Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: In-vivo transfection of pcDNA3.1-IGFBP7 inhibits melanoma growth in mice through apoptosis induction and VEGF downexpression

doi: 10.1186/1756-9966-29-13

Figure Lengend Snippet: Detection of IGFBP7, caspase-3, VEGF, and apoptosis expressed in homeograft tumors sections with original magnification × 100 in A-F, and ×400 in G-L . A shows significantly higher IGFBP7 expression in pcDNA3.1-IGFBP7. B demonstrates the successful transfection of pcDNA3.1 plasmid. C shows the physiological expression of IGFBP7 in melanoma (red color, as blue arrows indicate). D-F shows the effect of pcDNA3.1-IGFBP7 on caspase-3 expression in the cytoplasm of tumor sections, with strong expression in pcDNA3.1-IGFBP7 group seen in D, while weak expression in the pcDNA3.1-CONTROL and B16-F10 cell groups seen in E, F. G-I shows the expression of VEGF in vivo , with negative expression in most of cells in the pcDNA3.1-IGFBP7 group seen in G, while strong expression in the cytoplasm of pcDNA3.1-CONTROL and B16-F10 cell groups (red arrow represented) showed in H, I. J-L shows tumor apoptosis in vivo , with few apoptotic cells in pcDNA3.1-CONTROL (K) and B16-F10 cell groups (L), while increased apoptosis in the B16-F10 tissue in mice treated with pcDNA3.1-IGFBP7 (J) (red arrow shows deep blue cells).

Article Snippet: IGFBP7 cDNA was purified by Shanghai Biological Engineering Company.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, In Vivo

Journal: bioRxiv

Article Title: Nanchangmycin regulates FYN, FAK and ERK to control the fibrotic activity of hepatic stellate cells

doi: 10.1101/2021.10.08.463221

Figure Lengend Snippet: A. Expression of house-keeping genes in HSCs was plotted by RPKM as previously quantified . Three pairs of samples were used in this analysis: ceramide treatment vs control, nortriptyline treatment vs control, nortriptyline treatment vs control in TGF-β treated HSCs . Each dot represents the result from one sample, and bars represent mean ± standard deviation (sd). The value of sd is indicated above each mRNA. The fold change (fc) of EMC7 , VCP , PSMB2 and VPS29 in inactivated HSCs (ceramide or nortriptyline-treated) compared to corresponding control groups is indicated below the dots. Two groups of reference mRNAs were analyzed -- commonly used ones (left) and genes that are expressed uniformly across tissues . Red arrows indicate samples with sd of 0.15 or less. Blue arrows highlight the reference mRNAs with fold change of no more than 10% in inactivated HSCs and were selected for further analysis. GAPDH , which is used routinely as a reference control was also included. B. Quantification of expression of EMC7 , GAPDH , GUSB , POLR2A , and PSMB2 from HSC cDNA samples (left: n=14, right: n=53) that were reverse transcribed from equal amounts of total input RNA. All samples were normalized to the mean value of its own control group before they were combined for each of the reference mRNAs. Each dot represents the result from one sample, and bars represent mean ± standard deviation (sd) of all the tested samples. The value of sd is indicated above each mRNA.

Article Snippet: Reverse transcription was performed using iScript gDNA Clear cDNA Synthesis Kit (BIO-RAD, cat# 1725035) with 1 μg total RNA input, and quantitative real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, cat# 4305719) and TaqMan Real-time PCR Assays for specific genes listed below.

Techniques: Expressing, Control, Standard Deviation, Reverse Transcription

Journal: bioRxiv

Article Title: Nanchangmycin regulates FYN, FAK and ERK to control the fibrotic activity of hepatic stellate cells

doi: 10.1101/2021.10.08.463221

Figure Lengend Snippet: A-B. FYN was depleted in HSCs with two siRNAs (si1 and si2). The expression levels of COL1A1 ( A ) and FYN ( B ) were analyzed by qPCR after 72 hours in comparison to a non-targeting control siRNA (NTC). Error bars represent mean ± SEM (n=3) for each of three donor lines. ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001 (one-way ANOVA test). C. HSCs were treated with NCMC or 1-Naphthyl PP1 (PP1) for 48 hours. COL1A1 level was analyzed by qPCR. Error bars represent mean ± SEM (n=3 for donor 1, and n=6 for donor 3). Data are representative of three independent experiments for donor 1 and experiment for donor 3. **** indicates p < 0.0001 (one-way ANOVA test). D. Effect of FYN-depletion on collagen deposition in ECM. Top: representative images. Scale bar represents 100 µm. Bottom: quantified results. Error bars represent mean ± SEM (n=6). Data are representative of two independent experiments. ** indicates p < 0.01, and **** indicates p < 0.0001 (one-way ANOVA test). E. HSCs transduced with control virus or virus containing the cDNA encoding dominant negative mutant FYN (FYN-DN) were treated with DMSO or 100 nM NCMC for 48 hours. Expression of COL1A1 was quantified by qPCR. Error bars represent mean ± SEM (n=3). Data are representative of three independent experiments). ns indicates not significant (p > 0.05), * indicates p < 0.05, ** indicates p < 0.01, and **** indicates p < 0.0001 (two-way ANOVA test). F. Phospho-ERK and phospho-FAK levels were determined by Western blot in control HSCs and HSCs overexpressing DN-FYN. Left: representative Western blot results. Right: quantified results. Representative of two independent experiments. This figure has four supplements.

Article Snippet: Reverse transcription was performed using iScript gDNA Clear cDNA Synthesis Kit (BIO-RAD, cat# 1725035) with 1 μg total RNA input, and quantitative real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, cat# 4305719) and TaqMan Real-time PCR Assays for specific genes listed below.

Techniques: Expressing, Comparison, Control, Transduction, Virus, Dominant Negative Mutation, Western Blot

Journal: Cell reports

Article Title: A Two-Enzyme Adaptive Unit within Bacterial Folate Metabolism

doi: 10.1016/j.celrep.2019.05.030

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli K12 MG1655 CGSC CGSC#:7740 MG1655 with chromosomal dcas9 Gift from David Bikard N/A ER2566 ΔfolA ΔthyA Gift from S. Benkovic N/A Chemicals, Peptides, and Recombinant Proteins Purezol Bio-Rad Cat#7326890 Amicase Sigma Cat#65072-00-6 MOPS EZ Rich Defined Medium Teknova Cat#M2105 Critical Commercial Assays 300 cycle Illumina MiSeq kit V2 Illumina Cat#MS-102-2002 Luna Universal One-Step RT-qPCR kit NEB Cat#E3005S QIAamp DNA Minit Kit QIAGEN Cat#51304 Nextera XT DNA Library Prep kit Illumina Cat#FC-131-1096 50 cycle Illumina MiSeq kit V2 Illumina Cat#MS-102-2001 Deposited Data Evolved MG1655 genome sequencing reads NCBI BioProject PRJNA378892 Experimental Models: Organisms/Strains MG1655 with chromosomal dcas9 and sgRNA library (gAM-350) This study N/A Evolved MG1655 strains (5thy condition) This study N/A Evolved MG1655 strains (10thy condition) This study N/A Evolved MG1655 strains (50thy condition) This study N/A MG1655 reconstitution strains This study N/A MG1655 folA single mutants Gift from E. Toprak ( Palmer et al., 2015 ) N/A ER2566 ΔfolA ΔthyA pACYC folA/thyA library This study N/A Oligonucleotides Table S2 : Primers used for CRISPRi and qPCR This study N/A Recombinant DNA pAM-111 This study N/A pAM-112 This study N/A pAM-288/290 This study N/A pCRISPR Jiang et al., 2013 Addgene plasmid #42875 pgRNA Qi et al., 2013 Addgene plasmid #44251 pTKRED Kuhlman and Cox.

Techniques: Recombinant, Sequencing, Plasmid Preparation, Software