Journal: Annals of palliative medicine

Article Title: Down-regulated expression of transient receptor potential ankyrin 1 in lichen simplex chronicus.

doi: 10.21037/apm-20-1712

Figure Lengend Snippet: Figure 1 Protein expression of TRPA1 in the LSC group and control group. (A) Western blot results for TRPA1 expression in skin tissues

Article Snippet: The proteins in a sample were separated using sodium dodecyl sulfate-polyacrylamide gel (Beyotime Biotech, Shanghai, China) electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Beyotime Biotech, Shanghai, China) at a constant voltage of 80 V. After being blocked with a rapid blocking buffer for 10–15 minutes, and the membrane was incubated at 4 °C in an ice room overnight with primary antibodies against TRPA1 (1:1,000; ACC-037, Alomeno Labs, Israel) and β-actin (1:500; Wuhan Boster Biological Technology, China).

Techniques: Expressing, Control, Western Blot

Journal: Annals of palliative medicine

Article Title: Down-regulated expression of transient receptor potential ankyrin 1 in lichen simplex chronicus.

doi: 10.21037/apm-20-1712

Figure Lengend Snippet: Figure 2 Expression of TRPA1 in the LSC group and control group by Immunohistochemical analysis. (A) The expression of TRPA1 in skin tissue of

Article Snippet: The proteins in a sample were separated using sodium dodecyl sulfate-polyacrylamide gel (Beyotime Biotech, Shanghai, China) electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Beyotime Biotech, Shanghai, China) at a constant voltage of 80 V. After being blocked with a rapid blocking buffer for 10–15 minutes, and the membrane was incubated at 4 °C in an ice room overnight with primary antibodies against TRPA1 (1:1,000; ACC-037, Alomeno Labs, Israel) and β-actin (1:500; Wuhan Boster Biological Technology, China).

Techniques: Expressing, Control, Immunohistochemical staining

Journal: Annals of palliative medicine

Article Title: Down-regulated expression of transient receptor potential ankyrin 1 in lichen simplex chronicus.

doi: 10.21037/apm-20-1712

Figure Lengend Snippet: Figure 3 TRPA1 and inflammatory mediators in the skin tissue specimens mRNA expression in the LSC group and control group. (A)

Article Snippet: The proteins in a sample were separated using sodium dodecyl sulfate-polyacrylamide gel (Beyotime Biotech, Shanghai, China) electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Beyotime Biotech, Shanghai, China) at a constant voltage of 80 V. After being blocked with a rapid blocking buffer for 10–15 minutes, and the membrane was incubated at 4 °C in an ice room overnight with primary antibodies against TRPA1 (1:1,000; ACC-037, Alomeno Labs, Israel) and β-actin (1:500; Wuhan Boster Biological Technology, China).

Techniques: Expressing, Control

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

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: 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