Journal: Stem Cells (Dayton, Ohio)

Article Title: Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1‐Dependent Autophagy

doi: 10.1002/stem.3091

Figure Lengend Snippet: Sequences of RNA and DNA oligonucleotides

Article Snippet: Lentiviruses targeting PCK2 (sh1‐ PCK2 , sh2‐ PCK2 ) and negative control (NC) vectors (NC1/NC2) were purchased from GenePharma Co. (Suzhou, China).

Techniques: shRNA, Control, Negative Control

Journal: Stem Cells (Dayton, Ohio)

Article Title: Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1‐Dependent Autophagy

doi: 10.1002/stem.3091

Figure Lengend Snippet: PCK2 knockdown impairs the osteogenic capacity of hASCs in vitro. (A): Efficiency of PCK2 knockdown and overexpression was validated by Western blot analysis. GAPDH was used for normalization. (B, C): Knockdown of PCK2 decreased ALP staining (B) and activity (C) on the seventh day after osteogenic induction of hASCs. (D, E): After 7 days of osteogenic induction, relative mRNA expressions of osteogenic marker RUNX2 (D) and ALP (E) were decreased by the depletion of PCK2. (F, G): Extracellular matrix mineralization (F) and ARS quantification (G) were decreased in PCK2 knockdown cells on the 14th day of osteogenic induction. (H, I): Relative mRNA expression of RUNX2 (H) and OCN (I) were downregulated by depletion of PCK2 after 14 days of osteogenic induction. Data represent three independent experiments and values are presented as mean ± SD. **, p ≤ .01; *, p ≤ .05; Student's t test. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; hASCs, human adipose‐derived stem cells; NC1, negative control for sh1‐PCK2; OCN, osteocalcin; OM, osteogenic media; PCK2, mitochondrial phosphoenolpyruvate carboxykinase; PM, proliferation media; RUNX2, runt‐related transcription factor 2.

Article Snippet: Lentiviruses targeting PCK2 (sh1‐ PCK2 , sh2‐ PCK2 ) and negative control (NC) vectors (NC1/NC2) were purchased from GenePharma Co. (Suzhou, China).

Techniques: Knockdown, In Vitro, Over Expression, Western Blot, Staining, Activity Assay, Marker, Expressing, Derivative Assay, Negative Control

Journal: Stem Cells (Dayton, Ohio)

Article Title: Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1‐Dependent Autophagy

doi: 10.1002/stem.3091

Figure Lengend Snippet: PCK2 overexpression enhances the osteogenic capacity of hASCs in vitro. (A, B): Overexpression of PCK2 significantly increased ALP staining (A) and activity (B) after 7 days of osteogenic induction. (C, D): PCK2 overexpression upregulates relative mRNA expression of RUNX2 (C) and ALP (D) after 7 days of osteogenic differentiation. (E, F): The ARS staining (E) and quantification (F) showed an increasing trend after overexpression of PCK2. (G, H): Osteogenic gene markers RUNX2 (G) and OCN (H) were unregulated on the 14th day of osteogenic differentiation. (I, J): Knockdown of PCK2 downregulated the protein level of RUNX2 (I) while PCK2 overexpression upregulated RUNX2 expression (J). GAPDH was used for normalization. Data represent three independent experiments and values are presented as mean ± SD. **, p ≤ .01; *, p ≤ .05; Student's t test. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; OCN, osteocalcin; OM, osteogenic media; PCK2, mitochondrial phosphoenolpyruvate carboxykinase; PM, proliferation media; RUNX2, runt‐related transcription factor 2.

Article Snippet: Lentiviruses targeting PCK2 (sh1‐ PCK2 , sh2‐ PCK2 ) and negative control (NC) vectors (NC1/NC2) were purchased from GenePharma Co. (Suzhou, China).

Techniques: Over Expression, In Vitro, Staining, Activity Assay, Expressing, Knockdown

Journal: Stem Cells (Dayton, Ohio)

Article Title: Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1‐Dependent Autophagy

doi: 10.1002/stem.3091

Figure Lengend Snippet: PCK2 enhances hASC osteogenesis in vivo. The hASCs transfected with sh1‐ PCK2 , sh2‐ PCK2 , NC, PCK2 , and vector were mixed with β‐TCP carriers and were subcutaneously implanted into the dorsal side of the mice. After 8 weeks, the samples were harvested. (A, B, D, E): H&E staining (A, D), Masson's trichrome staining (B, E) and the histomorphometry analysis of the implanted hASC‐scaffold hybrids are presented. (C, F): Representative micro‐CT images and quantitative analysis of BV/TV (%) are shown. H&E staining of implanted hASCs‐TCP hybrids is presented. The black arrows in (D) point to microvascular formation. Scale bar = 100 μm. Images are representative of three independent experiments, each including 10 BALB/c nude mice. Results are presented as the mean ± SD, n = 3. *, p < .01; **, p < .05. Abbreviations: BV/TV, bone volume to total volume; hASCs, human adipose‐derived stem cells; H&E, hematoxylin and eosin; β‐TCP, beta‐tricalcium phosphate; NC, negative control for sh1‐PCK2 and sh2‐PCK2; PCK2, mitochondrial phosphoenolpyruvate carboxykinase.

Article Snippet: Lentiviruses targeting PCK2 (sh1‐ PCK2 , sh2‐ PCK2 ) and negative control (NC) vectors (NC1/NC2) were purchased from GenePharma Co. (Suzhou, China).

Techniques: In Vivo, Transfection, Plasmid Preparation, Staining, Micro-CT, Derivative Assay, Negative Control

Journal: Stem Cells (Dayton, Ohio)

Article Title: Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1‐Dependent Autophagy

doi: 10.1002/stem.3091

Figure Lengend Snippet: PCK2 positively regulates autophagy during osteogenesis by hASCs. (A): PCK2 knockdown and control cells were cultured in regular PM or PM containing 500 μM H 2 O 2 for 24 hours. (B): The control cells or PCK2 knockdown cells were cultured in PM, PM containing 500 μM H 2 O 2 , or PM containing 500 μM H 2 O 2 and 10 μg/ml pep + E64d for 24 hours. (C): The control vector cells or PCK2‐expressing and cells were cultured in PM or PM containing 500 μM H 2 O 2 for 24 hours. (D): The control cells or PCK2 knockdown cells were cultured in PM or PM without serum (SS medium) for 48 hours. (E): The sh1‐ PCK2 or NC1 cells were cultured in PM, SS medium, or SS medium containing 10 μg/ml pep + E64d for 48 hours. (F): The PCK2‐expressing and control vector cells were cultured in PM or SS medium for 7 days. (G, H): The control cells and PCK2 knockdown cells were cultured in PM or OM (G). The control vector cells and PCK2‐expressing cells were cultured in PM or OM (H). (I): The control cells and PCK2 knockdown cells were cultured in PM and OM, with or without 10 μg/ml pep + E64d for 7 days. (J): Confocal microscopy of LC3B with DAPI counterstaining on the seventh day of osteogenic induction. Scale bars = 100 μm. Images represent three independent experiments. GAPDH was used as loading control. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; LC3B, microtubule associated protein 1 light chain 3 β; NC1, negative control for sh1‐ PCK2 ; OM, osteogenic media; PCK2, mitochondrial phosphoenolpyruvate carboxykinase; p62, p62/SQSTM1; pep: pepstain A; PM, proliferation media; OM, osteogenic media; RUNX2, runt‐related transcription factor 2; SS, serum starvation.

Article Snippet: Lentiviruses targeting PCK2 (sh1‐ PCK2 , sh2‐ PCK2 ) and negative control (NC) vectors (NC1/NC2) were purchased from GenePharma Co. (Suzhou, China).

Techniques: Knockdown, Control, Cell Culture, Plasmid Preparation, Expressing, Confocal Microscopy, Negative Control

Journal: Stem Cells (Dayton, Ohio)

Article Title: Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1‐Dependent Autophagy

doi: 10.1002/stem.3091

Figure Lengend Snippet: Autophagy is required for the osteogenic differentiation of hASCs. (A, B): Depletion of ATG7 decreased ALP staining (A) and activity (B) on the seventh day after induction of osteogenic differentiation of hASCs. (C, D): Extracellular matrix mineralization (C) and ARS quantification (D) were decreased in ATG7 knockdown cells after 14 days of osteogenic induction. (E): Protein levels of RUNX2, p62, and LC3B are indicated as shown. GAPDH was used as internal loading control. (F–M): Treatment with 5 mM 3‐MA decreased the osteogenic capacity of PCK2‐expressing cells. Relative mRNA expression of RUNX2 (F) and ALP (G) on the seventh day of osteogenic induction are shown. Relative mRNA expression of RUNX2 (H) and OCN (I) after 14 days of osteogenic induction is shown. ALP staining (J) and activity (K) on the seventh day of osteogenic induction are shown. Extracellular matrix mineralization (L) and ARS quantification (M) on the 14th day of osteogenic induction are presented. (N): Protein expression patterns of PCK2, RUNX2, p62, and LC3B are shown. The control vector cells and PCK2‐expressing cells were cultured in PM, PM containing 5 mM 3‐MA, OM, or OM containing 5 mM 3‐MA for 5 days. GAPDH was used as internal loading control. Data are represented as mean ± SD. **, p ≤ .01; *, p ≤ .05, Student's t test. Abbreviations: 3‐MA, 3‐methyladenine; ALP, alkaline phosphatase; ARS, alizarin red S; ATG7, autophagy‐related‐gene‐7; LC3B, microtubule associated protein 1 light chain 3 β; OCN, osteocalcin; NC, negative control for sh‐ ATG7 ; NS, not significant; OM, osteogenic media; p62, p62/SQSTM1; PCK2, mitochondrial phosphoenolpyruvate carboxykinase; PM, proliferation media; RUNX2, runt‐related transcription factor 2.

Article Snippet: Lentiviruses targeting PCK2 (sh1‐ PCK2 , sh2‐ PCK2 ) and negative control (NC) vectors (NC1/NC2) were purchased from GenePharma Co. (Suzhou, China).

Techniques: Staining, Activity Assay, Knockdown, Control, Expressing, Plasmid Preparation, Cell Culture, Negative Control

Journal: Stem Cells (Dayton, Ohio)

Article Title: Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1‐Dependent Autophagy

doi: 10.1002/stem.3091

Figure Lengend Snippet: PCK2 enhances osteogenic differentiation of hASCs via modulating AMPK/ULK1‐dependent autophagy. (A): Protein expression levels of p‐ULK1 (ser 555), p‐ULK1 (ser 556), p‐ULK1 (ser 757), ULK1, p‐AMPK α, and AMPK in PCK2‐knockdown and control cells are shown. (B): Western blots of p‐ULK1 (ser 555), p‐ULK1 (ser 556), p‐ULK1 (ser 757), ULK1, p‐AMPK α, and AMPK in PCK2‐expressing and control vector cells. (C, D): Relative mRNA expression levels of ULK1 (C) and RUNX2 (D) in hASCs transfected with si1‐ ULK1 , si2‐ ULK1 , and negative control (si‐NC) after 7 days of osteogenic induction. Data represent three independent experiments. (E, F): Depletion of ULK1 decreases ALP staining (E) and activity (F) on the seventh day after induction of osteogenic differentiation in hASCs. (G, H): Knockdown of ULK1 decreases ARS staining (G) and activity (H) on the 14th day after induction of osteogenic differentiation in hASCs. (I): Inhibition of AMPK in PCK2‐expressing hASCs suppresses osteogenic ability and autophagy activity of hASCs, as shown by the protein expressions of RUNX2, LC3B, and p62. (J): PCK2‐knockdown cells expressing wild‐type AMPK increase the osteogenic capacity and autophagy activity of hASCs. The protein expression patterns of RUNX2, LC3B, and p62 are presented. GAPDH was used as internal loading control in (A, B) and (I, J). ( K–N): H&E staining, Masson's trichrome staining from implanted hASC‐scaffold hybrids. Scale bar = 100 μm, n = 10. Data in this figure represent three independent experiments and are presented as mean ± SD. **, p ≤ .01; *, p ≤ .05; Student's t test. Abbreviations: ALP, alkaline phosphatase; AMPK, AMP‐activated protein kinase; ARS, alizarin red S; ATG7, autophagy‐related‐gene‐7; hASCs, human adipose‐derived stem cells; H&E, hematoxylin and eosin; LC3B, Microtubule associated protein 1 light chain 3 β; NS, not significant; OM, osteogenic media; p62, p62/SQSTM1; PCK2, mitochondrial phosphoenolpyruvate carboxykinase; PM, proliferation media; RUNX2, runt‐related transcription factor 2; ULK1, unc‐51 like autophagy activating kinase 1.

Article Snippet: Lentiviruses targeting PCK2 (sh1‐ PCK2 , sh2‐ PCK2 ) and negative control (NC) vectors (NC1/NC2) were purchased from GenePharma Co. (Suzhou, China).

Techniques: Expressing, Knockdown, Control, Western Blot, Plasmid Preparation, Transfection, Negative Control, Staining, Activity Assay, Inhibition, Derivative Assay