Journal: Journal of Cellular and Molecular Medicine

Article Title: CircTLK1 modulates sepsis‐induced cardiomyocyte apoptosis via enhancing PARP1/HMGB1 axis–mediated mitochondrial DNA damage by sponging miR‐17‐5p

doi: 10.1111/jcmm.16738

Figure Lengend Snippet: Effect of LPS challenge on expression of PARP1, HMGB1 and circTLK1 oxidative stress and apoptosis in HCM cells. A, The viability of HCM cells after exposure to various concentrations of LPS. B, The mRNA expression of PARP1 and HMGB1 in LPS‐stimulated HCM cells was shown. C, Western blotting for PARP1 and HMGB1 protein levels in HCM cells induced by LPS. D, The expression of circTLK1 in HCM cells. E, The production of ROS in HCM cells. The lactate dehydrogenase (LDH) (F), creatine kinase (CK) (G) levels in the cellular supernatant, and the MDA level (H) and SOD activity (I) in HCM cells were evaluated. J, The apoptosis of HCM cells was assessed. All results from three independent experiments were expressed as mean ± SD. * P < .05; ** P < .01; *** P < .001 vs control group

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C.

Techniques: Expressing, Western Blot, Activity Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircTLK1 modulates sepsis‐induced cardiomyocyte apoptosis via enhancing PARP1/HMGB1 axis–mediated mitochondrial DNA damage by sponging miR‐17‐5p

doi: 10.1111/jcmm.16738

Figure Lengend Snippet: CircTLK1 or PARP1 inhibition restrains LPS‐induced apoptosis in HCM cells. The mRNA (A) and protein (B) levels of PARP1 and HMGB1 in HCM cells subjected to various treatments. C, Effect of PARP1 on the acetylation of HMGB1 was evaluated by Co‐IP assay. D, The viability of HCM cells from different groups was shown. The apoptosis of HCM cells was assessed by flow cytometer (E) and TUNEL (F). G, Protein levels of Bcl‐2, Bax and cleaved caspase‐3 in HCM cells. All results from three independent experiments were expressed as mean ± SD. * P < .05; ** P < .01; *** P < .001 vs the indicated group

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C.

Techniques: Inhibition, Co-Immunoprecipitation Assay, Flow Cytometry, TUNEL Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircTLK1 modulates sepsis‐induced cardiomyocyte apoptosis via enhancing PARP1/HMGB1 axis–mediated mitochondrial DNA damage by sponging miR‐17‐5p

doi: 10.1111/jcmm.16738

Figure Lengend Snippet: Effect of circTLK1 silencing or PARP1 suppression on LPS‐induced oxidative stress and mitochondrial dysfunction. A, ROS production in HCM cells. The MDA level (B) and SOD activity (C) in HCM cells were determined. D, Mitochondrial membrane potential (∆Ψm) of HCM cells was detected. E, The NAD+/NADH ratio was detected. The distribution of cytochrome C in HCM cells was determined by Western blotting (F) and immunofluorescence staining (G). All results from three independent experiments were expressed as mean ± SD. * P < .05; ** P < .01; *** P < .001 vs the indicated group

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C.

Techniques: Activity Assay, Membrane, Western Blot, Immunofluorescence, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircTLK1 modulates sepsis‐induced cardiomyocyte apoptosis via enhancing PARP1/HMGB1 axis–mediated mitochondrial DNA damage by sponging miR‐17‐5p

doi: 10.1111/jcmm.16738

Figure Lengend Snippet: Effect of PARP1 or circTLK1 suppression on mtDNA damage after exposure to LPS. A, The copy number of mtDNA. B, To assess mtDNA transcript levels, mtDNA‐encoded ND1, CytB and ATP6 expression was detected. C, The 8‐OHdG level in HCM cells was determined. D, The ATP level in HCM cells was assessed. All results from three independent experiments were expressed as mean ± SD. * P < .05; ** P < .01; *** P < .001 vs the indicated group

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircTLK1 modulates sepsis‐induced cardiomyocyte apoptosis via enhancing PARP1/HMGB1 axis–mediated mitochondrial DNA damage by sponging miR‐17‐5p

doi: 10.1111/jcmm.16738

Figure Lengend Snippet: Effect of circTLK1/PARP1 axis inhibition on oxidative mtDNA damage‐mediated apoptosis in LPS‐induced HCM cells. A, The apoptotic rate of HCM cells was detected. The MDA level (B) and SOD activity (C) in HCM cells were evaluated. D, The copy number of mtDNA was detected. E, The mtDNA transcript levels (ND1, CytB and ATP6) were assessed. All results from three independent experiments were expressed as mean ± SD. * P < .05; ** P < .01; *** P < .001 vs the indicated group

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C.

Techniques: Inhibition, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircTLK1 modulates sepsis‐induced cardiomyocyte apoptosis via enhancing PARP1/HMGB1 axis–mediated mitochondrial DNA damage by sponging miR‐17‐5p

doi: 10.1111/jcmm.16738

Figure Lengend Snippet: Effect of circTLK1/PARP1 axis on oxidative mtDNA damage–mediated cardiomyocyte apoptosis in septic rats. (A) and (B) PARP1 and HMGB1 expression levels in the myocardial tissues. C, The apoptosis in myocardial tissues. D, The protein levels of Bcl‐2, Bax and cleaved caspase‐3 in myocardial tissues were assessed. The levels of MDA (E) and 8‐OHdG (F) in myocardial tissues were detected. G, The mtDNA copy number in myocardial tissues was determined. H, The mtDNA transcript levels (ND1, CytB and ATP6) in myocardial tissues were determined. All results from three independent experiments were expressed as mean ± SD. * P < .05; ** P < .01; *** P < .001 vs the indicated group

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircTLK1 modulates sepsis‐induced cardiomyocyte apoptosis via enhancing PARP1/HMGB1 axis–mediated mitochondrial DNA damage by sponging miR‐17‐5p

doi: 10.1111/jcmm.16738

Figure Lengend Snippet: CircTLK1 promotes PARP1 expression via sponging miR‐17‐5p. A, The predicted complementarity between circTLK1 and miR‐17‐5p. B, Dual‐luciferase assay for verifying the relationship between circTLK1 and miR‐17‐5p. C, The direct binding between circTLK1 and miR‐17‐5p was confirmed by RIP assay. D, Expression of miR‐17‐5p in HCM cells was assessed. E, The mRNA expression of PARP1 in HCM cells with various treatments was detected. F, The putative region of complementarity between miR‐17‐5p and PARP1. G, The interaction between miR‐17‐5p and PARP1 was determined by dual‐luciferase assay. All results from three independent experiments were expressed as mean ± SD. ** P < .01; *** P < .001 vs the indicated group

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C.

Techniques: Expressing, Luciferase, Binding Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: CircTLK1 modulates sepsis‐induced cardiomyocyte apoptosis via enhancing PARP1/HMGB1 axis–mediated mitochondrial DNA damage by sponging miR‐17‐5p

doi: 10.1111/jcmm.16738

Figure Lengend Snippet: A schematic overview of the role of circTLK1/miR‐17‐5p/PARP1/HMGB1 axis in LPS‐induced mtDNA oxidative damage and subsequent cardiomyocyte apoptosis

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C.

Techniques:

Journal: Cells

Article Title: Lactate-Induced HBEGF Shedding and EGFR Activation: Paving the Way to a New Anticancer Therapeutic Opportunity

doi: 10.3390/cells13181533

Figure Lengend Snippet: Effect of cisplatin (CPL) in control and lactate-exposed cultures. ( A ) In MDA-MB-231 cells grown in Low-Glc DMEM, the antiproliferative effect of 50 µM CPL was increased by CRM197, given at 32 nM (* p < 0.05, as assessed by t -test). This effect was not observed in lactate-exposed cells. ( B ) Lactate-exposed cells were exposed to CPL to evaluate the effect of CRM197 and BC11 on the drug response. Data were analyzed by ANOVA, followed by Tukey’s post-test. @: a statistically significant difference was observed between cell samples treated with BC11 and those exposed to BC11+CRM197 ( p < 0.05). *: BC11 significantly increased the effect of CPL ( p < 0.001). ( C ) In HT-29 cells grown in Low-Glc DMEM, the antiproliferative effect of 50 µM CPL was not modified by CRM197. Lactate-exposed cells showed a reduced response to CPL and, again, this effect was not modified by CRM197. ( D ) The experiments shown in ( B ) were replicated in HT-29 cultures. #: no statistically significant difference was observed between cell samples treated with BC11 and those exposed to BC11 + CRM197. §: the increased antiproliferative effect observed in cell samples exposed to CPL/CRM197/BC11 did not reach the level of statistical significance, when compared to the single CPL treatment. In these experiments, no difference in the proliferation rate was observed between cells maintained in Low Glc DMEM and those cultured in Low Glc DMEM + lactate.

Article Snippet: CRM197 (Cross Reacting Material 197) was obtained in lyophilized form (SC-203924, Santa Cruz Biotechnology, Dallas, TX, USA); it was dissolved in ultra-pure water (W4502, Merck) and stored at −80 °C.

Techniques: Control, Modification, Cell Culture

Journal: Cells

Article Title: Lactate-Induced HBEGF Shedding and EGFR Activation: Paving the Way to a New Anticancer Therapeutic Opportunity

doi: 10.3390/cells13181533

Figure Lengend Snippet: ( A ) Glycolysis inhibition, assessed by quantifying the released lactate. Data were analyzed by ANOVA followed by Dunnett’s post-test; a statistically significant reduction of lactate release was observed in cell samples exposed to the combined CRM197/BC11 treatment, with p < 0.01. ( B ) According to the method described in , the antiproliferative effect caused by CRM197/BC11 suggests synergism by the two compounds. ( C ) Immunoblotting evaluation of EGFR-mediated signaling shutdown and of apoptosis induction (PUMA). Phospho-EGFR band intensities were normalized on the corresponding EGFR signal; for this immunoblotting analysis the same sample was used and was run in parallel experiments; gels and blots were processed in parallel. PUMA band intensities were normalized on the corresponding Actin level. The bar graph shows the effects caused by the two compounds, given individually or in association. Data were analyzed by one-sample t-tests. The combination CRM197/BC11 significantly reduced phospho-EGFR, which became barely detectable, and markedly increased the level of PUMA. * and ** indicate a statistically significant difference compared to the control cultures, with p < 0.05 and 0.01, respectively.

Article Snippet: CRM197 (Cross Reacting Material 197) was obtained in lyophilized form (SC-203924, Santa Cruz Biotechnology, Dallas, TX, USA); it was dissolved in ultra-pure water (W4502, Merck) and stored at −80 °C.

Techniques: Inhibition, Western Blot, Control

Journal: Cells

Article Title: Lactate-Induced HBEGF Shedding and EGFR Activation: Paving the Way to a New Anticancer Therapeutic Opportunity

doi: 10.3390/cells13181533

Figure Lengend Snippet: ( A ) Representative pictures of MDA-MB-231 cultures. The limits of the wound area have been outlined in red; repopulation was evaluated with the aid of the ImageJ software, as described in , and the percentage of healed wound over time is reported in ( B ). Data were analyzed by ANOVA followed by Tukey’s post-test. *: a statistically significant difference was observed between control and lactate-exposed cultures at all the considered time intervals ( p < 0.01). #: CRM197/BC11 significantly reduced the effects of lactate at 20 and 30 h ( p < 0.01).

Article Snippet: CRM197 (Cross Reacting Material 197) was obtained in lyophilized form (SC-203924, Santa Cruz Biotechnology, Dallas, TX, USA); it was dissolved in ultra-pure water (W4502, Merck) and stored at −80 °C.

Techniques: Software, Control

Journal: Cells

Article Title: Lactate-Induced HBEGF Shedding and EGFR Activation: Paving the Way to a New Anticancer Therapeutic Opportunity

doi: 10.3390/cells13181533

Figure Lengend Snippet: ( A ) Representative pictures of colonies formed by MDA-MB-231 cells, stained with CV. ( B ) Colorimetric evaluation of colonies. Data were evaluated by ANOVA, followed by Tukey’s post-test. *: a statistically significant difference was observed between lactate-exposed cells compared to controls and to all the applied treatments, with p values < 0.01–0.001. ( C ) High magnification pictures of colonies, showing the morphology changes induced by CRM197 (60×) and the immunostaining of E-cadherin (E-CAD) (600×). The green fluorescence indicative of E-CAD positive cells was clearly evident only in cells exposed to CRM197.

Article Snippet: CRM197 (Cross Reacting Material 197) was obtained in lyophilized form (SC-203924, Santa Cruz Biotechnology, Dallas, TX, USA); it was dissolved in ultra-pure water (W4502, Merck) and stored at −80 °C.

Techniques: Staining, Immunostaining, Fluorescence

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