Journal: Theranostics

Article Title: Overactivation of EGFR signaling in skeletal stem/progenitor cells promotes bone formation and repair

doi: 10.7150/thno.115406

Figure Lengend Snippet: Activation of EGFR signaling contributes to higher trabecular and cortical bone contents. (A) Tracing of Prx1 lineage cells on femoral frozen sections of 1- and 3-month-old Prx1-Cre; tdTomato mice. Red: tdTomato + cells; blue: nuclear staining by DAPI. Scale bars = 50 µm. (B) Micro-CT images of femurs from 3-month-old WT and HBEGF Over Prx1 mice. (C) Quantification of femur length and width of WT and HBEGF Over Prx1 mice at 3 months old; n = 8 per group. (D) Representative 3D reconstructed micro-CT images of trabecular architecture and cortical bone from 3-month-old WT and HBEGF Over Prx1 distal femurs. (E) Quantification of the bone parameters including trabecular bone volume fraction (BV/TV), trabecular number (TB. N), trabecular thickness (TB.TH), trabecular separation (TB. SP) and cortical thickness (CT. TH) based on micro-CT; n = 8 per group. (F) Representative H&E staining images of distal femurs from 3-month-old WT and HBEGF Over Prx1 mice. Magnified images of the boxed areas are shown in the panel below. Scale bar = 500 μm (upper image); 100 μm (lower image). (G) Serum PINP and CTX levels in WT and HBEGF Over Prx1 mice analyzed by ELISA; n = 8 per group. (CTX, P = 0.6441). (H) Immunohistochemical staining of OCN and TRAP staining of trabecular bone sections from distal femurs of WT and HBEGF Over Prx1 mice. OCN-positive cells or TRAP-positive cells in the distal femurs are indicated by red or black arrows, respectively; scale bar = 50 µm. (I) Representative 3D reconstructed micro-CT images of trabecular architecture and cortical bone at 4 weeks post OVX surgery from WT and HBEGF Over Prx1 distal femurs. (J) Quantification of cortical thickness (CT. TH) based on micro-CT; n = 6 per group. (K) Quantification of the bone parameters including BV/TV, TB. N, TB.TH and TB. SP based on micro-CT; n = 6 per group. (L) Immunohistochemical staining of OCN and TRAP staining on trabecular bone sections at 4 weeks post OVX surgery from distal femurs of WT and HBEGF Over Prx1 mice. OCN-positive cells or TRAP-positive cells in the distal femurs are indicated by red or black arrows; scale bar = 50 µm. Data are presented as means ± SD. Statistical analysis was performed using two-tailed Student's t test (C, E, G) and one-way ANOVA with Bonferroni's post-hoc test for multiple comparisons (J, K). ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The protein bands were detected using specific primary antibodies against EGFR (1:1000; CST, 4267), p-EGFR (1:1000; Abcam, ab40815), ERK (1:1000; CST, 4695), p-ERK (1:1000; CST, 4370), HBEGF (1:1000; Boster, A01759-3), β-Actin (1:4000; CST, 4970) and secondary antibodies.

Techniques: Activation Assay, Staining, Micro-CT, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Two Tailed Test

Journal: Theranostics

Article Title: Overactivation of EGFR signaling in skeletal stem/progenitor cells promotes bone formation and repair

doi: 10.7150/thno.115406

Figure Lengend Snippet: Activation of EGFR signaling accelerates fracture healing. (A) Immunofluorescence images of HBEGF distribution in intact and fractured Prx1-Cre; tdTomato mouse femurs. Boxed areas in the left panel are shown at higher magnification on the right. Red: tdTomato + cells; green: HBEGF + cells; blue: nuclear staining by DAPI. Scale bars = 500 µm or 50 µm. (B) Percentage of HBEGF + and tdTomato + HBEGF + over tdTomato + within callus were calculated; n = 3 per group. (C) Representative 3D reconstructions and coronal cross-sectional micro-CT images of fracture callus at 7, 10, 14 and 28 dpf. (D) The tissue volume (TV), bone volume (BV) and bone volume fraction (BV/TV) of fracture callus at 7, 10, 14 and 28 dpf were analyzed; n = 8 per group. (E) Fracture healing scores were quantified based on mRUST scoring criteria at 7, 10, 14 and 28 dpf; n = 8 per group. (F) Three-point bending test was performed on femurs at 6 weeks post-fracture; n = 6 per group. (G) Representative Safranin O/Fast green staining images of fracture calluses at 7, 10, 14 and 28 dpf; scale bar = 500 µm. (H) Callus area, cartilage area, and bone area were measured at 7, 10, 14 and 28 dpf; n = 6 per group. Data are presented as means ± SD. Statistical analysis was performed using two-tailed Student's t test (B, D, E, F, H). ns = not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: The protein bands were detected using specific primary antibodies against EGFR (1:1000; CST, 4267), p-EGFR (1:1000; Abcam, ab40815), ERK (1:1000; CST, 4695), p-ERK (1:1000; CST, 4370), HBEGF (1:1000; Boster, A01759-3), β-Actin (1:4000; CST, 4970) and secondary antibodies.

Techniques: Activation Assay, Immunofluorescence, Staining, Micro-CT, Two Tailed Test

Journal: Theranostics

Article Title: Overactivation of EGFR signaling in skeletal stem/progenitor cells promotes bone formation and repair

doi: 10.7150/thno.115406

Figure Lengend Snippet: HBEGF promotes survival, migration and differentiation of periosteal progenitors. (A) Western blot of HBEGF and EGFR downstream signals in periosteal progenitors derived from WT and HBEGF Over Prx1 mice. (B) Immunofluorescence staining of Ki67 in WT and HBEGF Over Prx1 periosteal progenitors; scale bar = 200 µm. (C) CFU-F assay using periosteal progenitors dissociated from WT and HBEGF Over Prx1 mice. (D) Percentages of Ki67 + cells or CFU + cells were quantified; n = 3 per group. (Ki67, P = 0.6297; CFU, P = 0.7096). (E) Apoptosis was measured by flow cytometry. (F) Percentages of apoptotic cells were quantified; n = 3 per group. (G) Periosteal progenitors were seeded into the upper chamber in 1% FBS medium. Migrated cells on the lower surface of the membrane were stained with crystal violet, and the number of migrated cells was quantified; n = 3 per group. Scale bar = 100 µm. (H) ALP staining of WT and HBEGF Over Prx1 periosteal progenitors after culture in osteogenic medium for 10 days. (I) Alizarin red S (ARS) staining of WT and HBEGF Over Prx1 periosteal progenitors after culture in osteogenic medium for 21 days. (J) ALP- or ARS-positive areas were measured using Image J; n = 3 per group. (K) Representative images of OCN immunostaining (green) in fracture callus at 10 dpf, counterstained with DAPI (blue); scale bar = 100 µm. (L) Alcian blue staining of WT and HBEGF Over Prx1 periosteal progenitors after culture in chondrogenic medium for 20 days. Alcian blue-positive areas were measured using Image J; n = 3 per group. (M) Western blot of RUNX2 and SOX9 in periosteal progenitors derived from WT and HBEGF Over Prx1 mice. (N) RT-PCR analysis of osteogenic marker gene expression and chondrogenic marker gene expression in WT and HBEGF Over Prx1 periosteal progenitors harvested after 2 weeks of culture in osteogenic or chondrogenic medium; n = 3 per group. Data are presented as means ± SD. Statistical analysis was performed using two-tailed Student's t test (D, F, G, J, K, L, N). ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The protein bands were detected using specific primary antibodies against EGFR (1:1000; CST, 4267), p-EGFR (1:1000; Abcam, ab40815), ERK (1:1000; CST, 4695), p-ERK (1:1000; CST, 4370), HBEGF (1:1000; Boster, A01759-3), β-Actin (1:4000; CST, 4970) and secondary antibodies.

Techniques: Migration, Western Blot, Derivative Assay, Immunofluorescence, Staining, Flow Cytometry, Membrane, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Marker, Gene Expression, Two Tailed Test

Journal: Theranostics

Article Title: Overactivation of EGFR signaling in skeletal stem/progenitor cells promotes bone formation and repair

doi: 10.7150/thno.115406

Figure Lengend Snippet: HBEGF activates EGFR/ERK signaling to promote survival, migration and differentiation of periosteal progenitors. (A) Western blot of HBEGF, RUNX2, SOX9, VEGFA and EGFR downstream signals in periosteal progenitors derived from WT and HBEGF Over Prx1 mice. HBEGF Over Prx1 periosteal progenitors were treated with or without gefitinib (10 μM) or U0126 (10 μM); n = 3 per group. (B) Apoptosis in the indicated periosteal progenitors, measured by flow cytometry. Percentages of apoptotic cells were quantified; n = 3 per group. (C) Periosteal progenitors were seeded into the upper chamber in 1% FBS medium. Migrated cells on the lower surface of the membrane were stained with crystal violet, and the number of migrated cells was quantified; n = 3 per group. Scale bar = 100 µm. (D) ALP, ARS and alcian blue staining of the indicated periosteal progenitors after culture in osteogenic or chondrogenic medium. (E) RT-PCR analysis of osteogenic marker gene expression and chondrogenic marker gene expression in the indicated periosteal progenitors harvested after 2 weeks of culture in osteogenic or chondrogenic medium. n = 3 per group. (F) Tube formation and HUVEC migration assays were performed with control or HBEGF Over Prx1 conditioned medium in the absence or presence of U0126 or gefitinib. Scale bar = 100 µm. (G) Quantifications of the tube formation number, branch points, tube formation area and migrated cell number; n = 3 per group. Data are presented as means ± SD. Statistical analysis was performed using one-way ANOVA with Bonferroni's post-hoc test for multiple comparisons (B, C, E, G). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The protein bands were detected using specific primary antibodies against EGFR (1:1000; CST, 4267), p-EGFR (1:1000; Abcam, ab40815), ERK (1:1000; CST, 4695), p-ERK (1:1000; CST, 4370), HBEGF (1:1000; Boster, A01759-3), β-Actin (1:4000; CST, 4970) and secondary antibodies.

Techniques: Migration, Western Blot, Derivative Assay, Flow Cytometry, Membrane, Staining, Reverse Transcription Polymerase Chain Reaction, Marker, Gene Expression, Control