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: IGF2BP3-RRM2 association.

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:

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. IGF2BP3 RIP assay. IGF2BP3 immunoprecipitation was performed and detected by Western blotting in TNF-α or IL-1β-treated MH7A cells. GAPDH and IgG was used as the negative control. B. RIP-qPCR assay demonstrated the enrichment of RRM2 mRNA in in anti-IGF2BP3 precipitates of TNF-α or IL-1β-treated MH7A cells(**p<0.01). C. MeRIP-qPCR assay. The m6A enrichment of RRM2 mRNA was shown by using anti-IgG and anti-m6A antibodies in TNF-α or IL-1β-treated MH7A cells after knocking down IGF2BP3. **P < 0.01.

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: Immunoprecipitation, Western Blot, Negative Control

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: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A, RRM2 mRNA expression in pancreatic cancer cell lines relative to expression identified in HPDE. Columns, mean of triplicate; bars, SD. n = 3. B, Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDa) in whole cell lysates of HPDE and pancreatic cancer cell lines. C, Expression of let- 7 family members in pancreatic cancer cell lines relative to expression in HPDE. Columns , mean of triplicate; bars , SD. n = 3; * P <0.05. D, RRM2 is a direct target of let-7 . 293TA and MIA PaCa-2 cells were virally infected for expression of precursors of let-7a-1 , let-7a-2 , let-7a-3 , let-7b , and miR-214 ( negative control ) and subsequently transfected with a RRM2 3′ UTR luciferase reporter construct. Luciferase activities measured 36 h after transfection (normalized relative to renilla activity) were plotted. Columns , mean of triplicate; bars , SD. n = 3. * p <0.05, ** p <0.01.

Article Snippet: RRM2 cDNA without the 3′ UTR region was constructed from RRM2 truclone (RRM2 (NM_001165931) Human cDNA Clone; Product ID: SC326997; Origene, MD) using PCR-based methods.

Techniques: Expressing, Western Blot, Infection, Negative Control, Transfection, Luciferase, Construct, Activity Assay

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A , Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDA) in whole cell lysates of MIA PaCa-2 overexpressing precursors of let-7 family members. Ratios of RRM2 to β-actin band intensities (normalized to control) from three experiments are indicated ( top ). Asterisks indicate significant reductions ( p <0.05) in RRM2 levels compared with control. B , Immunocytochemical detection of RRM2 in exponentially growing MIA PaCa-2 overexpressing pre- let-7 family members. Original magnification, x20. C , MIA PaCa-2 cells stably overexpressing pre- let-7 family members ( red ) or vector alone ( blue ) were treated with gemcitabine (0.1 nM to 100 µM), and percent inhibition of cellular proliferation was measured using an MTT assay. Points , mean of triplicate; bars , SE. n = 3. Gemcitabine IC 50 estimations indicated ( parentheses ).

Article Snippet: RRM2 cDNA without the 3′ UTR region was constructed from RRM2 truclone (RRM2 (NM_001165931) Human cDNA Clone; Product ID: SC326997; Origene, MD) using PCR-based methods.

Techniques: Western Blot, Stable Transfection, Plasmid Preparation, Inhibition, MTT Assay

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A, Western blotting analysis of hENT1 (∼50–55 kDa), hENT2 (50 kDa), hCNT1 (72 kDa), hCNT3 (77 kDa), CDA (55 kDa), dCK (30 kDa), RRM1 (94 kDa), RRM2 (45 kDa), and β-actin (45 kDA) levels in whole cell lysates of Capan-1 and Capan-1-GR cells. B , RRM2 protein increased in a gemcitabine dose-dependent fashion in Capan-1-GR cells. C, Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDA) levels in cells with acquired gemcitabine resistance. Ratios of RRM2 to β-actin band intensities (normalized to untreated cells) from three experiments are indicated ( top ). Asterisk indicates significantly higher RRM2 expression in gemcitabine-resistant cells ( p <0.05) compared with untreated cells. D and E, Relative expression of precursor and mature let-7a in Capan-1 ( D ) and L3.6pl ( E ) cells induced to acquire gemcitabine resistance. Columns, mean of triplicate; bars, SD. n = 3. F , Differential miRNA expression in Capan-1-GR compared with Capan-1. Putative RRM2-modulating miRNAs and their extent of reduction in Capan-1-GR cells are shown ( right ).

Article Snippet: RRM2 cDNA without the 3′ UTR region was constructed from RRM2 truclone (RRM2 (NM_001165931) Human cDNA Clone; Product ID: SC326997; Origene, MD) using PCR-based methods.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A and B, Relative expression of primary let-7a transcripts ( A ) and mature let-7a ( B ) in 2 normal pancreatic tissues and 10 PDAC samples representing various tumor stages. C , Ratios of mature to precursor let-7a transcripts calculated from data points in A and B . D, Western blotting analysis of RRM2 (45 kDa) in total lysates (50 µg) of 6 matched normal-PDAC pairs. Ratios of RRM2 band intensities in PDACs compared to matched normal tissues indicated ( top ). Asterisk indicates significantly higher RRM2 expression in PDAC tissues ( p <0.05) compared with matched normal tissues. E and F, Relative expression of primary let-7a transcripts ( E ) and mature let-7a ( F ) in matched normal-PDAC pairs. G , Ratios of mature to precursor let-7a transcripts calculated from data points in E and F . Asterisk indicates significantly lower mature to precursor let-7a ratio in PDAC tissues ( p <0.05) compared with matched normal tissues.

Article Snippet: RRM2 cDNA without the 3′ UTR region was constructed from RRM2 truclone (RRM2 (NM_001165931) Human cDNA Clone; Product ID: SC326997; Origene, MD) using PCR-based methods.

Techniques: Expressing, Western Blot