Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: A. The expression of RRM2 was detected via western blotting in synovial specimens from patients with RA and normal controls. The relative expression of RRM2 was shown in histogram. **p<0.01, compared to normal controls. B. MH7A cells were treated with TNF-α (10 ng/mL) and IL-1β (10 mg/L) for 24 h, and the expression of RRM2 was determined via western blotting. The relative expression of RRM2 was shown in histogram. **p<0.01, compared with untreated group. ##p<0.01, compared with untreated group. C. MH7A cells were treated with RRM2 shRNA lentivirus and lentiviral controls for 24 h. The relative expression of RRM2 was determined via western blotting and shown in histogram. **p<0.01, compared with control lentivirus group. D. MTT assay. MH7A cells were infected with RRM2 shRNA lentivirus and lentiviral controls for 24, 48, and 72 h, and cell viability was determined via MTT assay. **p<0.01. E. Clone formation assay. MH7A cells were infected with RRM2 shRNA and control shRNA lentivirus and cultured for 2 weeks. Colony formation assay was performed as described in Materials and Methods. The colony number was shown in histogram. **p<0.01, compared with control shRNA group. F. Migration and invasion assays. MH7A cells were infected with RRM2 shRNA and control shRNA lentiviruses and treated with 10 ng/mL of TNF-α. The migratory and invasive abilities were detected using transwell Boyden chamber coated without or with a Matrigel basement membrane matrix after 48 h. Original magnification ×100.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques: Expressing, Western Blot, shRNA, MTT Assay, Infection, Tube Formation Assay, Cell Culture, Colony Assay, Migration, Membrane

Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: A. MH7A cells were treated with TNF-α (10 ng/mL) and IL-1β (10 mg/L) for 24 h, and the expression of RRM2 was determined via western blotting. B. The relative expression of IGF2BP3, RRM2, MMP-9 and MMP-1 was shown in histogram. **p<0.01, compared with control group. C. MH7A cells were infected with IGF2BP3 shRNA and control shRNA lentiviruses and treated with 10 ng/mL of TNF-α for 24 h. The expression of IGF2BP3, RRM2, MMP-9, and MMP-1 was detected via western blotting. D. The relative expression of IGF2BP3, RRM2, MMP-9 and MMP-1 was shown in histogram. **p<0.01, compared with control shRNA group.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques: Expressing, Western Blot, Infection, shRNA

Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: A. MTT assay. MH7A cells were infected with IGF2BP3 shRNA lentivirus and control shRNA lentivirus was treated with TNF-α (10 ng/mL) for 24, 48, and 72 h. The cell viability was determined using MTT assay. **p<0.01, compared with control shRNA group. B. Clone formation assay. MH7A cells were infected with IGF2BP3 shRNA and control shRNA lentivirus and cultured for 2 weeks. The colony number was shown in histogram. **p<0.01, compared with control shRNA group. C. Migration and invasion assays. The effects of IGF2BP3 knockdown on invasion and migration were detected using transwell Boyden chamber coated with or without a Matrigel basement membrane matrix after 48 h. Original magnification ×100. D. MTT assay. MH7A cells were treated with IGF2BP3 shRNA lentivirus and overexpressed control lentivirus, control shRNA lentivirus and overexpressed RRM2 lentivirus, IGF2BP3 shRNA lentivirus and overexpressed control lentivirus, and IGF2BP3 shRNA lentivirus and overexpressed RRM2 lentivirus for 24, 48, and 72 h. Cell viability was determined using MTT assay. **p<0.01, compared with control group.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques: MTT Assay, Infection, shRNA, Tube Formation Assay, Cell Culture, Migration, Membrane

Journal: PLOS ONE

Article Title: IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway

doi: 10.1371/journal.pone.0303593

Figure Lengend Snippet: A. MH7A cells were infected with RRM2 shRNA or control shRNA lentiviruses for 48 h. The expression of RRM2, phosphorylated Akt, total Akt, and MMP-9 was detected via western blotting. The relative expression of RRM2, p-Akt, Akt and MMP-9 was shown in histogram. **p<0.01, compared with control shRNA group. B. The MH7A cells infected with RRM2 shRNA or control shRNA lentiviruses were treated with Akt inhibitor for 24 h. The expression of pAkt-S473, total Akt, and MMP-9 was detected via western blotting. The relative expression of RRM2, p-Akt, Akt, MMP-1 and MMP-9 was shown in histogram. **p<0.01, ##p<0.01, compared with control group.

Article Snippet: RRM2 Lentiviral copy DNA Open Reading Frame Clone, Human, C-GFPSpark® tag (Cat: HG18284-ACGLN) and pLV-C-GFPSpark lentivirus control plasmid (Cat: LVCV-35) were purchased from SinoBiological corporation (Beijing, China).

Techniques: Infection, shRNA, Expressing, Western Blot

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

Journal: Molecular Medicine Reports

Article Title: Downregulation of inhibitor of apoptosis-stimulating protein of p53 inhibits proliferation and promotes apoptosis of gastric cancer cells

doi: 10.3892/mmr.2015.3587

Figure Lengend Snippet: iASPP expression is significantly downregulated in gastric cancer cells following infection with iASPP-siRNA-lentivirus, while the control siRNA had no effect on iASPP protein levels. (A) iASPP expression in MKN-45 cells was inhibited following iASPP-siRNA-lentivirus infection. (B) iASPP expression in SGC-7901 cells was inhibited following iASPP-siRNA-lentivirus infection. Values are expressed as the mean ± standard deviation; * P<0.05. iASPP, inhibitor of apoptosis-stimulating protein of p53; siRNA, small interfering RNA.

Article Snippet: Downregulation of iASPP was achieved by infecting the cells with the iASPP-small interfering (si)RNA lentivirus (Genepharma Co., Ltd., Shanghai, China).

Techniques: Expressing, Infection, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Downregulation of inhibitor of apoptosis-stimulating protein of p53 inhibits proliferation and promotes apoptosis of gastric cancer cells

doi: 10.3892/mmr.2015.3587

Figure Lengend Snippet: Effects of iASPP expression on GC cells. (A) An MTT assay indicated that GC cell proliferation was significantly inhibited following iASPP-siRNA-lentivirus infection. (B) Inhibition of iASPP expression inhibited the colony-forming ability of GC cell lines. (C) Inhibition of iASPP promoted GC cell apoptosis. Values are expressed as the mean ± standard deviation; * P<0.05, ** P<0.01 vs. control. iASPP, inhibitor of apoptosis-stimulating protein of p53; siRNA, small interfering RNA; GC, gastric cancer; PE, phycoerythrin; UL, upper left; UR, upper right; LL, lower left; LR, lower right.

Article Snippet: Downregulation of iASPP was achieved by infecting the cells with the iASPP-small interfering (si)RNA lentivirus (Genepharma Co., Ltd., Shanghai, China).

Techniques: Expressing, MTT Assay, Infection, Inhibition, Standard Deviation, Small Interfering RNA

Journal: Cellular and Molecular Immunology

Article Title: The role of transcriptional factor D-site-binding protein in circadian CCL2 gene expression in anti-Thy1 nephritis

doi: 10.1038/s41423-018-0020-4

Figure Lengend Snippet: DBP regulated CCL2 expression via the target gene TRIM55. a siDBP inhibited TRIM55 mRNA expression, but not the expression of six other genes. b DBP bound to the TRIM55 promoter, as revealed by chromatin immunoprecipitation sequencing (ChIP)-PCR. c DBP bound to the TRIM55 promoter (-2984 bp), as revealed by the dual luciferase assay. d The TRIM55 promoter was divided into three subregions. e The dual luciferase assay indicated that DBP specifically bound to the -2116–2984-bp region. f, g: siTRIM55-1 and siTRIM55-2 significantly inhibited TNF-α and CCL2 expression at both the mRNA and protein levels in MCs. h The rescue assay showed that DBP-mediated CCL2 upregulation was blocked by si-TRIM55-1. si-TRIM55-1 and si-TRIM55-2, two siRNAs targeting TRIM55; Lv-Flag, lentivirus control; Lv-Flag-DBP: DBP-expressing lentivirus. *p < 0.05, **p < 0.01; n = 3. All assays were repeated at least three times

Article Snippet: DBP lentiviruses ( ≥ 1 × 10 9 TU/mL) expressing the Flag peptide (Lv-Flag-DBP and control Lv-Flag) were prepared by GenePharma.

Techniques: Expressing, ChIP-sequencing, Luciferase, Rescue Assay, Control