Journal: Journal of Virology

Article Title: SGIV envelope protein VP088 facilitated virus replication via interacting with other viral proteins and promoting p62-dependent autophagic degradation of TBK1

doi: 10.1128/jvi.01193-25

Figure Lengend Snippet: VP088 down-regulated host IFN response. ( A ) VP088 overexpression alone decreased the transcription levels of IFN- related genes. The mRNA levels of ISG15, ISG56, Viperin, and MX1 in pFlag-VP088-transfected GS cells were measured using qPCR. ( B ) The silencing effects of siRNA on VP088 protein synthesis during SGIV infection. GS cells transfected with siRNA-NC, si-VP088-1, si-VP088-2, or si-VP088-3 were infected with SGIV for 24 h and then collected for IB analysis using anti-VP088 antibody. ( C ) The transcription levels of IFN-related genes in VP088 silenced cells upon SGIV infection. GS cells were transfected with si-VP088 and then infected with SGIV for 24 h. Cells were collected for qPCR analysis to evaluate the mRNA levels of ISG15, ISG56, Viperin, and MX1. ( D–F ) VP088 decreased EccGAS-EcSTING-induced promoter activities of IFN1 ( D ), IFN3 ( E ), or ISRE ( F ). ( H–J ) VP088 reduced EcTBK1-induced promoter activities of IFN1 ( H ), IFN3 ( I ), or ISRE ( J ). Cells were co-transfected with the reporter gene plasmids (IFN1-Luc, IFN3-Luc, or ISRE-Luc), internal control plasmid pRL-SV40, pHA-VP088 and pFlag-EccGAS plus pGFP-EcSTING, or pGFP-EcTBK1 for 24 h, and then collected for dual fluorescence reporter gene assay. ( G and K ) VP088 decreased EccGAS-EcSTING ( G ) and EcTBK1 ( K ) induced IFN response. Cells were transfected with pHA-VP088 and pFlag-EccGAS plus pGFP-EcSTING or pGFP-EcTBK1 for 48 h, and then the mRNA levels of IFN-related genes were detected using qPCR. * P < 0.05.

Article Snippet: To evaluate the effect of VP088 knockdown on IFN immune response during SGIV infection, three specific small interfering RNA (siRNA) oligonucleotides targeting VP088 (si-VP088) and stealth RNAi negative control (siRNA-NC) were designed by Sangon Biotech (Shanghai), and the specific sequences were listed in .

Techniques: Over Expression, Transfection, Infection, Control, Plasmid Preparation, Fluorescence, Reporter Gene Assay

Journal: Journal of Virology

Article Title: SGIV envelope protein VP088 facilitated virus replication via interacting with other viral proteins and promoting p62-dependent autophagic degradation of TBK1

doi: 10.1128/jvi.01193-25

Figure Lengend Snippet: Involvement of Ecp62 in VP088-mediated autophagic degradation of EcTBK1. ( A ) VP088 degraded EcTBK1 in a dose-dependent manner. GS cells were co-transfected with pHA-EcTBK1 and pFlag-VP088 under different concentrations, and then the cells were collected for western blotting analysis. ( B ) The schematic diagram of the truncated mutants of EcTBK1. ( C ) The interaction between VP088 and different domains of EcTBK1. Cells were transfected with pGFP-VP088 and pHA-EcTBK, pHA-EcTBK1-∆C, or pHA-EcTBK1-∆N, and then the cells were collected for Co-IP assay and western blotting analysis. ( D ) Co-localization analysis between VP088 and different truncations of EcTBK1 in GS cells. Cells were transfected with pGFP-VP088 and pHA-EcTBK1-∆C or pHA-EcTBK1-∆N and then fixed at 48 h post-transfection for IFA. The samples were visualized under a confocal microscope. Scale bar, 5 µM. ( E–G ) The effects of different inhibitors, including MG132 ( E ), 3-MA ( F ), or NH 4 Cl ( G ), on the degradation effect of VP088 on exogenous EcTBK1. GS cells were co-transfected with pFlag-VP088 and pHA-EcTBK1, then treated without or with MG132, 3-MA, or NH 4 Cl, and collected for western blotting. ( H, I ) The interactions between Ecp62 and VP088 ( H ) or EcTBK1 ( I ) in vitro . GS cells were transfected with Ecp62 and VP088 or EcTBK1 and then collected for Co-IP assay and western blotting assay. ( J ) The silencing effect of siRNA on Ecp62 protein synthesis. GS cells were transfected with siRNA-NC, si-Ecp62-1, si-Ecp62-2, or si-Ecp62-3 and then collected for western blotting analysis. ( K ) Ecp62 was essential for the degradation effect of VP088 on EcTBK1. GS cells were co-transfected with si-Ecp62-1, pFlag-VP088, and pHA-EcTBK1, and then the cells were collected for western blotting analysis.

Article Snippet: To evaluate the effect of VP088 knockdown on IFN immune response during SGIV infection, three specific small interfering RNA (siRNA) oligonucleotides targeting VP088 (si-VP088) and stealth RNAi negative control (siRNA-NC) were designed by Sangon Biotech (Shanghai), and the specific sequences were listed in .

Techniques: Transfection, Western Blot, Co-Immunoprecipitation Assay, Microscopy, In Vitro

Journal: Genes to Cells

Article Title: The Protein Kinase aPKC as Well as the Small GTPases RhoA and Cdc42 Regulates Neutrophil Chemotaxis Partly by Recruiting the ROCK Kinase to the Leading Edge

doi: 10.1111/gtc.70002

Figure Lengend Snippet: Endogenous Cdc42 localizes to the pseudopod in migrating neutrophils and participates in correct cell polarization. (a, e) Representative confocal images of PLB‐985 cells. Cells transfected with negative control RNA or Cdc42‐specific small interfering RNAs (siRNAs) (Cdc42 siRNA‐1 and siRNA‐2) were uniformly stimulated for 90 s with 3 nM C5a (a), or placed for 10 min in a Zigmond chamber with or without a C5a gradient (e). Cells were then fixed with 10% TCA and stained with the anti‐Cdc42 and anti‐β‐actin antibodies and Hoechst. The corresponding differential interference contrast (DIC) images are also shown. Scale bar = 10 μm. (b) Immunoblot analysis of Cdc42 siRNA‐transfected PLB‐985 cells with the anti‐Cdc42 and GAPDH antibodies. Positions for marker proteins are indicated in kDa. (c) Quantification of PLB‐985 cells with polarized accumulation of endogenous Cdc42 at the pseudopod induced by a uniform C5a stimulation. (d, f) Quantification of PLB‐985 cells with a single pseudopod (left) or those with multiple protrusions (right) when cells were stimulated uniformly with 3 nM C5a (d) or in a C5a gradient (f). (g) Quantification of PLB‐985 cells with a single pseudopod facing the source of C5a (correct pseudopod orientation). Values in c, d, f, and g are means ± SD from three independent experiments ( n ≥ 100 cells/experiment). ** p < 0.01 and *** p < 0.001 (Tukey–Kramer test).

Article Snippet: Stealth RNAi siRNA Negative Control Med GC Duplex #2 (Thermo Fisher Scientific) was used as a negative control.

Techniques: Transfection, Negative Control, Staining, Western Blot, Marker

Journal: Genes to Cells

Article Title: The Protein Kinase aPKC as Well as the Small GTPases RhoA and Cdc42 Regulates Neutrophil Chemotaxis Partly by Recruiting the ROCK Kinase to the Leading Edge

doi: 10.1111/gtc.70002

Figure Lengend Snippet: Endogenous RhoA preferentially localizes to the pseudopod in neutrophils stimulated with a uniform concentration of C5a. (a, b) Representative confocal images of PLB‐985 cells. Cells on glass surfaces without coating (uncoated) or coated with FN (FN‐coated) were uniformly stimulated for 90 s with 3 nM C5a or 100 nM fMLP as indicated. Cells were then fixed with 10% TCA and stained with the anti‐RhoA antibody (26C4), Hoechst, and the anti‐β‐actin or anti‐phospho‐ERM (pERM) antibody. The corresponding DIC images are also shown. (c) Immunoblot analysis of RhoA siRNA‐transfected PLB‐985 cells with the anti‐RhoA and GAPDH antibodies. Proteins in the lysates of PLB‐985 cells transfected with negative control RNA or RhoA‐specific siRNAs (RhoA siRNA‐1 and siRNA‐2) were analyzed by immunoblot with the anti‐RhoA (26C4) and anti‐GAPDH antibodies. Positions for marker proteins are indicated in kDa. (d, e) Representative confocal images of PLB‐985 cells stimulated with a uniform concentration of C5a. Cells were fixed with 10% TCA and stained with the anti‐RhoA monoclonal antibody clone 26C4 (d) or clone 1B12 (e). (f) Quantification of PLB‐985 cells with a single pseudopod (left) or multiple protrusions (right). Values are means ± SD from three independent experiments ( n ≥ 100 cells/experiment). *** p < 0.001 (Tukey–Kramer test). Scale bar = 10 μm.

Article Snippet: Stealth RNAi siRNA Negative Control Med GC Duplex #2 (Thermo Fisher Scientific) was used as a negative control.

Techniques: Concentration Assay, Staining, Western Blot, Transfection, Negative Control, Marker

Journal: Genes to Cells

Article Title: The Protein Kinase aPKC as Well as the Small GTPases RhoA and Cdc42 Regulates Neutrophil Chemotaxis Partly by Recruiting the ROCK Kinase to the Leading Edge

doi: 10.1111/gtc.70002

Figure Lengend Snippet: Endogenous RhoA preferentially localizes to the pseudopod of migrating neutrophils in a C5a gradient. (a) Representative confocal images of PLB‐985 cells in a C5a gradient. Cells transfected with negative control RNA or RhoA‐specific siRNAs (RhoA siRNA‐1 and siRNA‐2) were placed in a Zigmond chamber with or without a C5a gradient. Cells were then fixed with 10% TCA and stained with the anti‐RhoA monoclonal antibody clone 26C4 (left) or clone 1B12 (right), anti‐β‐actin antibody, and Hoechst. The corresponding DIC images are also shown. (b) Representative super‐resolution images of migrating PLB‐985 cells in a C5a gradient. The areas outlined with dashed lines are further magnified (zoom). (c, d) Quantification of PLB‐985 cells with a single pseudopod (left in c), with multiple protrusions (right in c), or with a single pseudopod facing the source of C5a (d). Values are means ± SD from three independent experiments ( n ≥ 100 cells/experiment). * p < 0.05 and *** p < 0.001 (Tukey–Kramer test). The scale bars represent 10 μm, unless otherwise indicated.

Article Snippet: Stealth RNAi siRNA Negative Control Med GC Duplex #2 (Thermo Fisher Scientific) was used as a negative control.

Techniques: Transfection, Negative Control, Staining

Journal: Genes to Cells

Article Title: The Protein Kinase aPKC as Well as the Small GTPases RhoA and Cdc42 Regulates Neutrophil Chemotaxis Partly by Recruiting the ROCK Kinase to the Leading Edge

doi: 10.1111/gtc.70002

Figure Lengend Snippet: ROCK1 distributes to the rear of neutrophils polarized by uniform C5a stimulation in a manner independent of RhoA. (a) Immunoblot analysis of ROCK1/2 siRNA‐transfected PLB‐985 cells with the anti‐ROCK1, anti‐ROCK2, and anti‐GAPDH antibodies. Proteins in the lysates of PLB‐985 cells transfected with the indicated RNA were analyzed by immunoblot with the indicated antibodies. Positions for marker proteins are indicated in kDa. (b, c) Representative confocal images of PLB‐985 cells stimulated with 3 nM C5a. ROCK1/2‐depleted (b) or RhoA‐depleted (c) cells were fixed with 3.7% formaldehyde and stained with the anti‐ROCK1 antibody, phalloidin, and Hoechst. The corresponding DIC images are also shown. Scale bar = 10 μm. (d) Quantification of PLB‐985 cells with accumulation of endogenous ROCK1 at the uropod. Values are means ± SD from three independent experiments ( n ≥ 100 cells/experiment). ns, not significant (Tukey–Kramer test).

Article Snippet: Stealth RNAi siRNA Negative Control Med GC Duplex #2 (Thermo Fisher Scientific) was used as a negative control.

Techniques: Western Blot, Transfection, Marker, Staining

Journal: Genes to Cells

Article Title: The Protein Kinase aPKC as Well as the Small GTPases RhoA and Cdc42 Regulates Neutrophil Chemotaxis Partly by Recruiting the ROCK Kinase to the Leading Edge

doi: 10.1111/gtc.70002

Figure Lengend Snippet: The Cdc42‐downstream kinase aPKC participates in C5a‐induced neutrophil chemotaxis. (a) Representative confocal images of PLB‐985 cells expressing FLAG–aPKCι. Cells transfected with FLAG‐tagged aPKCι were placed in a Zigmond chamber with or without a C5a gradient. Cells were then fixed with 3.7% formaldehyde and stained with the anti‐FLAG antibody, phalloidin, and Hoechst. (b) Immunoblot analysis of aPKC siRNA‐transfected PLB‐985 cells with the anti‐aPKC and GAPDH antibodies. Proteins in the lysates of PLB‐985 cells transfected with negative control RNA or aPKC‐specific siRNAs (aPKC siRNA‐1 and siRNA‐2) were analyzed by immunoblot with the anti‐pan‐aPKC, anti‐aPKCι, and anti‐GAPDH antibodies. Positions for marker proteins are indicated in kDa. (c) Representative confocal images of aPKC‐depleted PLB‐985 cells in a C5a gradient. Cells were fixed with 3.7% formaldehyde and stained with phalloidin and Hoechst. (d, e) Quantification of aPKC‐depleted PLB‐985 cells with a single pseudopod (left in d), with multiple protrusions (right in d), or with a single pseudopod facing the source of C5a (e). Values are means ± SD from three independent experiments ( n ≥ 100 cells/experiment). (f, g) Chemotactic response of PLB‐985 cells. Migration of cells transfected with the indicated RNA (f) or cells treated with Y‐27632 (g) in response to C5a was measured in transwell chemotaxis chambers. The percentage of migrated cells was calculated by dividing the number of migrated cells by that of the input cells. Values are means ± SD from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; and ns, not significant (Tukey–Kramer test). Scale bar = 10 μm.

Article Snippet: Stealth RNAi siRNA Negative Control Med GC Duplex #2 (Thermo Fisher Scientific) was used as a negative control.

Techniques: Chemotaxis Assay, Expressing, Transfection, Staining, Western Blot, Negative Control, Marker, Migration

Journal: Biomolecules

Article Title: A Cell-Based Evaluation of the Tyrosinase-Mediated Metabolic Activation of Leukoderma-Inducing Phenols, II: The Depletion of Nrf2 Augments the Cytotoxic Effect Evoked by Tyrosinase in Melanogenic Cells

doi: 10.3390/biom15010114

Figure Lengend Snippet: Nrf2 knockdown enhances the reduction in viability of melanoma cells following their exposure to leukoderma-inducing phenolic compounds in a tyrosinase-dependent manner. B16BL6 melanoma cells were transfected with a negative control siRNA ( Ctrl ) or a siRNA directed against Nrf2 (#1, MSS275988) or Tyr (#1, MSS212191) for 24 h. ( A , D ) Levels of Nrf2 and Tyr mRNA following 24 h incubation in control medium. ( B , C , E , F ) The viability of cells following exposure to the indicated concentration of compounds for 24 h or 48 h. Data are presented as the means ± SD of independent experiments (n = 3 for ( A ) or as the number indicated for ( C , F )) or the means ± SD (n = 3 wells) with similar results in more than two independent experiments (for ( B , D , E )). *, p < 0.05, versus control siRNA-transfected cells (for ( A , D )) or between respective values. B16BL6 melanoma cells (for G – J ) were transfected with a siRNA directed against Nrf2 (#2, MSS207018), Tyr (#2, MSS212190), or a negative control siRNA ( Ctrl ) for 24 h. B16-4A5 melanoma cells (for ( K – M )) were transfected with Nrf2 (#1) and Tyr (#1). ( G , K , M ). Levels of Nrf2 or Tyr mRNA following 24 h incubation in control medium. ( H , I , J , L ) Viability of the cells following exposure to the indicated concentration of compounds for 24 h or 48 h. Data represent means ± SD (n = 3 wells). Similar results were obtained in two independent experiments (for ( H , I , J , L )). For M, data represent the means ± SD of independent experiments (as the number indicated). *, p < 0.05, between respective values or versus control siRNA-transfected cells (for ( G , K )).

Article Snippet: The following siRNAs were utilized: Stealth RNAi™ siRNA (Thermo Fisher Scientific) for mouse Nrf2 ( Nfe2l2 ) (#1, MSS275988; #2, MSS207018), mouse Tyr (#1, MSS212191; #2 MSS212190), mouse Slc7a11 (MSS218649), and mouse Nqo1 (MSS276039), and Stealth RNAi™ siRNA negative control hi GC (Thermo Fisher Scientific).

Techniques: Knockdown, Transfection, Negative Control, Incubation, Control, Concentration Assay

Journal: Biomolecules

Article Title: A Cell-Based Evaluation of the Tyrosinase-Mediated Metabolic Activation of Leukoderma-Inducing Phenols, II: The Depletion of Nrf2 Augments the Cytotoxic Effect Evoked by Tyrosinase in Melanogenic Cells

doi: 10.3390/biom15010114

Figure Lengend Snippet: Nrf2 knockdown reduces GSH , Slc7a11, and Nqo1 mRNA levels and effectively prevents their induction by phenolic compounds. B16BL6 melanoma cells were transfected with a negative control siRNA ( Ctrl ) or a siRNA directed against Nrf2 (#1) for 24 h. The cells were further incubated in the medium containing a vehicle or compounds (0.3 mM) for 24 h. ( A , B ) Cellular GSH levels. Data represent means ± SD (n = 3 wells). Similar results were obtained in two independent experiments. ( C , D ) Levels of Slc7a11 , Nqo1 , and Nrf2 mRNA. Data represent means ± SD of independent experiments (n = 3 for ( C ), or as the number indicated for ( D )). *, p < 0.05, versus control siRNA-transfected cells; #, p < 0.05, versus vehicle-treated cells.

Article Snippet: The following siRNAs were utilized: Stealth RNAi™ siRNA (Thermo Fisher Scientific) for mouse Nrf2 ( Nfe2l2 ) (#1, MSS275988; #2, MSS207018), mouse Tyr (#1, MSS212191; #2 MSS212190), mouse Slc7a11 (MSS218649), and mouse Nqo1 (MSS276039), and Stealth RNAi™ siRNA negative control hi GC (Thermo Fisher Scientific).

Techniques: Knockdown, Transfection, Negative Control, Incubation, Control

Journal: Biomolecules

Article Title: A Cell-Based Evaluation of the Tyrosinase-Mediated Metabolic Activation of Leukoderma-Inducing Phenols, II: The Depletion of Nrf2 Augments the Cytotoxic Effect Evoked by Tyrosinase in Melanogenic Cells

doi: 10.3390/biom15010114

Figure Lengend Snippet: Slc7a11 knockdown enhances the cytotoxicity induced by RD, RK, and pCRE, whereas Nqo1 knockdown or inhibition is ineffective for most compounds. B16BL6 melanoma cells were transfected with a negative control siRNA ( Ctrl ) or a siRNA directed against Slc7a11 or Nqo1 for 24 h. ( A ) Slc7a11 and Nqo1 mRNA levels following 24 h incubation in a control medium. ( B – D ) The viability of cells following exposure to the indicated concentration of compounds for 24 h or 48 h. ( E , F ) The viability of B16BL6 cells after exposure to the indicated concentration of phenolic compounds in the presence or absence of ES936 (500 nM) for 24 h. ( F ) Cells were pretreated with a vehicle or ES936 for 24 h before their exposure to the compounds with/without ES936. ( G ) Western blots and quantitation of tyrosinase and the loading control of β-actin in cells at 48 h after Nqo1 -siRNA transfection or 24 h after ES936 treatment. Data represent means ± SD (n = 3 wells) with similar results in two independent experiments (for ( A – F )) or means ± SD of independent experiments (n = 4 for RD in panel C, D or n = 3 for 4SCAP in panel D). *, p < 0.05, versus Ctrl -siRNA-transfected cells (for ( A )) or between respective values (for ( B – F )). B16-4A5 melanoma cells were transfected with a negative control siRNA ( Ctrl ) or a siRNA directed against Slc7a11 or Nqo1 for 24 h. ( H ) Levels of Slc7a11 and Nqo1 mRNA following 24 h incubation in a control medium. ( I ) The viability of cells following exposure to the indicated concentration of compounds for 24 h or 48 h. Data represent means ± SD (n = 3 wells). Similar results were obtained in two independent experiments (for ( I )). *, p < 0.05, versus Ctrl -siRNA-transfected cells.

Article Snippet: The following siRNAs were utilized: Stealth RNAi™ siRNA (Thermo Fisher Scientific) for mouse Nrf2 ( Nfe2l2 ) (#1, MSS275988; #2, MSS207018), mouse Tyr (#1, MSS212191; #2 MSS212190), mouse Slc7a11 (MSS218649), and mouse Nqo1 (MSS276039), and Stealth RNAi™ siRNA negative control hi GC (Thermo Fisher Scientific).

Techniques: Knockdown, Inhibition, Transfection, Negative Control, Incubation, Control, Concentration Assay, Western Blot, Quantitation Assay

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet:

Article Snippet: Stealth RNAi siRNA Negative Control Med GC , Thermo Fisher Scientific , 12935300.

Techniques: Virus, Recombinant, Protease Inhibitor, Transfection, Multiplex sample analysis, Protein Purification, Reporter Assay, Luciferase, Cell Isolation, Plasmid Preparation, Reverse Transcription, SYBR Green Assay, ChIP-qPCR, Sequencing, Negative Control, Software