Journal: Nature Communications

Article Title: Complement anaphylatoxin C3a is a potent inducer of embryonic chick retina regeneration

doi: 10.1038/ncomms3312

Figure Lengend Snippet: ( a ) Western blot analysis shows the level of phosphorylation of S727 STAT3 at 2 h PR in E4 developmental eyes (Dev), retinectomized eyes with no treatment (Ret), C3a-p-treated eyes and eyes treated with Scrambled C3a (Scr). P =0.03125 using the exact binomial test comparing C3a-p treatment and Scr treatment; n =5 biological samples. ( b ) Western analysis shows the level of phosphorylation of S727 STAT3 (pS727 STAT3) at 2 h PR in eyes treated with C3a-p+IgG and eyes treated with C3a-p+C3aR-Ab. P =0.03125 using the exact binomial test; n =5 biological samples. Error bars represent s.e.m. The mean ratio of pS727 STAT3 to actin, s.e.m., and range are provided in . ( c – f ) Histological analysis at 3 days (d) PR after treatment with either C3a-p+ Stattic ( c ), C3a-p+PD98059 ( d ), C3a-p+DMSO ( e ) or FGF2+PD98059 ( f ). ( g ) Quantification of the amount of regeneration at 3d PR shows that C3a-p-induced regeneration is significantly reduced in the presence of Stattic ( P =0.0054; n =10) and PD98059 ( P =0.0081; n =9) when compared with C3a-p+DMSO ( n =9). FGF2-induced regeneration is also significantly reduced in the presence of PD98059 ( n =5), P- value=0.0027 compared with FGF2-induced regeneration * P <0.05; ** P <0.01. A two-tailed permutation test was used for statistical analysis. Error bars represent s.e.m. The mean amount of regeneration, s.e.m. and range for each treatment are provided in . cr, regeneration from the ciliary margin; L, lens. Scale bar 100 μm ( c – f ).

Article Snippet: C3a-p inhibition studies were performed by injecting a 6-μl aliquot of either 1 μM of PD173074 (Cell Signaling Technology, Inc., Danvers, MA), 1 μM of PD98059 (Cell Signaling Technology, Inc., Danvers, MA), 2 μM of Stattic (Tocris, Minneapolis, MN), 10 μM CHIR 99021 (Stemgent, San Diego, CA), 6 μg of C3aR-Ab or 30 ng of IL-6 Ab (AbD Serotec, Kidlington, UK) into the vitreous chamber post retinectomy 30 min before adding 50 μg of C3a-p (1 nM), along with another dose of either 1 μM of PD173074, 1 μM of PD98059, 2 μM of Stattic, 10 μm CHIR 99021, 3 μg of C3aR-Ab or 30 ng of IL-6 Ab, respectively.

Techniques: Western Blot, Phospho-proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: Complement anaphylatoxin C3a is a potent inducer of embryonic chick retina regeneration

doi: 10.1038/ncomms3312

Figure Lengend Snippet: ( a )RT–qPCR shows the level of expression of IL-6 , Wnt2b , Six3 and Sox2 in the presence of Scr, C3a-p, C3a-p+PD98059, C3a+Stattic, C3a-p+DMSO, C3a-p+C3aR-Ab or C3a-p+IgG. The expression levels were normalized to that in the CM of retinectomized eyes with no other treatment. Significance was determined by comparing C3a-p and Scr ( P =0.0006, 0.000001, 0.0006, 0.0002 for IL-6, Wnt2b , Six3 and Sox2 , respectively); C3a-p+IgG and C3a-p+C3aR-Ab ( P =0.003, 0.002, 0.001, 0.0001 for IL-6 , Wnt2b , Six3 and Sox2 , respectively); C3a-p+DMSO and C3a-p+PD98059 ( P =0.01, 0.0006, 0.02, 0.0001 for IL-6 , Wnt2b , Six3 and Sox2 , respectively); and C3a-p+DMSO and C3a-p+Stattic ( P =0.0001, 0.0009, 0.00000006, 0.00009 for IL-6 , Wnt2b , Six3 and Sox2 , respectively); n =3 biological samples performed in triplicate. The Student’s t -test was used to determine significance. Error bars represent s.e.m. The mean ratio of the mRNA for each gene of interest relative to GAPDH along with the s.e.m. and range is provided in . * P <0.05; ** P <0.01; *** P <0.001. ( b – e ) Histological analysis shows the amount of regeneration at 3 days (d) PR in the presence of IL-6 ( b ), DPBS ( c ), C3a-p+IL-6 Ab ( d ) or C3a-p+IgG ( e ). ( f ) Graphical representation of the mean level of regeneration for each histological analysis. IL-6 ( n =10) versus DPBS ( n =8; P =0.0020); C3a-p+IL-6 Ab ( n =8) versus C3a-p+IgG ( n =8; P =0.026); and C3a-p+CHIR ( n =16) versus C3a-p+DMSO ( n =12; P =0.0041). Statistical analysis was done using the two-tailed permutation test. Error bars represent s.e.m. * P <0.05; ** P <0.01. The mean amount of regeneration, s.e.m. and range for each treatment are provided in . ( g ) Western analysis shows the amount of phosphorylation of STAT3 at Ser727 (pS727 STAT3) in the presence of IL-6 Ab ( P =0.03125; n =5 biological samples). * P <0.05. Statistical analysis was done using the exact binomial test. Error bars represent s.e.m. Mean ratio of pS727 STAT3 to actin, s.e.m, and range are provided in . ( h , i ) Histological analysis shows the amount of regeneration in the presence of C3a-p+CHIR ( h ) or C3a-p+DMSO ( i ). cr, regeneration from the ciliary margin; L, lens.

Article Snippet: C3a-p inhibition studies were performed by injecting a 6-μl aliquot of either 1 μM of PD173074 (Cell Signaling Technology, Inc., Danvers, MA), 1 μM of PD98059 (Cell Signaling Technology, Inc., Danvers, MA), 2 μM of Stattic (Tocris, Minneapolis, MN), 10 μM CHIR 99021 (Stemgent, San Diego, CA), 6 μg of C3aR-Ab or 30 ng of IL-6 Ab (AbD Serotec, Kidlington, UK) into the vitreous chamber post retinectomy 30 min before adding 50 μg of C3a-p (1 nM), along with another dose of either 1 μM of PD173074, 1 μM of PD98059, 2 μM of Stattic, 10 μm CHIR 99021, 3 μg of C3aR-Ab or 30 ng of IL-6 Ab, respectively.

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Phospho-proteomics

Journal: PLOS ONE

Article Title: Role of the JAK2/STAT3 pathway on infection of Francisella novicida

doi: 10.1371/journal.pone.0310120

Figure Lengend Snippet: J774.1 cells treated with 0.01 to 10 μM of cucurbitacin I (A, C) or Stattic (B, D) were infected with F . novicida (C, D) or GFP-expressing F. novicida (A, B). Cells were treated with gentamicin and incubated for 12 h and then observed with confocal microscopy (A, B), or the number of intracellular bacterial was counted (C, D). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Dunnett’s test) and indicated by asterisks, ** P < 0.01, * P < 0.05. Scale bar: 10μm.

Article Snippet: Cucurbitacin I (Merck, Darmstadt, Germany) and Stattic (Merck) were dissolved in dimethyl sulfoxide (DMSO) at 2 mM and then diluted to 200, 20, and 2 μM.

Techniques: Infection, Expressing, Incubation, Confocal Microscopy, Standard Deviation, Comparison

Journal: PLOS ONE

Article Title: Role of the JAK2/STAT3 pathway on infection of Francisella novicida

doi: 10.1371/journal.pone.0310120

Figure Lengend Snippet: F . novicida was cultured in BHIc with cucurbitacin I (A, C) or Stattic (B, D) and optical density (λ = 595 nm) was measured at the indicated time point (A, B). The number of CFU at 0 and 12 h was counted (C, D). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Tukey–Kramer method) (A, C) or Student t-test (B, D) and indicated by asterisks, * P < 0.05.

Article Snippet: Cucurbitacin I (Merck, Darmstadt, Germany) and Stattic (Merck) were dissolved in dimethyl sulfoxide (DMSO) at 2 mM and then diluted to 200, 20, and 2 μM.

Techniques: Cell Culture, Standard Deviation, Comparison

Journal: PLOS ONE

Article Title: Role of the JAK2/STAT3 pathway on infection of Francisella novicida

doi: 10.1371/journal.pone.0310120

Figure Lengend Snippet: F . novicida was cultured in BHIc with cucurbitacin I or Stattic. J774.1 cells were infected with inhibitor-treated F . novicida and observed at 12 h post infection. Scale bar: 10μm.

Article Snippet: Cucurbitacin I (Merck, Darmstadt, Germany) and Stattic (Merck) were dissolved in dimethyl sulfoxide (DMSO) at 2 mM and then diluted to 200, 20, and 2 μM.

Techniques: Cell Culture, Infection

Journal: PLOS ONE

Article Title: Role of the JAK2/STAT3 pathway on infection of Francisella novicida

doi: 10.1371/journal.pone.0310120

Figure Lengend Snippet: J774.1 cells treated with 1 μM of cucurbitacin I (A, C) or 10 μM Stattic (B, D) were infected with F . novicida (C, D) or GFP-expressing F . novicida (A, B). Cells were observed with confocal microscopy (A, B), or the intracellular bacterial number was counted (C, D) at 10 or 30 min post infection. The data represent the averages and standard deviations of three identical experiments. Differences were analyzed with Student t-test and indicated by asterisks, * P < 0.05. Scale bar: 10μm.

Article Snippet: Cucurbitacin I (Merck, Darmstadt, Germany) and Stattic (Merck) were dissolved in dimethyl sulfoxide (DMSO) at 2 mM and then diluted to 200, 20, and 2 μM.

Techniques: Infection, Expressing, Confocal Microscopy

Journal: PLOS ONE

Article Title: Role of the JAK2/STAT3 pathway on infection of Francisella novicida

doi: 10.1371/journal.pone.0310120

Figure Lengend Snippet: J774.1 cells treated with 1 μM of cucurbitacin I (A, C) or 10 μM of Stattic (B, D) were infected with F . novicida (C, D) or GFP-expressing F . novicida (A, B). Cells were treated with gentamicin and incubated for 1.5 or 12 h, Cells were then observed with confocal microscopy (A, B), or the intracellular bacterial number was counted (C, D). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with Student t-test and indicated by asterisks, ** P < 0.01, * P < 0.05. Scale bar: 10μm.

Article Snippet: Cucurbitacin I (Merck, Darmstadt, Germany) and Stattic (Merck) were dissolved in dimethyl sulfoxide (DMSO) at 2 mM and then diluted to 200, 20, and 2 μM.

Techniques: Infection, Expressing, Incubation, Confocal Microscopy, Standard Deviation

Journal: PLOS ONE

Article Title: Role of the JAK2/STAT3 pathway on infection of Francisella novicida

doi: 10.1371/journal.pone.0310120

Figure Lengend Snippet: J774.1 cells were infected with F . novicida (B, C) or GFP-expressing F . novicida (A). Cells were treated with gentamicin and cultured with 1 μM of cucurbitacin I (A, B) or 10 μM of Stattic (A, C) for 12 h. Cells were observed with confocal microscopy(A), or the intracellular bacterial number was counted (B, C). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Dunnett’s test) and indicated by asterisks, ** P < 0.01. Scale bar: 10μm.

Article Snippet: Cucurbitacin I (Merck, Darmstadt, Germany) and Stattic (Merck) were dissolved in dimethyl sulfoxide (DMSO) at 2 mM and then diluted to 200, 20, and 2 μM.

Techniques: Infection, Expressing, Cell Culture, Confocal Microscopy, Standard Deviation, Comparison

Journal: PLOS ONE

Article Title: Role of the JAK2/STAT3 pathway on infection of Francisella novicida

doi: 10.1371/journal.pone.0310120

Figure Lengend Snippet: J774.1 cells treated with 1 μM of cucurbitacin I (A, B) or 10 μM of Stattic (A, B) were infected with E . coli (B) or GFP-expressing E . coli (A). Cells were treated with gentamicin and incubated for 3 h. Cells were observed with confocal microscopy (A), or the intracellular bacterial number was counted (B). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Dunnett’s test) and indicated by asterisks, ** P < 0.01. Scale bar: 10μm.

Article Snippet: Cucurbitacin I (Merck, Darmstadt, Germany) and Stattic (Merck) were dissolved in dimethyl sulfoxide (DMSO) at 2 mM and then diluted to 200, 20, and 2 μM.

Techniques: Infection, Expressing, Incubation, Confocal Microscopy, Standard Deviation, Comparison

Journal: PLOS ONE

Article Title: Role of the JAK2/STAT3 pathway on infection of Francisella novicida

doi: 10.1371/journal.pone.0310120

Figure Lengend Snippet: J774.1 cells treated with 1 μM of cucurbitacin I or 10 μM of Stattic were infected with GFP-expressing F . novicida for the indicated time. Cells were stained with phalloidin-rhodamine and observed by confocal microscopy. Scale bar: 10μm.

Article Snippet: Cucurbitacin I (Merck, Darmstadt, Germany) and Stattic (Merck) were dissolved in dimethyl sulfoxide (DMSO) at 2 mM and then diluted to 200, 20, and 2 μM.

Techniques: Infection, Expressing, Staining, Confocal Microscopy

Journal: International Journal of Nanomedicine

Article Title: Chitosan-Coated-PLGA Nanoparticles Enhance the Antitumor and Antimigration Activity of Stattic – A STAT3 Dimerization Blocker

doi: 10.2147/IJN.S337093

Figure Lengend Snippet: Stattic released in PBS and plasma. Cumulative percentage of Stattic released from S@PLGA NPs and S@C-PLGA NPs in different solution at 0–48 h ( A ) and 0–2 h ( B ). Data represent mean ± SEM from three independent experiments; ****Indicate p < 0.0001, as assessed by two-way ANOVA with Tukey’s post-hoc test.

Article Snippet: In this study, the changes in the antimigration properties of Stattic before and after its entrapment into the C-PLGA NPs (ie, Stattic vs S@C-PLGA) were assessed using human metastatic MDA-MB-231 cells and murine metastatic 4T1 cells., Stattic entrapped in the C-PLGA nanocarrier exhibited better in vitro antimigration effects than S@PLGA and Stattic, whereby S@C-PLGA at 1 μM Stattic equivalent concentration suppressed the migration of MDA-MB-231 ( ) and 4T1 ( ) cells by 65.1% and 47.3%, respectively ( ).

Techniques: Clinical Proteomics

Journal: International Journal of Nanomedicine

Article Title: Chitosan-Coated-PLGA Nanoparticles Enhance the Antitumor and Antimigration Activity of Stattic – A STAT3 Dimerization Blocker

doi: 10.2147/IJN.S337093

Figure Lengend Snippet: In vitro anti-migration properties of Stattic, S@PLGA and S@C-PLGA NPs in Scratch Assay. Microscopic images of the Scratch Assay on MDA-MB-231 ( A ) and 4T1 ( B ) at different time points. ( C ) The normalized migration rate of MDA-MB-231 and 4T1 cells after being treated with Stattic, S@PLGA and S@C-PLGA NPs at 1 µM Stattic equivalent concentration for 24 h. Data represent mean ± SEM from three independent experiments; ** and ****Indicate p < 0.01, 0.0001, respectively, as assessed by two-way ANOVA with Tukey’s post-hoc test.

Article Snippet: In this study, the changes in the antimigration properties of Stattic before and after its entrapment into the C-PLGA NPs (ie, Stattic vs S@C-PLGA) were assessed using human metastatic MDA-MB-231 cells and murine metastatic 4T1 cells., Stattic entrapped in the C-PLGA nanocarrier exhibited better in vitro antimigration effects than S@PLGA and Stattic, whereby S@C-PLGA at 1 μM Stattic equivalent concentration suppressed the migration of MDA-MB-231 ( ) and 4T1 ( ) cells by 65.1% and 47.3%, respectively ( ).

Techniques: In Vitro, Migration, Wound Healing Assay, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Chitosan-Coated-PLGA Nanoparticles Enhance the Antitumor and Antimigration Activity of Stattic – A STAT3 Dimerization Blocker

doi: 10.2147/IJN.S337093

Figure Lengend Snippet: In vitro anti-migration properties of Stattic, S@PLGA and S@C-PLGA NPs in transwell assay. Microscopic images of the transwell migration assay in MDA-MB-231 and 4T1 after treated with 1 μM of Stattic, S@PLGA NPs and S@C-PLGA NPs. The percentage of migrated cells relative to control were quantified using ImageJ software version 2.0. Data represent mean ± SEM from three independent experiments; ** and ****Indicate p < 0.01, 0.0001, respectively, as assessed by two-way ANOVA with Tukey’s post-hoc test.

Article Snippet: In this study, the changes in the antimigration properties of Stattic before and after its entrapment into the C-PLGA NPs (ie, Stattic vs S@C-PLGA) were assessed using human metastatic MDA-MB-231 cells and murine metastatic 4T1 cells., Stattic entrapped in the C-PLGA nanocarrier exhibited better in vitro antimigration effects than S@PLGA and Stattic, whereby S@C-PLGA at 1 μM Stattic equivalent concentration suppressed the migration of MDA-MB-231 ( ) and 4T1 ( ) cells by 65.1% and 47.3%, respectively ( ).

Techniques: In Vitro, Migration, Transwell Assay, Transwell Migration Assay, Control, Software

Journal: International Journal of Nanomedicine

Article Title: Chitosan-Coated-PLGA Nanoparticles Enhance the Antitumor and Antimigration Activity of Stattic – A STAT3 Dimerization Blocker

doi: 10.2147/IJN.S337093

Figure Lengend Snippet: ( A ) In vivo toxicity profiling and antitumor efficacy study of Stattic and S@C-PLGA NPs. In vivo toxicity profile of Stattic and S@C-PLGA NPs in 4T1 tumor-bearing mice. Data represents the mean body weights ± SEM (n = 3) for each group. ( B ) In vivo antitumor efficacy of Stattic and S@C-PLGA NPs in 4T1 tumor-bearing mice. S@C-PLGA NPs (24 mg Stattic eqv./kg) showed greater suppression in 4T1 tumor growth compared to Stattic (24 mg/kg) and saline. The day of treatment was indicated as black arrows. Data represents the mean tumor volume ± SEM (n = 4) for each group; ** and **** indicate p < 0.01, 0.0001, respectively, as assessed by one-way ANOVA with Dunnett’s post-hoc test.

Article Snippet: In this study, the changes in the antimigration properties of Stattic before and after its entrapment into the C-PLGA NPs (ie, Stattic vs S@C-PLGA) were assessed using human metastatic MDA-MB-231 cells and murine metastatic 4T1 cells., Stattic entrapped in the C-PLGA nanocarrier exhibited better in vitro antimigration effects than S@PLGA and Stattic, whereby S@C-PLGA at 1 μM Stattic equivalent concentration suppressed the migration of MDA-MB-231 ( ) and 4T1 ( ) cells by 65.1% and 47.3%, respectively ( ).

Techniques: In Vivo, Saline

Journal: International Journal of Nanomedicine

Article Title: Chitosan-Coated-PLGA Nanoparticles Enhance the Antitumor and Antimigration Activity of Stattic – A STAT3 Dimerization Blocker

doi: 10.2147/IJN.S337093

Figure Lengend Snippet: In vivo antimetastatic effects of S@C-PLGA NPs. Histopathological analysis and quantification of metastatic foci for lung, liver, and lymph node of untreated control mice ( A ) and 4T1 tumor-bearing mice treated with multiple doses of saline ( B ), Stattic (24 mg/kg) ( C ) and S@C-PLGA NPs (24 mg Stattic eqv./kg) ( D ). No metastatic foci were observed in heart, kidney and spleen. Quantification of number of metastatic foci found in lung ( E ) and liver ( F ) by observing 5 and 8 images in lung and liver, respectively, for each mouse in all the treatment groups. Average sizes of metastatic foci found in lung ( G ) and liver ( H ) for each treatment group. Data represent the mean number of metastatic foci ± SEM (n=4); *, **, *** and ****Indicate p <0.05, 0.01, 0.001 and 0.0001, respectively as assessed by One-way ANOVA with Dunnett’s post-hoc test. Yellow arrows indicate metastatic foci.

Article Snippet: In this study, the changes in the antimigration properties of Stattic before and after its entrapment into the C-PLGA NPs (ie, Stattic vs S@C-PLGA) were assessed using human metastatic MDA-MB-231 cells and murine metastatic 4T1 cells., Stattic entrapped in the C-PLGA nanocarrier exhibited better in vitro antimigration effects than S@PLGA and Stattic, whereby S@C-PLGA at 1 μM Stattic equivalent concentration suppressed the migration of MDA-MB-231 ( ) and 4T1 ( ) cells by 65.1% and 47.3%, respectively ( ).

Techniques: In Vivo, Control, Saline