitored under MMP inhibition with batimastat. Cultured skin explants from E12.5 embryos of eGFP-Col4a2 mice were used, corresponding to , under MMP-inhibitor conditions. Live imaging was performed under a TCS SP8X (Leica) confocal microscope. Frames were collected approximately every 16 min and 13 s during the 18-h and 54-min time-lapse. The video is 1 min and 14 s in total, with the first and last frames each held for 2 s to indicate regions of interest. The remaining frames are displayed at one frame per second.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Inhibition, Cell Culture, Imaging, Microscopy

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Col4a2 mRNA expression in developing hair follicles. (A) Confocal images of Col4a2 mRNA expression in control and MMP inhibitor-treated developing hair follicles, obtained via RNAscope. The signals were detected 16 h after the addition of the MMP inhibitor batimastat. Scale bar: 20 μm. (B) Quantitative analysis of Col4a2 mRNA expression in control and MMP inhibitor-treated developing hair follicles in different tissue regions. ( n = 9 follicles from five explants, each from an independent experiment for the control, and n = 7 follicles from six explants, each from an independent experiment for the inhibitor-treated). Scale bar: 20 μm. Values: means ± SD. DP: dermal papilla. Two-tailed unpaired t tests were used. Data distribution was assumed to be normal, but this was not formally tested.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Expressing, Control, RNAscope, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Matrix metalloproteinase inhibition alters the angle of daughter cell allocation. (A) Snapshot images and cell division tracking data from developing hair follicles in skin explants from eGFP-Col4a2; mem-tdTomato mouse embryos cultured under control or MMP-inhibited conditions. The BM and cell membrane were visualized with eGFP-COL4A2 (green) and mem-tdTomato (magenta), respectively. Scale bars: 20 μm. (B) Frequency of cell division in epithelial basal progenitors in the experiments in A ( n = 6 follicles from five explants, each from an independent experiment for the control, n = 6 follicles, each from a separate explant in an independent experiment for the inhibitor-treated). A two-tailed unpaired t test was used. (C) EdU incorporation assays. Dividing cells were labeled with EdU (magenta). All the cells were counterstained with DAPI (blue). Scale bar: 20 μm. (D) Percentage of EdU-positive basal epithelial cells in the tip, lower stalk, and upper stalk regions ( n = 7 follicles from three 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). Values: means ± SD. Two-way ANOVA with Tukey’s post-hoc tests was used. (E) Daughter cell allocation angle of proliferating basal epithelial progenitors relative to the BM zone in the experiments in A. Images of perpendicular and horizontal divisions are shown. Scale bar: 5 μm. (F and G) Pie graphs and a plot illustrating the angle of daughter cell allocation relative to the BM under control and MMP inhibitor treatment conditions ( n = 48 cells from 11 follicles from seven explants, each from an independent experiment for the control, and n = 36 cells from eight follicles from eight explants, each from an independent experiment for the inhibitor-treated). All statistical tests in this figure assumed normal data distribution, but this was not formally tested.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Inhibition, Cell Culture, Control, Membrane, Two Tailed Test, Labeling

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Representative time-lapse video of daughter cells undergoing perpendicular or horizontal division during hair follicle development. This time-lapse video shows tracked epithelial basal progenitors that undergo perpendicular or horizontal division in cultured skin explants from eGFP-Col4a2; mem-tdTomato mouse embryos. The BM and cell membrane were visualized with eGFP-COL4A2 (green) and mem-tdTomato (magenta), corresponding to . Live imaging was performed under an LSM980 (Carl Zeiss) with a two-photon laser unit Chameleon Discovery NX (Coherent). Frames were collected approximately every 30 min during the 2-h time lapse. The last frame of each part is displayed at 2 s per frame. The remaining frames are displayed at 0.67 frames per second.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Cell Culture, Membrane, Imaging

Journal: The Journal of Cell Biology

Article Title: An eGFP-Col4a2 mouse model reveals basement membrane dynamics underlying hair follicle morphogenesis

doi: 10.1083/jcb.202404003

Figure Lengend Snippet: Model depicting the role of basement membrane dynamics in hair follicle morphogenesis. This model illustrates the spatially distinct molecular and tissue-level dynamics of the BM, highlighting its pivotal role in regulating cell and tissue dynamics during hair follicle morphogenesis. The green lines indicate BMs. Magenta cells and BM zones are highlighted tracer epithelial basal cells and BM zones, respectively. The blue cells are epithelial progenitors that divide horizontally relative to the BM, and the red cells are epithelial progenitors that divide perpendicularly to the BM. In normal hair follicle development, the following key features are observed: (1) the rates of COL4A2 turnover and BM expansion exhibit spatial gradients, and these processes are coupled; (2) epithelial progenitors undergo both horizontal and perpendicular divisions; (3) basal epithelial cells move in unison with the directionally expanding underlying BM; and (4) the epithelial tube results in directional elongation. In MMP inhibitor-treated hair follicles: (1) COL4A2 turnover and BM expansion are restrained; (2) progenitors alter the angle of daughter cell allocation from horizontal to perpendicular; and (3) directional epithelial elongation is halted, resulting in a wider organ shape.

Article Snippet: To quantify the number of EdU-positive nuclei, a freehand line was drawn on the BM (eGFP-COL4A2 signal) using Imaris (version 8.4.2; Bitplane).

Techniques: Membrane

Journal: Scientific Reports

Article Title: Conditional Deletion of Smad1 Ameliorates Glomerular Injury in Progressive Glomerulonephritis

doi: 10.1038/srep31216

Figure Lengend Snippet: The microscopic lesions in NTN mice appeared as the diffuse proliferation of mesangial matrix and expansion of the mesangial area. Immunohistochemical staining with anti-Col4 antibodies revealed the overexpression of Col4 in the expanded mesangial area of glomeruli from kidneys of NTN mice. NTN mics also showed significantly enhanced expression of PDGF-BB and PDGFβR in the glomeruli. Representative microscopic appearance of the glomerulus is shown. ( a – d ) PAM. ( e – h ) Col 4. ( i – l ) PDGF-BB. ( m – p ) PDGFβR. Original magnification for all panels was ×400. ( q ) PAM staining-positive area of the glomeruli. We collected at least 20 glomeruli in each sample. The expression levels of Col4 (Col4a1 and Col4a2) ( r ) and PDGF signals ( s ) in the glomeruli 21 d after the initial NTS injection were analysed by qPCR and normalized to the expression of Rn18s. The values are expressed as the means ± SD. *P < 0.05. ( t ) Time course of changes in urinary albumin excretion in the two groups – control IgG- (○) and APB5- (●) treated mice, and serum Cre and BUN levels at day 21. Statistical analysis for comparison was performed using unpaired two-tailed Student’s t -test. NS, not significant, *P < 0.05 (n = 5 mice/group).

Article Snippet: The other TaqMan gene expression assays used were as follows: Mm00484723_m1 (Smad1), Mm00483888_m1 (Col1 alpha 2 chain), Mm01210125_m1 (Col4 alpha 1 chain), Mm00802386_m1 (Col4 alpha 2 chain), Mm01298578_m1 (PDGF-B), Mm00435546_m1 (PDGFR-β), Mm00711781_m1 (Id2), and Mm00437306_m1 (VEGF-A).

Techniques: Immunohistochemical staining, Staining, Over Expression, Expressing, Injection, Two Tailed Test

Journal: Scientific Reports

Article Title: Conditional Deletion of Smad1 Ameliorates Glomerular Injury in Progressive Glomerulonephritis

doi: 10.1038/srep31216

Figure Lengend Snippet: Immunohistochemical staining of Smad1 and pSmad1 in glomeruli in NTN. NTN also showed significantly enhanced expression of Smad1 and pSmad1 in the glomeruli. APB5 treatment led to a significant decrease in expressions of these proteins. ( a – d ) Smad1. ( e – h ) pSmad1. ( i ) The expression levels of Smad1 in the glomeruli 21 d after the initial NTS injection were analysed by qPCR and normalized to the expression of Rn18s. The values are expressed as the mean ± SD. *P < 0.05. ( j ) The number of cells positive for pSmad1. Fifty glomeruli were analysed for each sample. The values are expressed as the mean ± SD. *P < 0.05. Regression analysis comparing the changes in pSmad1 levels with changes in Col4a1 ( k ) and Col4a2 ( l ) expression mRNA levels in NTN model mice treated with control IgG (○) and APB5 (●).

Article Snippet: The other TaqMan gene expression assays used were as follows: Mm00484723_m1 (Smad1), Mm00483888_m1 (Col1 alpha 2 chain), Mm01210125_m1 (Col4 alpha 1 chain), Mm00802386_m1 (Col4 alpha 2 chain), Mm01298578_m1 (PDGF-B), Mm00435546_m1 (PDGFR-β), Mm00711781_m1 (Id2), and Mm00437306_m1 (VEGF-A).

Techniques: Immunohistochemical staining, Staining, Expressing, Injection

Journal: Scientific Reports

Article Title: Conditional Deletion of Smad1 Ameliorates Glomerular Injury in Progressive Glomerulonephritis

doi: 10.1038/srep31216

Figure Lengend Snippet: Smad1 -CKO and WT mice treated with NTS-injection and control mice were dissected 21 weeks after treatment (n = 6 in each group). Light microscopic and immunohistochemical analyses were performed. Representative microscopic appearance of the glomerulus is shown. ( a – d ) PAM. ( e – h ) Col 4. ( i – l ) pSmad1. ( m – p ) Col1. ( q – t ) αSMA. Original magnification for all panels was ×400. ( u ) PAM staining-positive area of the glomeruli. We collected at least 20 glomeruli in each sample. ( v ) The expression levels of Col4 (Col4a1 and Col4a2) in the glomeruli 21 d after the initial NTS injection were analysed by qPCR and normalized to the expression of Rn18s. The values are expressed as the mean ± SD. *P < 0.05. ( w ) The number of cells positive for pSmad1. Fifty glomeruli were analysed for each sample. The values are expressed as the mean ± SD. *P < 0.05. The expression levels of αSMA ( x ) and Col1a2 ( y ) in the glomeruli 21 d after the initial NTS injection were analysed by qPCR and normalized to the expression of Rn18s. The values are expressed as the mean ± SD. *P < 0.05. ( z ) Time course of changes in urinary albumin excretion in the two groups – WT mice (○) and Smad1 -CKO mice (●) – after injection of NTS, and serum Cre and BUN levels at day 21.. Statistical analysis for comparison between CKO and control mice was performed using unpaired two-tailed Student’s t -test. NS, not significant, *P < 0.05 (n = 4–5 mice/group).

Article Snippet: The other TaqMan gene expression assays used were as follows: Mm00484723_m1 (Smad1), Mm00483888_m1 (Col1 alpha 2 chain), Mm01210125_m1 (Col4 alpha 1 chain), Mm00802386_m1 (Col4 alpha 2 chain), Mm01298578_m1 (PDGF-B), Mm00435546_m1 (PDGFR-β), Mm00711781_m1 (Id2), and Mm00437306_m1 (VEGF-A).

Techniques: Injection, Immunohistochemical staining, Staining, Expressing, Two Tailed Test

Journal: iScience

Article Title: HucMSCs can alleviate abnormal vasculogenesis induced by high glucose through the MAPK signaling pathway

doi: 10.1016/j.isci.2024.111354

Figure Lengend Snippet: Generation of BVOs and exploration of the effects of high-glucose conditions on angiogenesis in BVOs (A) Schematic representation of the process for generating BVOs derived from human ESCs. (B) BVOs displayed CD31 staining as a marker of endothelial cells, PDGFR-β staining as a marker of pericytes, and Col IV staining as a marker of the basement membrane. Moreover, BVOs were able to take up DiI-Ac-LDL. (C) The gene expression levels of Pecam1, VE-Cadherin, Pdgfr, α-Sma, Col 4a1 and Col 4a2 in BVOs were higher than those in ESCs. (D) HE staining visualized the lumen (∗) of BVOs. (E) Inverted phase contrast images of VI-BVOs on day 3 after embedding in the hydrogel: control medium group (BVOs-Control) and high-glucose medium group (BVOs-HG). (F) Compared to those in the BVOs-Control group, the CD 31 expression levels and acLDL uptake function of endothelial cells in the BVOs-HG group were significantly decreased. (G) Compared to those in the BVOs-Control group, the expression of the endothelial cell marker genes Pecam1/VE-cadherin and the pericyte marker genes Pdgfr/α-sma in the BVOs-HG group was significantly downregulated in the BVOs-HG group. For (C) and (G), data are represented as mean ± SD. ( n = 3 replicates per condition from 3 independent experiments, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 from two-tailed Student’s t test)(Scale bar: A, B, E, F = 100 μm, D (left) = 50 μm, D (right) = 20 μm).

Article Snippet: After they were thoroughly rinsed with PBS, they were incubated with the following primary antibodies for 12 h at 4°C: rabbit anti-PDGFRβ (1:200, Cell Signaling, Danvers, Massachusetts, USA, 3169S), rabbit anti-COL 4A2 (1:200, Proteintech, 55131-1-AP) Alexa Fluor647 mouse anti-human CD31 (1:100, BD Biosciences, USA, 558094), rabbit anti-Oct-4 Antibody (1:200, Cell Signaling, 2750), mouse anti-SSEA-4 antibody (1:200, Santa Cruz Biotechnology, sc-59368), rabbit anti-PDGFR alpha antibody (1:200, Abcam, ab203491), rabbit anti-VEGF Receptor 2 (FLK1, 1:200, HUABIO, Hangzhou, China, ET1608-33).

Techniques: Derivative Assay, Staining, Marker, Membrane, Gene Expression, Control, Expressing, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Loss of function of Colgalt1 disrupts collagen post-translational modification and causes musculoskeletal defects

doi: 10.1242/dmm.037176

Figure Lengend Snippet: Collagen IV accumulates intracellularly and exhibits decreased stability and molecular mass in Colgalt1 mutant MEFs. Immunoblot detection of the α2 chain of type IV collagen in lysates (A) and conditioned media (B) from wild-type and Colgalt1 fosse/fosse MEF cultures. Ponceau S staining is shown as a loading control. Arrows indicate the band corresponding to full-length collagen IV. Graphs show densitometric quantitation of COL4A2 signal intensities normalized to Ponceau S. (C) Immunoblot detection of monomer and dimers of the NC1 domain of COL4A2 in collagenase digests of the ECM isolated from wild-type and fosse MEFs. The graph shows densitometric quantitation of the total (monomers and dimers) signal intensity from five different samples in each MEF group. Results are shown as mean (s.d.) and P -values ≤0.05 and ≤0.01 are indicated by one (*) or two (**) asterisks, respectively. (D) Immunofluorescent detection of type IV collagen in muscle tissue from wild-type and fosse mice. Magnification: 40×.

Article Snippet: Slides were then incubated with primary antibodies against COL4A2 (1:1000, rat monoclonal H22 antibody, Chondex, cat. # 7071,) or COL6A1 (1:100, Abcam, #6588).

Techniques: Mutagenesis, Western Blot, Staining, Control, Quantitation Assay, Isolation

Journal: Theranostics

Article Title: COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells

doi: 10.7150/thno.35914

Figure Lengend Snippet: B-dECM/P-dECM distinct protein.

Article Snippet: Next, COL4A2 Lentiviral Activation Particles (h) (Santa Cruz, sc-401462-LAC) were used to upregulate COL4A2 protein expression.

Techniques:

Journal: Theranostics

Article Title: COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells

doi: 10.7150/thno.35914

Figure Lengend Snippet: Proliferation and osteogenic differentiation ability of PDLSCs. PDLSCs were cultured on B-dECM, P-dECM or no-ECM. To control the expression of COL4A2 in dECMs, COL4A2 in the P-dECM group was overexpressed via lentivirus-mediated transduction of PDLCs. COL4A2 in the B-dECM group was downregulated via transfection of BMCs with siRNA. ( A ) Expression levels of COL4A2 in B-dECM and P-dECM determined by Western blotting. ( B ) Expression of Ki67, Cyclin B1, and Cyclin D1 in PDLSCs cultured on specific dECMs and no-ECM. ( C ) Alizarin red staining to detect the osteogenic differentiation ability of PDLSCs in five groups. Quantification of positive staining (right panel). ( D ) ALP activity was assessed to identify the osteogenic differentiation ability of PDLSCs in five groups. Quantification of positive staining (right panel). ( E ) Western blotting was used to detect the expression of the indicated proteins to assess osteogenic differentiation of PDLSCs cultured on specific dECMs and no-ECM. The data are presented as the means ± SD; n = 5. * P < 0.05, ** P < 0.01 and *** P < 0.001 represent significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. ## P < 0.01 and ### P < 0.001 represent significant differences between the P-dECM and B-dECM groups. ++ P < 0.01 and +++ P < 0.001 represent significant differences in the P-dECM or B-dECM group compared with the P-dECM + LV-COL4A2 or B-dECM + COL4A2-siRNA group.

Article Snippet: Next, COL4A2 Lentiviral Activation Particles (h) (Santa Cruz, sc-401462-LAC) were used to upregulate COL4A2 protein expression.

Techniques: Cell Culture, Control, Expressing, Transduction, Transfection, Western Blot, Staining, Activity Assay

Journal: Theranostics

Article Title: COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells

doi: 10.7150/thno.35914

Figure Lengend Snippet: B-dECM and P-dECM protein MS with regulation of COL4A2.

Article Snippet: Next, COL4A2 Lentiviral Activation Particles (h) (Santa Cruz, sc-401462-LAC) were used to upregulate COL4A2 protein expression.

Techniques:

Journal: Theranostics

Article Title: COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells

doi: 10.7150/thno.35914

Figure Lengend Snippet: Formation of new bone-like tissue in immunocompromised mice with cell tracing of PDLSCs. ( A ) Schematic diagram of PDLSC implantation in the back of immunocompromised mice. ( B ) PDLSCs transfected with YFP fluorescence. ( C ) Cell tracing and comparison of different effects of implantation of dECM, Bio-Oss, and PDLSCs. Four PDLSC/dECM/Bio-Oss combinations were implanted into immunocompromised mice. Double labeling of PDLSCs and Col-I was used to examine osteogenic ability and trace cells. White bar, 200 µm. Quantitative analysis of Col-I expression (right panel). * P < 0.05 and *** P < 0.001 represent significant differences in the indicated columns. ( D ) H&E staining revealed more bone-like tissue and insertion of PDL-like fibers in B-dECM and P-dECM than in the no-ECM group. This effect was increased in the P-dECM + LV-COL4A2 group compared with the P-dECM group. B-dECM + COL4A2-siRNA exhibited the opposite result. Black arrow , newly formed bone-like tissue. Green arrow , newly formed vessels. ( E ) Higher magnification of H&E staining. Green box represents the enlarged area. ( F ) Quantitative analysis of the new bone area in H&E staining images was carried out using Image-Pro Plus 6.0 software. ( G ) Cell tracing study in immunocompromised mice. ( H ) Higher magnification of cell tracing study. BM , bone meal from Bio-Oss. Black bar and white bar, 200 µm. The data are presented as the means ± SD; n = 6. *** P < 0.001 represents significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. ### P < 0.001 represents significant differences between the P-dECM and B-dECM groups. +++ P < 0.001 represents significant differences between the P-dECM or B-dECM group and the P-dECM + LV-COL4A2 or B-dECM + COL4A2-siRNA group.

Article Snippet: Next, COL4A2 Lentiviral Activation Particles (h) (Santa Cruz, sc-401462-LAC) were used to upregulate COL4A2 protein expression.

Techniques: Transfection, Fluorescence, Comparison, Labeling, Expressing, Staining, Software

Journal: Theranostics

Article Title: COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells

doi: 10.7150/thno.35914

Figure Lengend Snippet: Formation of new bone-like tissue around Bio-Oss bone powder in immunocompromised mice following PDLSC implantation. ( A ) Masson staining of new bone and fibers. ( B ) Sirius red staining for identification of collagen I to IV fibers. Type I collagen shows red birefringence, type II collagen weak red light, type III collagen green birefringence, and type IV collagen weak yellow birefringence. ( C, D ) Immunohistochemical staining of Col-I and quantification of the Col-I-positive area. ( E, F ) Immunohistochemical staining of OCN and quantification of the OCN-positive area. BM , bone meal from Bio-Oss. Black bar and white bar, 200 µm. The data are presented as the means ± SD; n = 6. *** P < 0.001 represents significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. ### P < 0.001 represents significant differences between the P-dECM and B-dECM groups. +++ P < 0.001 represents significant differences between the P-dECM or B-dECM group and the P-dECM + LV-COL4A2 or B-dECM + COL4A2-siRNA group.

Article Snippet: Next, COL4A2 Lentiviral Activation Particles (h) (Santa Cruz, sc-401462-LAC) were used to upregulate COL4A2 protein expression.

Techniques: Staining, Immunohistochemical staining

Journal: Theranostics

Article Title: COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells

doi: 10.7150/thno.35914

Figure Lengend Snippet: The Wnt pathway is downstream of COL4A2 and negatively regulates osteogenic differentiation of PDLSCs. ( A ) PDLSCs were cultured on specific dECMs and no-ECM in osteogenic induction medium. With the regulation of COL4A2, the protein levels of nuclear β-catenin, total β-catenin, GSK-3β, and p-GSK-3β were measured via Western blotting in PDLSCs following 7 days of culture in osteogenic medium. Quantification of blots (right panel). ( B ) DKK-1 (inhibitor, 100 ng/ml) and Wnt3a (activator, 100 ng/ml) were used to downregulate and upregulate the expression of β-catenin, respectively, in the P-dECM, B-dECM and P-dECM + LV-COL4A2 groups. Alizarin red staining showing quantitative evaluation of osteogenic differentiation ability through modulation of the Wnt pathway. Quantification of positive staining (right panel). ( C, D ) The expression levels of nuclear β-catenin, total β-catenin, GSK-3β, p-GSK-3β, Col-I, ALP, and Runx2 in the presence of Wnt3a or DKK-1 in PDLSCs grown in osteogenic medium in the P-dECM + LV-COL4A2 group ( C ) or the B-dECM + COL4A2-siRNA group ( D ) were measured via Western blotting. The data are presented as the means ± SD; n = 5. *** P < 0.001 represents significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. # P < 0.05 and ### P < 0.001 represent significant differences between the P-dECM and B-dECM groups. + P < 0.05, ++ P < 0.01 and +++ P < 0.001 represent significant differences between the indicated columns.

Article Snippet: Next, COL4A2 Lentiviral Activation Particles (h) (Santa Cruz, sc-401462-LAC) were used to upregulate COL4A2 protein expression.

Techniques: Cell Culture, Western Blot, Expressing, Staining

Journal: Theranostics

Article Title: COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells

doi: 10.7150/thno.35914

Figure Lengend Snippet: Effects of PDLSCs cultured on specific dECMs and no-ECM on the repair of alveolar bone defects at 8 weeks following transplantation. ( A ) Representative images of micro-CT 3D images and horizontal cross section. Black arrow , the size of the alveolar bone defects after implantation. Yellow dots , the size of the alveolar bone defects after implantation in the horizontal cross section. Black bar, 1 mm. ( B ) Bone morphology analysis of the BMD, BV/TV, Tb.Th and Tb.Sp values in alveolar bone defects in rats treated with PDLSCs cultured on specific dECMs and no-ECM at 8 weeks following transplantation. ( C ) H&E staining of bone defects in these five groups. Black dots , the area of new bone in the defects. Green arrow , newly formed blood vessels. Black bar, 200 µm. ( D ) Quantitative analysis of the new bone area; H&E staining analysis was carried out using Image-Pro Plus 6.0 software. ( E ) Masson staining for new bone and fibers. NB , new bone. Red dots , the area of new bone in the defects. Black bar, 200 µm. ( F, G ) Immunohistochemical staining of Col-I in alveolar bone defects in rats treated with PDLSCs cultured on specific dECMs and no-ECM after 8 weeks of transplantation and quantification of the Col-I-positive area. NB , new bone. Red dots , the area of new bone in the defects. Black bar, 200 µm. The data are presented as the means ± SD; n = 6. * P < 0.05, ** P < 0.01 and *** P < 0.001 represent significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. ### P < 0.001 represents significant differences between the P-dECM and B-dECM groups. ++ P < 0.001 and +++ P < 0.001 represent significant differences between the P-dECM or B-dECM group and the P-dECM + LV-COL4A2 or B-dECM + COL4A2-siRNA group.

Article Snippet: Next, COL4A2 Lentiviral Activation Particles (h) (Santa Cruz, sc-401462-LAC) were used to upregulate COL4A2 protein expression.

Techniques: Cell Culture, Transplantation Assay, Micro-CT, Staining, Software, Immunohistochemical staining