Journal: PLoS ONE

Article Title: RNAi Screen Reveals Potentially Novel Roles of Cytokines in Myoblast Differentiation

doi: 10.1371/journal.pone.0068068

Figure Lengend Snippet: ( A ) A flow chart of the screening process. For the primary screen, lentiviruses expressing shRNAs against 134 genes, packaged in 96-well plates, were used to transduce C2C12 myoblasts seeded in 96-well plates. After 2 days of puromycin selection, the cells were induced to differentiate for 3 days, at the end of which they were fixed and stained for MHC and DAPI. shRNAs that induced morphological changes detectable by visual inspection were subjected to secondary screen with C2C12 cells seeded in 12-well plates, following the procedure described above. Quantification of myotube formation was then performed. ( B ) As a positive control, Cxcl12 was included in the screen and recovered as a positive hit. Shown are results of the secondary screen with two independent shRNAs. Cells were stained for MHC and DAPI, pseudo-colored green and red, respectively. A non-targeting shRNA served as a negative control. ( C ) Myotube formation in B was quantified for differentiation index, fusion index and average nuclei number per myotube (see Material and Methods for definition). Data shown are mean ± SD (n = 3). Paired t test was performed to compare data to control. *P<0.05. Scale bar: 100 µm.

Article Snippet: A non-targeting shRNA (Addgene plasmid 1864; ) was included as a negative control.

Techniques: Expressing, Transduction, Selection, Staining, Positive Control, shRNA, Negative Control, Control

Journal: Science signaling

Article Title: VEGF–Neuropilin-2 signaling promotes stem-like traits in breast cancer cells by TAZ-mediated repression of the Rac GAP β2-chimaerin

doi: 10.1126/scisignal.aao6897

Figure Lengend Snippet: A.) TAZ expression was diminished in MES cells and the impact on β2-chimaerin mRNA expression was quantified by qPCR. Data are mean ± SEM of n = 3 biological replicates. (B and C) β2-chimaerin abundance (B) and Rac1 activity (C) were assessed in TAZ knockdown MES cells. Representative blots are shown. Densitometric values are provided as bar graphs (mean ± SEM) of n = 3 biological replicates. D.) TAZ expression was diminished in MDA-MB-435 cells and the impact on β2-chimaerin mRNA expression was quantified by qPCR. Data are mean ± SEM of n = 3 biological replicates. E.) TAZ expression was diminished in MDA-MB-231 cells and the impact on β2-chimaerin mRNA expression was quantified by qPCR. Data are mean ± SEM of n = 3 biological replicates. F.) MDA-MB-231 cells were transfected with TAZ and the impact on β2-chimaerin mRNA expression was quantified by qPCR. Data are mean ± SEM of n = 3 biological replicates. G.) MES cells were treated with the indicated concentrations of the Rac inhibitor EHT1864 for 2 hours and β2-chimaerin mRNA expression was quantified by qPCR. Data are mean ± SEM of n = 3 biological replicates. * p ≤ 0.05 by two-tailed t test.

Article Snippet: EHT1864 was purchased from Tocris, NSC23766 was purchased from Selleckchem, FAK14 was purchased from Sigma, Sunitinib and Pazopanib were purchased from LC Laboratories, human VEGFA165 was purchased from R&D Systems and the function-blocking Neuropilin-2 antibody was provided by Genentech ( 11 ).

Techniques: Expressing, Activity Assay, Knockdown, Transfection, Two Tailed Test

Journal: Science signaling

Article Title: VEGF–Neuropilin-2 signaling promotes stem-like traits in breast cancer cells by TAZ-mediated repression of the Rac GAP β2-chimaerin

doi: 10.1126/scisignal.aao6897

Figure Lengend Snippet: A.) Expression of VEGF was diminished in MES cells and the impact on Rac1 activity was assessed using a GST fusion protein containing the Rac/cdc42 binding domain of PAK (PBD). Representative blots are shown. Densitometric values are provided as bar graphs (mean ± SEM) of n = 3 biological replicates. B.) MES cells were treated with 3 μg/mL of a function blocking NRP2 antibody for 6 hours and assayed for Rac1 activity. Representative blots are shown. Densitometric values are provided as bar graphs (mean ± SEM) of n = 3 biological replicates. C.) NRP2 expression was diminished in MDA-MB-231 cells and the impact on Rac1 activity was assessed. Representative blots are shown. Densitometric values are provided as bar graphs (mean ± SEM) of n = 3 biological replicates. D.) MDA-MB-231 cells were serum-starved for 24 hours, treated with 50 ng/mL of VEGF for 30 minutes and assayed for Rac1 activity. Representative blots are shown. Densitometric values are provided as bar graphs (mean ± SEM) of n = 3 biological replicates. E.) MES cells were treated with 2 μM of FAK14 for the indicated time points and assayed for Rac1 activity. Representative blots are shown. Densitometric values are provided as bar graphs (mean ± SEM) of n = 3 biological replicates of MES cells treated with FAK14 for 6 hours. F.) NRP2 expression was diminished in MDA-MB-231 cells that were then transfected with constitutively active V12 Rac1-GST. GST expression was quantified by immunoblotting. mRNA expression of the indicated TAZ target genes was quantified by qPCR. Data are mean ± SEM of n = 3 biological replicates. G.) MES cells were treated with 50 μM of the Rac inhibitors EHT1864 or NSC23766 and assayed for self-renewal by serial passage mammosphere formation (P1: passage 1; P2: passage 2). Data are mean ± SEM of n = 3 biological replicates. * p ≤ 0.05 by two-tailed t test.

Article Snippet: EHT1864 was purchased from Tocris, NSC23766 was purchased from Selleckchem, FAK14 was purchased from Sigma, Sunitinib and Pazopanib were purchased from LC Laboratories, human VEGFA165 was purchased from R&D Systems and the function-blocking Neuropilin-2 antibody was provided by Genentech ( 11 ).

Techniques: Expressing, Activity Assay, Binding Assay, Blocking Assay, Transfection, Western Blot, Two Tailed Test

Journal: Science signaling

Article Title: VEGF–Neuropilin-2 signaling promotes stem-like traits in breast cancer cells by TAZ-mediated repression of the Rac GAP β2-chimaerin

doi: 10.1126/scisignal.aao6897

Figure Lengend Snippet: A.) MES cells were treated with the indicated concentrations of the Rac inhibitor EHT1864 for 2 hours and the impact on pSer89 TAZ and TAZ abundance was quantified by immunoblotting. Representative blots are shown. Densitometric values are provided as bar graphs (mean ± SEM) of n = 3 biological replicates. B.) MES cells were treated with the Rac inhibitor EHT1864 (100 μM), lysed at the indicated time points and the impact on pThr1079 LATS1, LATS1 and TAZ abundance was quantified by immunoblotting. Data are representative of n = 2 biological replicates. C.) MES cells were transfected with dominant negative N17 Rac1-HA and the impact on TAZ abundance was quantified by immunoblotting. Data are representative of n = 2 biological replicates. D.) MES cells were treated with the indicated concentrations of a function-blocking NRP2 antibody for 6 hours and the impact on pSer89 TAZ and TAZ abundance was quantified by immunoblotting. Representative blots are shown. Densitometric values are provided as bar graphs (mean ± SEM) of n = 3 biological replicates of MES cells treated with 3 μg/mL of the NRP2 function-blocking antibody. E.) NRP2 expression was diminished in MDA-MB-231 cells and the impact on pSer89 TAZ and TAZ abundance was quantified by immunoblotting. Representative blots are shown. Densitometric values are provided as bar graphs (mean ± SEM) of n = 3 biological replicates. F.) NRP2 expression was diminished in MDA-MB-231 cells and the impact on pThr1079 LATS1 and LATS1 was quantified by immunoblotting. Data are representative of n = 2 biological replicates. G.) NRP2-depleted MDA-MB-231 cells were transfected with empty vector, wild-type TAZ or S89A TAZ and assayed for self-renewal by serial passage mammosphere formation. Data are mean ± SEM of n = 3 biological replicates. H.) VEGF-depleted MES cells were transfected with empty vector, wild-type TAZ or S89A TAZ and assayed for self-renewal by serial passage mammosphere formation. Error bars indicate standard deviation from 3 technical replicates. Data are representative of n = 2 biological replicates. * p ≤ 0.05 by two-tailed t test.

Article Snippet: EHT1864 was purchased from Tocris, NSC23766 was purchased from Selleckchem, FAK14 was purchased from Sigma, Sunitinib and Pazopanib were purchased from LC Laboratories, human VEGFA165 was purchased from R&D Systems and the function-blocking Neuropilin-2 antibody was provided by Genentech ( 11 ).

Techniques: Western Blot, Transfection, Dominant Negative Mutation, Blocking Assay, Expressing, Plasmid Preparation, Standard Deviation, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Identification and Characterization of Buffalo 7SK and U6 pol III Promoters and Application for Expression of Short Hairpin RNAs

doi: 10.3390/ijms15022596

Figure Lengend Snippet: Construction and transfection of dual EGFP/shRNA expression vectors. ( A ) Buffalo, bovine and human 7SK / U6 promoter fragments were amplified with restriction enzymes sites Xba I/ Sac I introduced by the primers, and ligated into the 5′ MCS (multi-cloning site) of the pMCS-shEGFP and pMCS-sh1864 vectors. The core fragments in the vectors, featuring the MCS and sense, loop, antisense and pol III terminator (TTTTTT), were chemically synthesized and ligated with framework plasmid pMD18-T; ( B ) Fluorescence microscopy images of buffalo fetal fibroblasts (BFF) cells transfected with the reporter vector (pEGFP-N1) only, or co-transfected with the reporter and various shEGFP expression plasmids as indicated in each image.

Article Snippet: To test the expression competence of the cloned bu 7SK and bu U6 promoters, the pMCS-shEGFP and pMCS-sh1864 base vectors were constructed from pMD18T by ligating a 65 bp synthetic copy of an EGFP-specific shRNA or a control scrambled shRNA (Addgene plasmid #1864; Raleigh, NC, USA) sequence produced by chemical synthesis (GenScript, Nanjing, China) between the Bam HI and Hin dIII sites (Takara, Dalian, China), respectively.

Techniques: Transfection, shRNA, Expressing, Amplification, Cloning, Synthesized, Plasmid Preparation, Fluorescence, Microscopy

Journal: Stroke

Article Title: Mammalian Target of Rapamycin Cell Signaling Pathway Contributes to the Protective Effects of Ischemic Postconditioning Against Stroke

doi: 10.1161/strokeaha.114.005406

Figure Lengend Snippet: Figure 1. Effects of ischemic postconditioning (IPC) on protein expression in the mammalian target of rapamycin (mTOR) pathway. A, Diagram showing the major proteins in the mTOR cell signaling pathway. P indicates phosphorylation. B, Brain schematic showing the ischemic penumbra and core regions. The ischemic penumbra, which represents tissue spared by IPC, was dissected for Western blot analysis. The core is the area of infarct that received IPC. C, Representative protein bands of a Western blot for p-mTOR, p-S6K1, p-S6, and p-4EBP1 at 1, 5, 9, and 24 h poststroke. β-Actin indicates equal protein loading. D, Relative optical densities of protein bands, normalized to those in the sham group and expressed as percentages. *, **vs sham; P<0.05, 0.01, respectively; #P<0.05, between the 2 indicated groups; n=8 per group.

Article Snippet: In addition, we used lentiviral vectors containing mTOR shRNA to inhibit mTOR expression and a scramble shRNA gene as a negative control for mTOR shRNA (mTOR shRNA 1856, scramble shRNA 1864; Addgene, Cambridge, MA).

Techniques: Expressing, Phospho-proteomics, Western Blot

Journal: Stroke

Article Title: Mammalian Target of Rapamycin Cell Signaling Pathway Contributes to the Protective Effects of Ischemic Postconditioning Against Stroke

doi: 10.1161/strokeaha.114.005406

Figure Lengend Snippet: Figure 3. Effects of rapamycin on protein levels in the mammalian target of rapamycin (mTOR) pathway. A, Representative protein bands of major proteins in the mTOR pathway. B, Quantified levels of each protein. *, **vs sham brain, P<0.05, 0.01, respectively; &vs con (nonischemic control with rapamycin injection), P<0.05; #between the 2 indicated groups, P<0.05, 0.01, respectively; n=8 per group. IPC indicates ischemic postconditioning; and rapa, rapamycin.

Article Snippet: In addition, we used lentiviral vectors containing mTOR shRNA to inhibit mTOR expression and a scramble shRNA gene as a negative control for mTOR shRNA (mTOR shRNA 1856, scramble shRNA 1864; Addgene, Cambridge, MA).

Techniques: Control, Injection

Journal: Stroke

Article Title: Mammalian Target of Rapamycin Cell Signaling Pathway Contributes to the Protective Effects of Ischemic Postconditioning Against Stroke

doi: 10.1161/strokeaha.114.005406

Figure Lengend Snippet: Figure 4. Detrimental effects of the mammalian target of rapamycin (mTOR) short hairpin RNA (shRNA) lentiviral vector in stroke. A, Schematic backbone of lentiviral vector of pLKO.1 puro with shRNA construct. B, Effects of gene transfer in primary neuronal cul- tures on protein expression of p-mTOR and p-4EBP1 in the mTOR pathway. mTOR shRNA was confirmed to reduce protein levels of p-mTOR and p-4EBP1. C, Effects of mTOR shRNA gene transfer on neuronal death in the oxygen–glucose deprivation (OGD) model measured by lactate dehydrogenase (LDH) release. shRNA exacerbated neuronal death in cell culture that underwent OGD and abol- ished the protective effect of hypoxic postconditioning (HPC). *vs OGD group with scramble shRNA, P<0.05; &vs post group with scramble shRNA, P<0.05; #, ##P<0.05, 0.01, between indicated 2 groups, respectively; n=16 per group. D, Effects of mTOR shRNA on infarct sizes in ischemic brains with or without ischemic postconditioning (IPC). Top: Representative coronal brain sections for infarc- tion stained by cresyl violet. Bottom: Bar graphs show infarct sizes. mTOR shRNA enlarged infarction in animals treated with control stroke and abolished the protective effects of IPC. **vs scramble shRNA, ischemic group, P<0.01; ##P<0.01, between indicated 2 groups; n=8 per group.

Article Snippet: In addition, we used lentiviral vectors containing mTOR shRNA to inhibit mTOR expression and a scramble shRNA gene as a negative control for mTOR shRNA (mTOR shRNA 1856, scramble shRNA 1864; Addgene, Cambridge, MA).

Techniques: shRNA, Plasmid Preparation, Construct, Expressing, Cell Culture, Staining, Control

Journal: Stroke

Article Title: Mammalian Target of Rapamycin Cell Signaling Pathway Contributes to the Protective Effects of Ischemic Postconditioning Against Stroke

doi: 10.1161/strokeaha.114.005406

Figure Lengend Snippet: Figure 5. Effects of mammalian target of rapamycin (mTOR) short hairpin RNA on protein levels in the mTOR pathway. A, Representative protein bands in the mTOR pathway measured by Western blot. Brain tissues from penumbra were collected and analyzed at 1, 5, 9, and 24 h poststroke with or without ischemic postconditioning (IPC). B, Bar graphs show quantified levels of each protein. *vs sham brain, P<0.05; #P<0.05, between the 2 indicated groups; n=8 per group.

Article Snippet: In addition, we used lentiviral vectors containing mTOR shRNA to inhibit mTOR expression and a scramble shRNA gene as a negative control for mTOR shRNA (mTOR shRNA 1856, scramble shRNA 1864; Addgene, Cambridge, MA).

Techniques: shRNA, Western Blot

Journal: Frontiers in Plant Science

Article Title: Harnessing the Tet-Off system for effective gene silencing in Magnaporthe oryzae

doi: 10.3389/fpls.2025.1641730

Figure Lengend Snippet: Generation of Tet-Off-mediated knockdown transformants in M. oryzae . (A) RT-qPCR analysis of the expression levels of MoVPS35 in MoVPS35 -Tet-Off mutants (Student’s t -test: **, P < 0.01). (B) RT-qPCR analysis of the expression levels of MoVMA1 in MoVMA1 -Tet-Off mutants (Student’s t -test: **, P < 0.01). (C) Analysis of MoVps35 abundance in MoVPS35 -Tet-Off mutants by immunoblotting (Actin was used as an internal reference). (D) Analysis of MoVma1 abundance in MoVMA1 -Tet-Off mutants by immunoblotting (Actin was used as an internal reference). (E) Confocal microscopic examination of GFP fluorescence in GFP- MoVPS35 -Tet-Off mutants. (F) Confocal microscopic examination of GFP signal in GFP- MoVMA1 -Tet-Off mutants. (G) Comparison of GFP fluorescence intensity in GFP- MoVPS35 -Tet-Off mutants (Student’s t -test: **, P < 0.01). (H) Comparison of GFP fluorescence intensity in GFP- MoVMA1 -Tet-Off mutants (Student’s t -test: **, P < 0.01). (I) Co-localization of GFP-MoVps35 and mScarlet-MoRab7 in vegetative hyphae. (J) Co-localization of GFP-MoVma1 and mScarlet-MoRab7 in vegetative hyphae.

Article Snippet: Sequence data from this article are available under the following accession numbers: MoVPS35 (MGG_05089); MoVMA1 (MGG_08087); MoTOR (MGG_15156); pFGL1252_TetOFF (Hyg) (Addgene ID 118992); pFGL1252_TetGFP (Hyg) (Addgene ID 118993).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Fluorescence, Comparison

Journal: Frontiers in Plant Science

Article Title: Harnessing the Tet-Off system for effective gene silencing in Magnaporthe oryzae

doi: 10.3389/fpls.2025.1641730

Figure Lengend Snippet: Phenotypic analyses of MoVPS35 - and MoVMA1 -Tet-Off mutants. (A) Vegetative growth of MoVPS35- and MoVMA1- Tet-Off mutants on CM media containing different concentrations of Doxycycline. (B) Colony diameters of MoVPS35- and MoVMA1- Tet-Off mutants on CM media supplemented with different concentrations of Doxycycline, along with their non-linear regression curves (Student’s t -test: **, P < 0.01). (C) Nomenclature of the Tet-Off mutants upon doxycycline addition. (D) Vegetative growth of MoVPS35 KD mutant on CM medium. (E) Vegetative growth of MoVMA1 KD mutant on CM medium. (F) Colony diameters of the wild-type (Guy11), MoVPS35 KD and MoVPS35 KD -C strains on CM media supplemented with Doxycycline (Student’s t -test: **, P < 0.01). (G) Colony diameters of the wild-type (Guy11), MoVMA1 KD and MoVMA1 KD -C strains on CM media supplemented with Doxycycline (Student’s t -test: **, P < 0.01). (H) Pathogenicity of the wild-type (Guy11), MoVPS35 KD and MoVPS35 KD -C strains on rice leaves at 5 days post infection (dpi). (I) Pathogenicity of the wild-type (Guy11), MoVMA1 KD and MoVMA1 KD -C strains on rice leaves at 5 dpi. (J) Number of lesions on 5 cm from the leaf tip of rice leaves 5 days after infection with wild-type (Guy11), MoVPS35 KD and MoVPS35 KD -C (Student’s t -test: ** P < 0.01). (K) Number of lesions on 5 cm from the leaf tip of rice leaves 5 days after infection with wild-type (Guy11), MoVMA1 KD and MoVMA1 KD -C (Student’s t -test: ** P < 0.01).

Article Snippet: Sequence data from this article are available under the following accession numbers: MoVPS35 (MGG_05089); MoVMA1 (MGG_08087); MoTOR (MGG_15156); pFGL1252_TetOFF (Hyg) (Addgene ID 118992); pFGL1252_TetGFP (Hyg) (Addgene ID 118993).

Techniques: Mutagenesis, Infection