human gene control primers  (PrimerDesign Inc)

Journal: Mediterranean Journal of Rheumatology

Article Title: Evaluation of IRF7 mRNA and its Association with Promoter Methylation in Kashmiri (North-Indian) Patients with Systemic Sclerosis: A Case-Control Study

doi: 10.31138/mjr.040124.eoi

Figure Lengend Snippet: (A) Methylation-specific PCR results of IRF-7 promoter using specific primers. Lane MM: 50bp in DNA molecular weight marker; Lane M: Methylated band (196bp); Lane U: Unmethylated band (208bp); Lane Mc: Human Methylation (+) DNA Control; Lane Uc: Human Unmethylation (−) DNA Control. (B) Association of methylation status with cases and controls in systemic sclerosis. (C) Effect of IRF7 promoter methylation on its mRNA Expression. **P=<0.01; ***P=<0.001.

Article Snippet: To verify the PCR results, representative bands from each target and housekeeping gene Human methylated DNA control and Human non-methylated control (ZYMO Research) were gel-purified on a 2% agarose gel yielding a band of 196bp for methylated and 208bp for an unmethylated product and visualised under UV illumination.

Techniques: Methylation, Molecular Weight, Marker, Control, Expressing

Journal: NPJ Regenerative Medicine

Article Title: Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration

doi: 10.1038/s41536-022-00228-5

Figure Lengend Snippet: rhPRG4 treatment results in near total closure of the ear wound 4 weeks-post injury, while Prg4 −/− demonstrate no wound closure. This is rescued by the addition of rhPRG4 a . Photographs of the representative ears 4 weeks post-injury b – e . The black dashed line represents the plane of histological sectioning, the parallel black bars represent the original injury diameter b – e , while the white dashed box indicates the location of the histological image presented b – e . Representative histological images stained with Safranin O, 4 weeks post-injury f – i . Mainly fibrotic-like tissue was observed in the injury site of C57BL/6 and Prg4 −/− mice treated with DMSO f , h . In Prg4 −/− mice treated with rhPRG4, new cartilage like-tissue can be observed in the injury site (arrow g). New cartilage bridging the injury site can be observed in C57BL/6 mice treated with rhPRG4 and only small areas are present that still lack cartilage (arrows i). The black dashed line represents site of original injury Scale bar equals 50 µm. n.s. = not significant. Sample sizes: a-i, n = 15 per group. Error bars equal mean ± SD (a - 2 way ANNOVA).

Article Snippet: RNA was converted to cDNA using High Capacity Reverse Transcriptase cDNA kit (Thermo-Fisher). mRNA levels were analyzed using TaqMan® Universal PCR Master Mix using TaqMan® Gene Expression Assay primers for mouse or human Vegf , Hif1α , Tgf β, Prg4 and 18 S (endogenous control) on Quantstudio 6 Real-Time PCR System (all Thermo-Fisher).

Techniques: Staining

Journal: NPJ Regenerative Medicine

Article Title: Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration

doi: 10.1038/s41536-022-00228-5

Figure Lengend Snippet: A 3 mm secondary biopsy was generated around the primary 2 mm wound injury at various timepoints post-injury and the isolated cells were purified and examined by qPCR a . Live, tdTomato + b , c , Hic1 + progenitors were assayed for Prg4 , Tgfβ and Vegf expression d – f . Both Prg4 and Tgfβ levels were upregulated by rhPRG4, while no Vegf response was observed to rhPRG4. Live, CD11b + , F4/80 + macrophages g were assayed for Prg4 , Tgfβ and Vegf expression h – j . Prg4 was not detected in macrophages, however both Tgfβ and Vegf levels were upregulated by rhPRG4. The number of Hic1 + and F4/80 + cells were quantified post-injury and it was observed that F4/80 + macrophage numbers increased immediately after rhPRG4 treatment k ; while Hic1 + cells increased at day 5 post-rhPRG4 treatment/injury l . All experiments were undertaken on at least 3 biological and 3 technical replicates unless otherwise stated. n.s. = not significant. Error bars equal mean ± SD ( d , e , f , h , I , j , k , l – t-test).

Article Snippet: RNA was converted to cDNA using High Capacity Reverse Transcriptase cDNA kit (Thermo-Fisher). mRNA levels were analyzed using TaqMan® Universal PCR Master Mix using TaqMan® Gene Expression Assay primers for mouse or human Vegf , Hif1α , Tgf β, Prg4 and 18 S (endogenous control) on Quantstudio 6 Real-Time PCR System (all Thermo-Fisher).

Techniques: Generated, Isolation, Purification, Expressing

Journal: NPJ Regenerative Medicine

Article Title: Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration

doi: 10.1038/s41536-022-00228-5

Figure Lengend Snippet: Bone marrow derived macrophages from C57BL/6, Prg4 − / − and Tlr4 − / − mice were stimulated with LPS or IL-4 to induce pro(CD38 + )/anti(CD206 + )-inflammatory polarization respectively. rhPRG4 was supplemented to LPS or IL-4 conditions to observe the effects of exogenous PRG4 on CD38 + /CD206 + macrophages. Both C57BL/6 and Prg4 − / − macrophages were sensitive to CD38 + polarization by LPS, while Tlr4 − / − macrophages were not. While supplementing rhPRG4 in LPS conditions had no effects on Tlr4 − / − macrophage CD38 + polarization; rhPRG4 reduced CD38 + polarization in C57BL/6 and Prg4 − / − macrophages. All macrophages were responsive to IL-4 polarization to CD206 + macrophages, however, Tlr4 − / − macrophages demonstrated robust CD206 + polarization. C57BL/6 and Prg4 − / − macrophages demonstrated an increased CD206 + polarization in the presence of rhPRG4, while it had no effect on Tlr4 − / − macrophage CD206 + polarization. All experimental were undertaken on at least 3 biological and 3 technical replicates unless otherwise stated. Error bars equal mean ± SD ( c , d , – t-test). n.s. = not significant.

Article Snippet: RNA was converted to cDNA using High Capacity Reverse Transcriptase cDNA kit (Thermo-Fisher). mRNA levels were analyzed using TaqMan® Universal PCR Master Mix using TaqMan® Gene Expression Assay primers for mouse or human Vegf , Hif1α , Tgf β, Prg4 and 18 S (endogenous control) on Quantstudio 6 Real-Time PCR System (all Thermo-Fisher).

Techniques: Derivative Assay

Journal: Disease Models & Mechanisms

Article Title: 17-DMAG regulates p21 expression to induce chondrogenesis in vitro and in vivo

doi: 10.1242/dmm.033662

Figure Lengend Snippet: Characterization of drug 70 in vivo in a mouse model of cartilage injury. (A-C) C57BL/6 and p21 −/− mice were treated with DMSO or drug 70 and examined at 7, 14 and 28 days postinjury with Safranin O staining. In DMSO-treated C57BL/6 mice, little cartilage (bright red tissue) within the injury site was observed. (E-G) However, with drug 70 treatment (0.1 µM), cartilage could be observed within the defect site at all time points examined. (I-K) In p21 −/− mice treated with DMSO, at 28 days postinjury, the wound was almost completely closed and a new cartilage scaffold was present. (M-O) Following drug 70 treatment, the injury site demonstrated near complete regeneration and integration with the native tissue. (D,H,L,P) At 28 days postinjury, the Safranin O staining was validated with collagen type I (green) and collagen type II (red) staining. (Q) The amount of cartilage tissue was quantified within the treatment groups and it was found that drug 70 significantly increased cartilage formation after injury to similar levels as those observed in DMSO-treated p21 −/− mice. Scale bars: 200 µm. Data are mean±s.d.; ns, nonsignificant; *** P <0.01.

Article Snippet: RNA (5 µg) was converted to cDNA using a High Capacity Reverse Transcriptase cDNA kit. mRNA levels were then analyzed using TaqMan ® Universal PCR Master Mix using TaqMan ® Gene Expression Assay primers for human CDKN1A ( p21 ), SOX9 , ACAN , COL2A1 , COL1A1 , COL1A2 and 18S (endogenous control) (all Thermo Fisher Scientific) on a 7900HT Fast-Real-Time PCR System.

Techniques: In Vivo, Staining

Journal: Disease Models & Mechanisms

Article Title: 17-DMAG regulates p21 expression to induce chondrogenesis in vitro and in vivo

doi: 10.1242/dmm.033662

Figure Lengend Snippet: Characterization of cells expressing MSC markers 3 days postinjury. (A,B) Injured C57BL/6 mice treated with DMSO demonstrated little to no cartilage at the site of injury (arrow). (C,D) Fibrotic-like tissue was observed adjacent to the cartilage scaffold (C) that stained positive for collagen type I (D). (E-G) Cells expressing Sca1 (F), CD140a (G) or both (E) were present within the wound site. (I,J) With drug 70 treatment, a large amount of new cartilage could be observed within the wound site (arrow). (K,L) Fibrotic-like tissue was not observed adjacent to the cartilage scaffold (K) with absence of collagen type I staining (L). (N-P) Sca1 (N), CD140a (O) or double-positive cells (P) were present. In DMSO-treated animals, Ki67-positive cells were observed within the epidermis and also within an area of fibrotic tissue (arrow, H), while in drug 70-treated animals, Ki67 staining was present within the epidermis and cartilage scaffold of the ear (arrow, P). (Q,R) Flow analysis of the wound area in both DMSO- and drug 70-treated animals demonstrated that significantly more chondrocytes (collagen type II + ) were present with drug treatment (Q), and that almost all chondrocytes were proliferating in vivo , compared with almost no proliferating chondrocytes observed with DMSO treatment (R). Data are mean±s.d; * P <0.05, *** P <0.01. Scale bars: 200 µm in A and I; 50 µm in B-D, H, J-L and P; 100 µm in E and M.

Article Snippet: RNA (5 µg) was converted to cDNA using a High Capacity Reverse Transcriptase cDNA kit. mRNA levels were then analyzed using TaqMan ® Universal PCR Master Mix using TaqMan ® Gene Expression Assay primers for human CDKN1A ( p21 ), SOX9 , ACAN , COL2A1 , COL1A1 , COL1A2 and 18S (endogenous control) (all Thermo Fisher Scientific) on a 7900HT Fast-Real-Time PCR System.

Techniques: Expressing, Staining, In Vivo

Journal: Disease Models & Mechanisms

Article Title: 17-DMAG regulates p21 expression to induce chondrogenesis in vitro and in vivo

doi: 10.1242/dmm.033662

Figure Lengend Snippet: Characterization of cells expressing MSC markers 3 days postinjury in p21 −/− mice. (A,B) Injured p21 −/− mice treated with DMSO demonstrated robust cartilage present at the site of injury (arrow). (C,D) The cartilage scaffold was also positive for collagen type II. (E-G) Few cells expressing Sca1 (F), CD140a (G) or both (E) were present within the wound site. (I-L) With drug 70 treatment (I), new cartilage could be observed within the wound site (arrow, J) that was positive for Safranin O (K) and collagen type II (L). (M-O) Sca1 (N), CD140a (O) or double-positive cells (M) were present within the cartilage tissue (arrow). (H,P) In DMSO- (H) and drug 70- (P) treated animals, Ki67 was staining is present within the epidermis and cartilage scaffold of the ear (arrows). (Q,R) Flow analysis of the wound area in both DMSO- and drug 70-treated animals demonstrated that significantly more chondrocytes (collagen 2 + ) (Q) were present with drug 70 treatment, and that almost all chondrocytes were proliferating in vivo in both treatment groups (R). Data are mean±s.d.; NS, nonsignificant; *** P <0.01. Scale bars: 200 µm in A and I; 50 µm in B-D, H, J-L and P; 100 µm in E and M.

Article Snippet: RNA (5 µg) was converted to cDNA using a High Capacity Reverse Transcriptase cDNA kit. mRNA levels were then analyzed using TaqMan ® Universal PCR Master Mix using TaqMan ® Gene Expression Assay primers for human CDKN1A ( p21 ), SOX9 , ACAN , COL2A1 , COL1A1 , COL1A2 and 18S (endogenous control) (all Thermo Fisher Scientific) on a 7900HT Fast-Real-Time PCR System.

Techniques: Expressing, Staining, In Vivo

Journal: Disease Models & Mechanisms

Article Title: 17-DMAG regulates p21 expression to induce chondrogenesis in vitro and in vivo

doi: 10.1242/dmm.033662

Figure Lengend Snippet: Effect of drug 70 on chondrocyte proliferation and maintenance of the chondrogenic phenotype. (A,B) Primary chondrocytes were obtained from embryonic limb buds of C57BL/6 and p21 −/− mice. They were treated with DMSO, drug 70 or an additional HSP90 inhibitor demonstrated not to affect p21 levels (drug 129) (A). Drug 70 induced proliferation in both C57BL/6 and p21 −/− chondrocytes, whereas DMSO and drug 129 had no effect (A). Maintenance of chondrogenic phenotype was assessed using the chondrogenic markers Sox9 and Acan . Treatment of C57BL/6 and p21 −/− chondrocytes with drug 70 increased the expression of both markers, whereas drug 129 decreased Acan expression in p21 −/− chondrocytes. Overall, p21 −/− chondrocytes demonstrated increased marker expression than C57BL/6 chondrocytes regardless of treatment group (B). (C) It was also observed that drug 70, but not drug 129, treatment increased/maintained the expression of Col2a1 , with a corresponding decrease in Col1a1 and Col1a2 . (D) In human articular chondrocytes (hAC), neither drug 70 nor drug 129 had any effect on proliferation in passaged chondrocytes. (E) However, drug 70 treatment maintained chondrocyte marker gene expression in passaged chondrocytes compared with fresh chondrocytes, whereas DMSO or drug 129 treatment did not. (F) A similar effect to that observed in mouse cells was observed in human chondrocytes, with drug 70 treatment increasing the expression of COL2A1 , with a corresponding decrease in COL1A1 and COL1A2 . Data are mean±s.d.; ns, nonsignificant; * P <0.05.

Article Snippet: RNA (5 µg) was converted to cDNA using a High Capacity Reverse Transcriptase cDNA kit. mRNA levels were then analyzed using TaqMan ® Universal PCR Master Mix using TaqMan ® Gene Expression Assay primers for human CDKN1A ( p21 ), SOX9 , ACAN , COL2A1 , COL1A1 , COL1A2 and 18S (endogenous control) (all Thermo Fisher Scientific) on a 7900HT Fast-Real-Time PCR System.

Techniques: Expressing, Marker

Journal: The Journal of Biological Chemistry

Article Title: A Small Ubiquitin-related Modifier-interacting Motif Functions as the Transcriptional Activation Domain of Kr?ppel-like Factor 4 *

doi: 10.1074/jbc.M110.101717

Figure Lengend Snippet: The KLF4 SIM is crucial for transcriptional activation in mammalian cells. Dual luciferase reporter assays were performed with C/EBPβ or Lefty1-luciferase reporter plasmids and expression constructs of KLF4 or its mutants in HEK293T cells as described under “Experimental Procedures”. A and B, shown is co-transfection of vector (Vec) alone, pCMV-Myc-KLF4 (WT), or pCMV-Myc-L101A/I106A (LI) with C/EBPβ-luciferase (A) or Lefty1-luciferase (B). The expression levels of Myc-KLF4 (WT) and Myc-KLF4-L101A/I106A (LI) were shown by Western blotting against Myc and β-actin. C and D, shown is co-transfection of vector (Vec) alone, pMT3-KLF4 (WT), pMT3-KLF4-E93V/E95V/E96V (EEE), or pMT3-D99V/D102V/D104V (DDD) with C/EBPβ-luciferase (C) and Lefty1-luciferase (D). The expression levels of both the EEE and DDD mutants have previously been documented (34). Shown are the means and S.D. of four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; by two-tailed t test. Asterisks not associated with brackets are comparisons to vector alone. RLU is relative luciferase unit.

Article Snippet: Three days later, total RNA was isolated with TRIzol (Invitrogen; catalog #15596-018), and quantitative real-time RT-PCR was performed in triplicate with primer sets specific for human SUMO-1, C/EBPβ, Lefty1, and the control gene β-actin (Qiagen; QT00014280, QT00237580, QT01667421, and QT00095431).

Techniques: Activation Assay, Luciferase, Expressing, Construct, Cotransfection, Plasmid Preparation, Western Blot, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: A Small Ubiquitin-related Modifier-interacting Motif Functions as the Transcriptional Activation Domain of Kr?ppel-like Factor 4 *

doi: 10.1074/jbc.M110.101717

Figure Lengend Snippet: Reduction of SUMO-1 inhibits KLF4 transcriptional activity. HEK293T cells were transfected with the C/EBPβ-luciferase (A) or Lefty1-luciferase (B) reporter, vector (Vec) or pCMV-Myc-KLF4 (KLF4), nonspecific siRNA (NS) or the dual siRNA mixture against SUMO-1 (Si), and the control renilla luciferase plasmid. Dual luciferase assays were performed, and the normalized luciferase activity presented as relative luciferase units (RLU). Shown are the means and S.D. of three independent experiments. ***, p < 0.001 by two-tailed t test. C, a fraction of lysates from the corresponding cells was subjected to Western blotting with mouse antibodies against SUMO-1 (upper panel), SUMO-2/3 (middle panel), and ubiquitin (lower panel). β-actin was used as a loading control. The arrows indicate free SUMO-1 (upper panel), SUMO-2/3 (middle panel), and ubiquitin (lower panel). A short exposure of free SUMO-2/3 in the middle panel is also provided.

Article Snippet: Three days later, total RNA was isolated with TRIzol (Invitrogen; catalog #15596-018), and quantitative real-time RT-PCR was performed in triplicate with primer sets specific for human SUMO-1, C/EBPβ, Lefty1, and the control gene β-actin (Qiagen; QT00014280, QT00237580, QT01667421, and QT00095431).

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Two Tailed Test, Western Blot