Journal: Journal of materials chemistry. B

Article Title: Injectable biocompatible hydrogels with tunable strength based on crosslinked supramolecular polymer nanofibers.

doi: 10.1039/d4tb01873g

Figure Lengend Snippet: Fig. 11 Transmitted light microscopy images of L929 mouse fibroblast cells incubated on the BTP-C6 hydrogel for 72 hours: (a) image taken on the hydrogel, and (b) image of cells reseeded after 48 hours of incubation, collected from the top of the hydrogel. Optical magnification is 10 and the scale bar is 100 mm.

Article Snippet: L929 cells (CLS Cell Lines Service GmbH, Eppelheim, Germany) were cultured in TC-treated cell culture flasks (Greiner Bio-One International GmbH and Labsolute Th.

Techniques: Light Microscopy, Incubation

Journal: Journal of materials chemistry. B

Article Title: Injectable biocompatible hydrogels with tunable strength based on crosslinked supramolecular polymer nanofibers.

doi: 10.1039/d4tb01873g

Figure Lengend Snippet: Fig. 12 Relative metabolic activity (%) of L929 mouse fibroblast cells growing on the BTP-C6 hydrogel was determined using the PrestoBluet assay. The experiments were conducted as triplicate for four different passages represented as P1, P2, P3, and P4. The calculated mean values were depicted using lines.

Article Snippet: L929 cells (CLS Cell Lines Service GmbH, Eppelheim, Germany) were cultured in TC-treated cell culture flasks (Greiner Bio-One International GmbH and Labsolute Th.

Techniques: Activity Assay

Journal: Nucleic Acids Research

Article Title: NFAT5 binds to the TNF promoter distinctly from NFATp, c, 3 and 4, and activates TNF transcription during hypertonic stress alone

doi: 10.1093/nar/gki701

Figure Lengend Snippet: NFAT family members bind portions of the TNF promoter with different affinities. ( A ) EMSA using recombinant NFATp, NFATc, NFAT3 and NFAT4 with portions of the TNF promoter. Three probes (−76, κ3L and κ3LL) were end-labeled with 32 P and incubated with recombinant protein, as indicated. Free probe marked at the bottom of the gel indicates equal loading. ( B ) EMSA using recombinant DBD5 or DBD5-GST and probes with portions of the TNF promoter. A diagram of the proximal human TNF promoter with probes for EMSA is just below. Known binding elements are boxed. Putative NFAT5 binding sites, based on the known consensus sequence, are bracketed. ( C ) EMSA using recombinant NFATp, NFATc, NFAT3, NFAT4 and NFAT5 with portions of the TNF promoter. Three probes (κ3L, κ3LL and −180) were end-labeled with 32 P and incubated with recombinant protein, as indicated. Free probe marked at the bottom of the gel indicates equal loading. ( D ) EMSA using raffinose-stimulated L929 nuclear extracts and the κ3LL probe. Nuclear extracts were prepared from L929 cells unstimulated (−) or stimulated (+) with raffinose (200 mM) for 18 h. An oligonucleotide probe containing the −76 NFAT, κ3-NFAT and the CRE binding sites (κ3LL) was used and the binding assay was performed in the presence or absence of antibodies to NFAT5, NFATc, ATF-2, c-jun or isotype control (normal rabbit serum) as indicated in the figure. The complexes containing NFAT5 or ATF-2 alone are indicated by the gray arrows, and the composite NFAT5/ATF-2/c-jun complexes are indicated by the black arrows in the figure.

Article Snippet: EMSA was performed, as described previously , using oligonucleotides end-labeled with [γ- 32 P]ATP and DBD5 and DBD5-GST recombinant proteins or nuclear extracts from L929 cells left untreated or treated with 200 mM raffinose for 18 h. In supershift assay, the appropriate antibodies (Santa Cruz) were pre-incubated with nuclear extracts for 30 min before adding the labeled probe.

Techniques: Recombinant, Labeling, Incubation, Binding Assay, Sequencing, Control

Journal: Nucleic Acids Research

Article Title: NFAT5 binds to the TNF promoter distinctly from NFATp, c, 3 and 4, and activates TNF transcription during hypertonic stress alone

doi: 10.1093/nar/gki701

Figure Lengend Snippet: Hypertonicity induces TNF transcription and NFAT5 expression. ( A ) Induction of TNF mRNA by hypertonic stress. L929 cells were cultured in medium supplemented with 200 mM raffinose for 18 h. RNA was harvested and submitted to RNase protection assay. ( B ) Hypertonic stress up-regulates NFAT5 expression. L929 cells were cultured to ∼95% confluence in a 6-well plate and stimulated with complete medium or medium supplemented with 200 mM raffinose for 18 h, as indicated. Whole cell extracts were collected and submitted to western blot analysis. Results are representative of four independent experiments. ( C ) L929 cells do not express NFATp. Whole cell extract (30 μg) from 68 to 41 cells were submitted to western blot analysis as a positive control for NFATp expression. L929 cells were stimulated with hypertonic stress as above. Results are representative of two independent experiments. Lamin B1 was used as a loading control. ( D ) Induction of TNF luciferase activity by hypertonic stress in L929 fibroblasts. A total of 2 × 10 6 L929 cells were transfected with 1 μg −200 TNF-Luc or −982 TNF-Luc and 0.4 μg pTK-RL control vectors by DEAE-dextran. Cells were stimulated with complete medium supplemented with 200 mM raffinose 24 h after transfection. Cells were harvested 18 h after stimulation and luciferase activities were assayed. Results are from four independent experiments. Error bars are the standard deviation.

Article Snippet: EMSA was performed, as described previously , using oligonucleotides end-labeled with [γ- 32 P]ATP and DBD5 and DBD5-GST recombinant proteins or nuclear extracts from L929 cells left untreated or treated with 200 mM raffinose for 18 h. In supershift assay, the appropriate antibodies (Santa Cruz) were pre-incubated with nuclear extracts for 30 min before adding the labeled probe.

Techniques: Expressing, Cell Culture, Rnase Protection Assay, Western Blot, Positive Control, Control, Luciferase, Activity Assay, Transfection, Standard Deviation

Journal: Nucleic Acids Research

Article Title: NFAT5 binds to the TNF promoter distinctly from NFATp, c, 3 and 4, and activates TNF transcription during hypertonic stress alone

doi: 10.1093/nar/gki701

Figure Lengend Snippet: Mutations in the −76 NFAT and κ3 site, but not in the −84-Ets site, decrease binding of recombinant NFAT5 and hypertonic induction of TNF. ( A ) DNase I footprinting assay of mutated TNF promoter with NFAT5 (DBD5-GST). The 3′M and 5′M sites are in the κ3 element and the −76 mutation is in the −76 NFAT site. The −84 mutation is between these two sites. For a diagram, see the bottom of the figure. ( B ) Relative activity of TNF luciferase constructs containing mutations in known binding sites of transcriptional activators. A total of 5 × 10 6 L929 cells were transfected with 1 μg −200 TNF-Luc and 0.2 μg pTK-RL control vectors by DEAE-dextran. Cells were stimulated with complete medium supplemented with 200 mM raffinose 24 h after transfection. Cells were harvested 18 h after stimulation and Photinus luciferase and Renilla activities were assayed. ( C ) DNase I footprinting assay of competitive binding of NFAT5 and ATF-2/c-jun. The binding of ANF-2/c-jun to the CRE element was not blocked by increasing amounts of DBD5-GST (see lanes 13–17).

Article Snippet: EMSA was performed, as described previously , using oligonucleotides end-labeled with [γ- 32 P]ATP and DBD5 and DBD5-GST recombinant proteins or nuclear extracts from L929 cells left untreated or treated with 200 mM raffinose for 18 h. In supershift assay, the appropriate antibodies (Santa Cruz) were pre-incubated with nuclear extracts for 30 min before adding the labeled probe.

Techniques: Binding Assay, Recombinant, Footprinting, Mutagenesis, Activity Assay, Luciferase, Construct, Transfection, Control

Journal: Nucleic Acids Research

Article Title: NFAT5 binds to the TNF promoter distinctly from NFATp, c, 3 and 4, and activates TNF transcription during hypertonic stress alone

doi: 10.1093/nar/gki701

Figure Lengend Snippet: A vector expressing a shRNA targeting NFAT5 and a dominant negative NFAT5 down-regulate hypertonic induction of TNF luciferase activity. ( A ) Diagram of the shRNA-expressing construct. The murine U6 small nuclear RNA (snRNA) promoter was cloned into pBluescript followed by a sequence coding for the shRNA targeting murine NFAT5. See Materials and Methods for details. ( B ) Diagram of the shRNAs expressed from the scrambled sequence (U6-N) and the sequence targeting exon 8 of murine NFAT5 (U6-N5 ex8). ( C and D ) A vector expressing an shRNA against NFAT5 depresses hypertonic induction of TNF-Luc activity in L929 cells and lowers NFAT5 levels. A total of 3 × 10 5 cells were plated in 6-wells plates (∼95% confluent) and were transfected the next day with 0.7 μg TNF-Luc, 0.3 μg of TK-RL and 1 μg of either the U6-N or the U6-N5 ex8 vector. The cells were stimulated as indicated 36 h later with complete medium or medium supplemented with 200 mM raffinose for 18 h. Cells were harvested and assayed as above. Error bars are the SD. The P -value was calculated with an unpaired two-tailed t -test. Results are from four independent experiments. ( D ) Nuclear extracts from mock or raffinose treated L929 cells transfected with the U6 constructs were also analyzed by western blot analysis with an antibody to NFAT5, which demonstrates specific knockdown of NFAT5 protein levels by the U6-N5 ex8 vector as shown. ( E ) A vector expressing a dominant negative NFAT5 decreases hypertonic induction of TNF-Luc activity. L929 cells were co-transfected by DEAE-dextran with TNF-Luc, pTK-RL, and different amounts of either pcDNA3, N5-DN or both vectors as indicated. Cells were stimulated and extracts were assayed as above. The P -value was calculated with an unpaired two-tailed t -test. Results are from four independent experiments.

Article Snippet: EMSA was performed, as described previously , using oligonucleotides end-labeled with [γ- 32 P]ATP and DBD5 and DBD5-GST recombinant proteins or nuclear extracts from L929 cells left untreated or treated with 200 mM raffinose for 18 h. In supershift assay, the appropriate antibodies (Santa Cruz) were pre-incubated with nuclear extracts for 30 min before adding the labeled probe.

Techniques: Plasmid Preparation, Expressing, shRNA, Dominant Negative Mutation, Luciferase, Activity Assay, Construct, Clone Assay, Sequencing, Transfection, Two Tailed Test, Western Blot, Knockdown