Journal: Journal of Functional Biomaterials

Article Title: Cytocompatibility Assessment of L-PBF-Manufactured Zinc–Silver–Copper Alloys for Customized Biodegradable Medical Implants

doi: 10.3390/jfb17030146

Figure Lengend Snippet: ( A ) Representative images of the direct cytocompatibility contact test with SAOS-2 cells are presented. The top row ( a ) shows the tested sample plates (black) at the top of the image, with cells growing in direct contact on the bottom of the six-well plate (five times magnification). ( b ) The cell density at a 12 mm distance from the test plates (ten times magnification). ( B , D ) Three different extract concentrations (undiluted, diluted 1:3 and diluted 1:10) from the three L-PBF-manufactured Zn alloys (ZnAgCu, ZnAgCuMn, and ZnAgCuTi), rolled Zn, Cu, and Ti controls were tested, and the proliferation of L929 ( B ) and SAOS-2 ( D ) cells was determined after 24 h. ( C , E ) Live/dead staining of L929 ( C ) and SAOS-2 ( E ) cells growing directly on top of the samples is shown. Red and yellow staining indicate dead or dying cells, respectively, while living cells appear green. Each test was carried out four times with four replicates per sample. Shown are the mean values with their corresponding standard deviations. All results were normalized to control cells grown in cell culture medium without extract (proliferation equals 100%). To calculate statistical significance, a one-way ANOVA followed by a Tukey test was carried out (** p < 0.01 and **** p < 0.0001).

Article Snippet: To test the biocompatibility, the mouse fibroblast cell line L929 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany), cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS SUPERIOR, Bio&SELL GmbH, Feucht, Germany) and 1% L-glutamine (Thermo Fisher Scientific Inc.) was used.

Techniques: Staining, Control, Cell Culture

Journal: The Journal of investigative dermatology

Article Title: Expression Differences in BCL2 Family Members between Uveal and Cutaneous Melanomas Account for Varying Sensitivity to BH3 Mimetics.

doi: 10.1016/j.jid.2021.11.035

Figure Lengend Snippet: Figure 2. BFL1 contributes to UM’s sensitivity to MCL1i. (a, b) Immunoblot showing the endogenous expression of (a) antiapoptotic and (b) BH3-only proapoptotic BCL2 family of proteins in a panel of CM and UM cell lines. (c) Quantification of the immunoblot data of a and b. (d) ATP assay data of single-drug treatments of S63845 on control and BFL1 OE UM cell lines. Y-axis shows the percentage of relative viability, and the X-axis indicates the dosages of the drug in micrometer. (e) Immunoblots confirming the overexpression. Error bars represent SEM for all figures. * indicates P < 0.05; ** indicates P < 0.01. CM, cutaneous melanoma; MCL1i, MCL1 inhibitor; OE, overexpressed; UM, uveal melanoma.

Article Snippet: Creation of BFL1-overexpressed cell lines UM cells were transduced with BFL1 (NM_004049) Human Tagged ORF Clone Lentiviral Particles (#RC201965L4V) or Lenti-ORF Con- trol Particles (#PS100093V) (Origene Technologies, Rockville, MD) per the manufacturer’s instructions.

Techniques: Western Blot, Expressing, ATP Assay, Control, Over Expression

Journal: The Journal of investigative dermatology

Article Title: Expression Differences in BCL2 Family Members between Uveal and Cutaneous Melanomas Account for Varying Sensitivity to BH3 Mimetics.

doi: 10.1016/j.jid.2021.11.035

Figure Lengend Snippet: Figure 4. TCGA data indicate the clinical relevancy of differential expression of BCL2 proteins between CM and UM and lower BFL1 and higher PUMA in UM than in other cancers. (a) The expression data for all the eight genes analyzed to compare the expressions between UM and CM. Of all eight genes listed, the first seven genes are significantly different between the two subtypes. Expression data of (b) BFL1 and (c) PUMA across various cancers are shown. For b and c, the mRNA level was compared for UM with those for each of the other types of cancers. *** indicates the least significant P-value of the comparison at P < 0.001. For b, pheochromocytoma, diffuse glioma, and prostate adenocarcinoma are additionally significant (P < 0.01) compared with UM. CM, cutaneous melanoma; TCGA, The Cancer Genome Atlas; UM, uveal melanoma.

Article Snippet: Creation of BFL1-overexpressed cell lines UM cells were transduced with BFL1 (NM_004049) Human Tagged ORF Clone Lentiviral Particles (#RC201965L4V) or Lenti-ORF Con- trol Particles (#PS100093V) (Origene Technologies, Rockville, MD) per the manufacturer’s instructions.

Techniques: Quantitative Proteomics, Expressing, Comparison

Journal: PLoS ONE

Article Title: Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR

doi: 10.1371/journal.pone.0164250

Figure Lengend Snippet: (A) Gene expression levels in MDA-MB-231 (Parent), MDA-MB-231SCP2 (SCP2), and MDA-MB-231BR (BR). Candidate genes for drug resistance were screened by RNA sequencing. The intensity of gene expression in the heat map is shown relative to that in the Parent. (B) COX-2 and BCL2A1 mRNA and protein expression levels in Parent, SCP2, and BR. mRNA expression levels in the SCP2 and BR are shown relative to those in the Parent. Data represent the mean with SEM of at least three independent samples (*** P <0.001 vs . Parent and SCP2) for mRNA expressions.

Article Snippet: pCMV-Myc-DDK-Entry vector or pCMV-Myc-DDK- BCL2A1 vector (Origene Technologies, Inc., Rockville, MD) was transfected into MDA-MB-231 and T-47D using Lipofectamine ® LTX, PLUS ™ reagent, and Opti-MEM reduced serum media, according to the manufacturer's protocol (ThermoFisher Scientific Inc.).

Techniques: Expressing, RNA Sequencing Assay

Journal: PLoS ONE

Article Title: Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR

doi: 10.1371/journal.pone.0164250

Figure Lengend Snippet: (A) Cytotoxicity of 5-FU to MDA-MB-231BR in the presence of 1 μM BAY11-7082. Blue broken lines parallel to x-axis represent the cell viability of MDA-MB-231 (Parent) treated with 30 μM 5-FU. Data represent the mean with SEM of four independent samples. (B) Changes in protein expressions of COX-2 and BCL2A1 in MDA-MB-231BR in response to 1 μM BAY11-7082.

Article Snippet: pCMV-Myc-DDK-Entry vector or pCMV-Myc-DDK- BCL2A1 vector (Origene Technologies, Inc., Rockville, MD) was transfected into MDA-MB-231 and T-47D using Lipofectamine ® LTX, PLUS ™ reagent, and Opti-MEM reduced serum media, according to the manufacturer's protocol (ThermoFisher Scientific Inc.).

Techniques:

Journal: PLoS ONE

Article Title: Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR

doi: 10.1371/journal.pone.0164250

Figure Lengend Snippet: (A) Cytotoxicity of 5-FU to MDA-MB-231BR after transient transfection with BCL2A1 siRNA. Blue broken lines parallel to x-axis represent the cell viability of MDA-MB-231 (Parent) treated with 30 μM 5-FU. Data represent the mean with SEM of three independent samples (* P <0.05, BR treated with 30 μM 5-FU and siControl vs . BR treated with 30 μM 5-FU and si BCL2A1 ). (B) Changes in protein expression of BCL2A1 in MDA-MB-231BR in response to BCL2A1 siRNA.

Article Snippet: pCMV-Myc-DDK-Entry vector or pCMV-Myc-DDK- BCL2A1 vector (Origene Technologies, Inc., Rockville, MD) was transfected into MDA-MB-231 and T-47D using Lipofectamine ® LTX, PLUS ™ reagent, and Opti-MEM reduced serum media, according to the manufacturer's protocol (ThermoFisher Scientific Inc.).

Techniques: Transfection, Expressing

Journal: PLoS ONE

Article Title: Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR

doi: 10.1371/journal.pone.0164250

Figure Lengend Snippet: BCL2A1 protein expression of (A) MDA-MB-231 and (C) T-47D after transfection with a pCMV-Myc-DDK Entry vector (Vector) or a pCMV-Myc-DDK BCL2A1 vector ( BCL2A1 ). Cytotoxicity of 5-FU toward (B) MDA-MB-231 transfected with a BCL2A1 vector and (D) T-47D transfected with a BCL2A1 vector. The insets show the IC 50 of 5-FU against cell lines transfected with vectors. Data represent the mean with SEM of at least three independent samples (*** p <0.001 vs . MDA-MB-231 transfected with a control vector; * p <0.05 vs . T-47D transfected with a control vector).

Article Snippet: pCMV-Myc-DDK-Entry vector or pCMV-Myc-DDK- BCL2A1 vector (Origene Technologies, Inc., Rockville, MD) was transfected into MDA-MB-231 and T-47D using Lipofectamine ® LTX, PLUS ™ reagent, and Opti-MEM reduced serum media, according to the manufacturer's protocol (ThermoFisher Scientific Inc.).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR

doi: 10.1371/journal.pone.0164250

Figure Lengend Snippet: Protein expression of BCL2A1 in various human cell lines.

Article Snippet: pCMV-Myc-DDK-Entry vector or pCMV-Myc-DDK- BCL2A1 vector (Origene Technologies, Inc., Rockville, MD) was transfected into MDA-MB-231 and T-47D using Lipofectamine ® LTX, PLUS ™ reagent, and Opti-MEM reduced serum media, according to the manufacturer's protocol (ThermoFisher Scientific Inc.).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR

doi: 10.1371/journal.pone.0164250

Figure Lengend Snippet: Celecoxib acts on COX-2 but did not regulate BCL2A1 expression, while BAY11-7082 acts directly on NF-κB and moderately regulated BCL2A1 expression, and neither of these sensitized MDA-MB-231BR to 5-FU. Both ABT-263 and BCL2A1 siRNA directly regulate BCL2A1, and were effective for the sensitization of MDA-MB-231BR to 5-FU.

Article Snippet: pCMV-Myc-DDK-Entry vector or pCMV-Myc-DDK- BCL2A1 vector (Origene Technologies, Inc., Rockville, MD) was transfected into MDA-MB-231 and T-47D using Lipofectamine ® LTX, PLUS ™ reagent, and Opti-MEM reduced serum media, according to the manufacturer's protocol (ThermoFisher Scientific Inc.).

Techniques: Expressing

Journal: Nature Communications

Article Title: A loss-of-adhesion CRISPR-Cas9 screening platform to identify cell adhesion-regulatory proteins and signaling pathways

doi: 10.1038/s41467-022-29835-y

Figure Lengend Snippet: a Log2 fold changes of the guides (blue lines) targeting all significant proximal BCR-integrin signaling hits. Significant drop-out genes are identified from the αIgM/PMA statistics of the Namalwa screen. The RRA scores are the αRRA depletion scores. The graphs show the results for αIgM-induced adhesion relative to pre-adhesion (left), PMA-induced adhesion relative to pre-adhesion (middle), and αIgM-induced adhesion relative to PMA-induced adhesion (right). b The median fold changes of the 8 guides from genes represented in the kinome-centered Brunello library from the PMA-induced adhesion arm were plotted against the ones from the αIgM-induced adhesion arm of the Namalwa screen performed with 3 independent replicates. The significant genes are shown in black, and the size of the dots corresponds to the inverse of the αRRA depletion scores. c The fold changes of the individual guides from the Namalwa screen (DESeq2 calculates the maximum likelihood estimation (MLE) to determine the fold changes from 3 independent replicates) were plotted against the average adhesion level of the validation assays ( n is shown in Fig ). The dark grey area represents the 95% confidence interval, and the light grey area the 95% prediction interval. sgNT represents a non-targeting guide, and sgNEG a guide against the non-essential/non-expressed gene BRDT. d Namalwa and JeKo-1 cells with indicated knockouts or 1 h pretreatment with indicated drugs, were stimulated with αIgM or PMA, and allowed to adhere to fibronectin-coated surfaces. The average inhibition of adhesion (∆ adhesion) compared to DMSO treatment or control guide (sgNEG) of independent adhesion assays is presented in a heatmap ( n and P values are shown in Fig ). Source data are provided as a Source data file.

Article Snippet: Namalwa (DSMZ #ACC24), JeKo-1 (DSMZ #ACC553) and BCWM.1 (kindly provided by S.P.

Techniques: Biomarker Discovery, Inhibition, Control

Journal: Heliyon

Article Title: miR-122/PPARβ axis is involved in hypoxic exercise and modulates fatty acid metabolism in skeletal muscle of obese rats

doi: 10.1016/j.heliyon.2024.e26572

Figure Lengend Snippet: Primers used for RT-qPCR analysis.

Article Snippet: The protein samples were then transferred to a PVDF membrane (0.45-μm pore size) at a constant pressure of 100 V for 10 min, followed by blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST), which contained 5% bovine serum albumin, and then probed overnight at 4 °C with primary antibodies against PPARβ (1:2000; GTX113250) and FAS (1:1000; GTX13550) (both from GeneTex, Irvine, CA, USA) and ACC2 (1:500; A03668-2) and CPT1b (1:500; PB9491) (both from Boster Bio, Pleasanton, CA, USA).

Techniques: Amplification

Journal: Heliyon

Article Title: miR-122/PPARβ axis is involved in hypoxic exercise and modulates fatty acid metabolism in skeletal muscle of obese rats

doi: 10.1016/j.heliyon.2024.e26572

Figure Lengend Snippet: Expression of the lipid metabolism regulator PPARβ and downstream effectors (CPT1b, FAS, and ACC2). mRNA levels in obese rats under hypoxic conditions, with hypoxic training, and with regulated miR-122 expression were determined using qRT-PCR. OE, obese rats with miR-122 overexpression and hypoxic training; IE, obese rats with miR-122 depletion and hypoxic training; CE, obese rats with hypoxic training only; H, obese sedentary rats without regulation of miR-122 expression. Data are presented as mean ± SD. *p < 0.05, **p < 0.01 vs rats in group H; # p < 0.05, ## p < 0.01 vs rats in group CE; & p < 0.05, && p < 0.01 vs rats in group OE (1-way analysis of variance).

Article Snippet: The protein samples were then transferred to a PVDF membrane (0.45-μm pore size) at a constant pressure of 100 V for 10 min, followed by blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST), which contained 5% bovine serum albumin, and then probed overnight at 4 °C with primary antibodies against PPARβ (1:2000; GTX113250) and FAS (1:1000; GTX13550) (both from GeneTex, Irvine, CA, USA) and ACC2 (1:500; A03668-2) and CPT1b (1:500; PB9491) (both from Boster Bio, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Over Expression

Journal: Heliyon

Article Title: miR-122/PPARβ axis is involved in hypoxic exercise and modulates fatty acid metabolism in skeletal muscle of obese rats

doi: 10.1016/j.heliyon.2024.e26572

Figure Lengend Snippet: Expression of the lipid metabolism regulator PPARβ and downstream effectors (CPT1b, FAS, and ACC2). Protein levels in obese rats under hypoxic conditions, with hypoxic training, and with regulated miR-122 expression were determined using Western blot analysis. OE, obese rats with miR-122 overexpression and hypoxic training; IE, obese rats with miR-122 depletion and hypoxic training; CE, obese rats with hypoxic training only; H, obese sedentary rats without regulation of miR-122 expression. Data are presented as mean ± SD. *p < 0.05, **p < 0.01 vs rats in group H; # p < 0.05, ## p < 0.01 vs rats in group CE; & p < 0.05, && p < 0.01 vs rats in group OE (1-way analysis of variance).

Article Snippet: The protein samples were then transferred to a PVDF membrane (0.45-μm pore size) at a constant pressure of 100 V for 10 min, followed by blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST), which contained 5% bovine serum albumin, and then probed overnight at 4 °C with primary antibodies against PPARβ (1:2000; GTX113250) and FAS (1:1000; GTX13550) (both from GeneTex, Irvine, CA, USA) and ACC2 (1:500; A03668-2) and CPT1b (1:500; PB9491) (both from Boster Bio, Pleasanton, CA, USA).

Techniques: Expressing, Western Blot, Over Expression

Journal: Nature Communications

Article Title: Comparative optimization of combinatorial CRISPR screens

doi: 10.1038/s41467-022-30196-9

Figure Lengend Snippet: a Three main combinatorial CRISPR systems evaluated: eight distinct combinations of alternative spCas9 tracrRNAs sequences (orange boxes represent libraries generated), enCas12a, and orthologous spCas9-saCas9 system. b Schematic representation of the CRISPR screen pipeline. Ten distinct libraries are screened in IPC298. spCas9 - saCas9, enCas12a, VCR1-WCR3, and WCR2-WCR3 libraries are screened in PK1 and MELJUSO. c Schematic of sgRNA constant regions with indicated alterations compared to original spCas9 tracrRNA. Dark blue represents canonical sequence; light blue represents regions where insertions are introduced; orange represents base changes.

Article Snippet: The MELJUSO (DSMZ; #ACC74), PK1 (RIKEN; #RCB1972), and IPC298 (DSMZ; #ACC251) cell lines were collected by the CCLE .

Techniques: CRISPR, Generated, Sequencing

Journal: Nature Communications

Article Title: Comparative optimization of combinatorial CRISPR screens

doi: 10.1038/s41467-022-30196-9

Figure Lengend Snippet: a – c The single-gene receiver operating characteristic (ROC)—area under the curve (AUC) curves derived from pan-essential and nonessential genes across distinct libraries in ( a ) IPC298, ( b ) MELJUSO, and ( c ) PK1. AUC values are indicated in parentheses. d Separation between gene scores for common essential genes and nonessential genes computed using null-normalized median difference (NNMD). e Correlation between the average LFC of right versus left sgRNAs coupled with sgAAVS1 cutting control in IPC298. Pearson’s correlation coefficient is indicated ( r value) in black. Percentage of pan-essential genes with LFC less than −1 for sgRNAs in both right and left positions are indicated in red. Pan-essential genes are annotated by red dots. Source data are provided in the Source Data file.

Article Snippet: The MELJUSO (DSMZ; #ACC74), PK1 (RIKEN; #RCB1972), and IPC298 (DSMZ; #ACC251) cell lines were collected by the CCLE .

Techniques: Derivative Assay, Control

Journal: Nature Communications

Article Title: Comparative optimization of combinatorial CRISPR screens

doi: 10.1038/s41467-022-30196-9

Figure Lengend Snippet: a Evaluation of tracrRNA effect calculated by the difference in LFC between sgRNAs utilizing the VCR1 and WCR3 tracrRNAs from the VCR1-WCR3 and WCR3-VCR1 screens in IPC298. Analyses separated by promoters expressing the sgRNA. b Evaluation of the promoter effect by correlation between the LFC from the U6 and H1 promoter. For ten genes, same crRNA sequences are used on both left and right position to remove crRNA variability. Analyses separated by tracrRNA used. c Schematic illustration evaluating the recombination rate by next-generation sequencing (NGS) reads. crRNA and tracrRNA in both positions were sequenced by NextSeq paired-end 150 bp. Reads were either mapped to crRNA only or crRNA+tracrRNA (sgRNA). d Recombination rate calculated by percent of reads mapped between sgRNA to crRNA in pDNA for WCR2-WCR3 (green) and VCR1-WCR3 (blue). e LFC calculated by reads mapped to crRNA or sgRNA. The percentage of pan-essential genes with LFC less than −3 are indicated in red. Source data are provided in the Source Data File.

Article Snippet: The MELJUSO (DSMZ; #ACC74), PK1 (RIKEN; #RCB1972), and IPC298 (DSMZ; #ACC251) cell lines were collected by the CCLE .

Techniques: Expressing, Next-Generation Sequencing

Journal: Nature Communications

Article Title: Comparative optimization of combinatorial CRISPR screens

doi: 10.1038/s41467-022-30196-9

Figure Lengend Snippet: a – c The ROC-AUC curves derived from pan-essential and nonessential paralog pairs across distinct libraries in ( a ) IPC298, ( b ) MELJUSO, and ( c ) PK1. AUC values are indicated in parentheses. d Correlation between the average LFC of dual knockouts with inverted positions of sgRNAs in IPC298. Pearson’s correlation coefficient is indicated ( r value) in black. Percent of pan-essential paralog pairs with LFC less than −1 for both sgRNAs in positions are indicated in red. Pan-essential paralog pairs are annotated by red dots. Source data are provided in the Source Data File.

Article Snippet: The MELJUSO (DSMZ; #ACC74), PK1 (RIKEN; #RCB1972), and IPC298 (DSMZ; #ACC251) cell lines were collected by the CCLE .

Techniques: Derivative Assay

Journal: Nature Communications

Article Title: Comparative optimization of combinatorial CRISPR screens

doi: 10.1038/s41467-022-30196-9

Figure Lengend Snippet: a Manhattan plot of FDRs corresponding to GEMINI synergy scores and color-coded by the LFC of dependencies for paralog pair knockouts in IPC298. The dotted line represents the FDR of 1e-3. b Scatter plot of GEMINI synergy score and LFC of dual knockout in IPC298. Curated paralog pairs of the MAPK pathway are annotated by red dots. Pan-essential paralog pairs or genes are annotated by blue or yellow dots, respectively. c LFC of single or combinatorial gene knockouts of paralog pairs associated with MAPK signaling pathway in IPC298 ( n = 3 biological replicates; 6 independent sgRNAs per gene and 18 independent sgRNA combinations per gene pair). The centerline, lower hinge, and upper hinge correspond to the 50th, 25th, and 75th percentiles, respectively. The upper and lower whiskers extend from the upper and lower hinges to the largest and smallest values no further than 1.5 * IQR (interquartile range). Source data are provided in the Source Data File.

Article Snippet: The MELJUSO (DSMZ; #ACC74), PK1 (RIKEN; #RCB1972), and IPC298 (DSMZ; #ACC251) cell lines were collected by the CCLE .

Techniques: Knock-Out