Journal: Cancers

Article Title: Resveratrol and AG490 Overcome Glioblastoma Cells’ Resistance to Monotherapy by Inhibiting JAK2/STAT3 Signalling Pathway

doi: 10.3390/cancers18050794

Figure Lengend Snippet: RES and AG490 suppress STAT3 signaling pathway activation in GBM cells. ( a ) Representative immunofluorescence images showing pSTAT3 (green) and nuclear staining with Hoechst (blue) in LN428 and U251 cells treated with RES, AG490, or RES + AG490. Merged images display the overlay of pSTAT3 and nuclear signals. ( b ) Representative immunocytochemistry results demonstrating pSTAT3 protein expression. ( c ) Representative Western blot results showing total STAT3 and pSTAT3 protein expression in both cells treated with RES, AG490, and RES + AG490. GAPDH serves as the loading control. ( d ) Densitometric quantification of STAT3 and pSTAT3 protein expression levels normalized to GAPDH in LN428 and U251 cells. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bar: 100 µm.

Article Snippet: Afterword, the membranes were blocked with 5% skim milk for 2 h, and washed thrice with Tris-buffered saline (TBS-T, 8 min each), and incubated overnight at 4 °C with primary antibodies, Rabbit polyclonal anti STAT3 (1:1000, Protein Tech, Rosemont, IL, USA 10253-2-AP), Rabbit polyclonal anti pSTAT3 (1:1000, abs118973), Rabbit polyclonal anti BAX (1:1000, Protein Tech, USA 50599-2-lg), Rabbit polyclonal anti BCL-2 (1:1000, Protein Tech, USA 26593-1-AP), and Rabbit polyclonal anti- GAPDH (1:5000, Proteintech, Wuhan, China 10494-1-AP).

Techniques: Activation Assay, Immunofluorescence, Staining, Immunocytochemistry, Expressing, Western Blot, Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 1. Average total fold intensity of phospho-protein signal as compared to median control value. Colored bars (dark blue = 0.5 Gy, light blue = 0.1 Gy and green = 0.05 Gy) indicate values significantly different from controls (yellow). Significance bars are shown for all significant differences between doses and 0 (p ≤0.05). Average fold intensity levels are shown at 2 h post radiation for γH2AX (A), pATF2 (B) and pSMC1 (C). Persistent effects are shown at 24 h for γH2AX (D), pATF2 (E) and pSMC1 (F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques: Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 3. Average total fold pATF2 intensity over median control level versus fluence for various radiation qualities. Average fold intensity levels are shown at 2 h post radiation for Si ions (A),Fe ions (C) and Ti ions (E). Persistent effects are shown at 24 h for Si ions (B), Fe ions (D) and Ti ions (F).

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques: Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 6. Average total fold intensity of pATF2 signal intensity divided by fluence and graphed versus LET. Average fold intensity levels are shown at 2 h post radiation for 0.05 Gy (A), 0.1 Gy (B) and 0.5 Gy (C). Persistent effects are shown at 24 h for 0.05 Gy (D), 0.1 Gy (E) and 0.5 Gy (F).

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques:

Journal: International immunology

Article Title: A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways.

doi: 10.1093/intimm/dxf046

Figure Lengend Snippet: Fig. 6. Expression of IRAK1 was severely reduced in R-848-pretreated macrophages. (A) Macrophages were treated with the indicated stimuli for 24 h. Whole-cell lysates were prepared and subjected to Western blot analysis using antibodies for MyD88, IRAK1, TRAF6 and Tollip. (B) Cell lysates were immunoprecipitated with anti-IRAK1 antibody. The kinase activity of IRAK1 in the immunoprecipitates was measured by in vitro kinase assay (upper panel). The same lysates were blotted with anti-IRAK1 antibody to monitor the expression level of IRAK1 (lower panel). The results are representative of three independent experiments that had similar results. L, LPS; R, R-848; P, poly(I:C).

Article Snippet: Rabbit anti-MyD88 antibody was purchased from ProSci (Poway, CA).

Techniques: Expressing, Western Blot, Immunoprecipitation, Activity Assay, In Vitro, Kinase Assay

Journal: International immunology

Article Title: A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways.

doi: 10.1093/intimm/dxf046

Figure Lengend Snippet: Fig. 7. Pretreatment with poly(I:C), but not R-848, led to the impaired LPS-induced expression of IFN-inducible genes. (A and B) Peritoneal macrophages were prepared from wild-type (A) or MyD88±/± (B) mice, and then pretreated with the indicated stimuli for 24 h. Cells were washed twice and stimulated with LPS for 4 h. Total RNA was extracted and subjected to Northern blot analysis using cDNA probes for the indicated genes. (C) Peritoneal macrophages were pretreated with poly(I:C) or LPS for 24 h. Cells were washed twice and then stimulated with LPS for the indicated periods. Cell lysates were prepared and subjected to native PAGE. Monomeric (arrow) and dimeric (arrowhead) forms of IRF-3 were detected by Western blotting. The results are representative of three independent experiments that had similar results. L, LPS; R, R-848; P, poly(I:C).

Article Snippet: Rabbit anti-MyD88 antibody was purchased from ProSci (Poway, CA).

Techniques: Expressing, Northern Blot, Clear Native PAGE, Western Blot

Journal: Antioxidants

Article Title: Liproxstatin-1 Attenuates Retinal Ischemia–Reperfusion Injury by Suppressing EGR1-Mediated Ferroptosis

doi: 10.3390/antiox15030391

Figure Lengend Snippet: Lip-1 halts the oxidative stress-to-ferroptosis cascade in retinal I/R injury. ( A ) Immunofluorescence co-staining of Tuj1-positive RGCs (green) with GPX4 (red), FSP1 (red), and ACSL4 (red) and DAPI (blue) at 24 h post-I/R in retinal sections from sham and I/R mice treated with Vehicle or Lip-1. ( B ) Immunofluorescence co-staining of Tuj1-positive RGCs (green) with 4HNE (red), FHC (red), and DHE (red) and DAPI (blue) at 24 h post-I/R in retinal sections from sham and I/R mice treated with Vehicle or Lip-1. ( C ) Immunofluorescence co-staining of FerroOrange (red) with DAPI (blue) at 24 h post-I/R in retinal sections from sham and I/R mice treated with Vehicle or Lip-1. ( D ) Quantification of the relative immunofluorescence intensity of GPX4, FSP1, ACSL4, 4-HNE, FHC, DHE, and FerroOrange from ( A – C ) ( n = 6 biologically independent experiments). ( E ) Immunofluorescence co-staining of C11-BODIPY (green/red) staining with DAPI (blue) at 24 h post-I/R in retinal sections from sham and I/R mice treated with Vehicle or Lip-1. ( F ) Quantification of the C11 fluorescence ratio (green/red) from ( E ) ( n = 6 biologically independent experiments). ( G ) Western blot analysis of GPX4, FSP1, and FHC expression from sham and I/R mice treated with Vehicle or Lip-1. β-Actin served as a loading control. ( H ) Quantification of GPX4, FSP1, and FHC protein levels from ( E ) ( n = 3 biologically independent experiments). Scale bars: 50 μm. Total magnification: 400×. Data were analyzed by two-way ANOVA with Tukey’s post hoc test. All data are shown as mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: FSP1 , Rabbit , Proteintech , 20886-AP , 1:1000 (WB).

Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing, Control

Journal: Antioxidants

Article Title: Liproxstatin-1 Attenuates Retinal Ischemia–Reperfusion Injury by Suppressing EGR1-Mediated Ferroptosis

doi: 10.3390/antiox15030391

Figure Lengend Snippet: Lip-1 attenuates OGD/R-induced ferroptosis in primary RGCs by restoring antioxidant defenses and suppressing lipid peroxidation. ( A ) Western blot analysis of GPX4 expression in primary RGCs at the indicated time points (3, 6, 12, 24 h) after OGD/R. β-Actin served as a loading control. ( B ) Quantification of GPX4 relative band density from ( A ) ( n = 3 biologically independent experiments). ( C ) Western blot analysis of GPX4, FSP1, and FHC expression in RGCs under different conditions (Control, OGD/R + Vehicle, OGD/R + Lip-1). β-Actin served as a loading control. ( D ) Quantification of GPX4, FSP1, and FHC protein levels from ( C ) ( n = 3 biologically independent experiments). ( E ) Immunofluorescence co-staining of Tuj1-positive RGCs (red) with GPX4 (green) and DAPI (blue) under different conditions. ( F ) Immunofluorescence co-staining of Tuj1-positive RGCs (red) with ferroptosis markers (FSP1, ACSL4, 4-HNE, FHC, all in green) and DAPI (blue) under different conditions. ( G ) Immunofluorescence co-staining of Tuj1-positive RGCs (purple) with FerroOrange staining (red), DCFH-DA (green) and DAPI (blue) under different conditions. ( H ) Quantification of the relative immunofluorescence intensity of GPX4, FSP1, ACSL4, 4-HNE, FHC, FerroOrange, and DCFH-DA from ( E – G ) ( n = 6 biologically independent experiments). ( I ) Immunofluorescence co-staining of Tuj1-positive RGCs (purple) with lipid peroxidation (detected by oxidized C11-BODIPY581/591, green/red), mitochondrial membrane potential (assessed by JC-1 monomer/aggregate ratio, green/red) and DAPI (blue) under different conditions. ( J ) Quantification of the C11 and JC-1 fluorescence ratio (green/red) from ( I ) ( n = 6 biologically independent experiments). ( E – G , I ) Scale bars: 50 μm. Total magnification: 400×. Data were analyzed by two-way ANOVA with Tukey’s post hoc test. All data are shown as mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: FSP1 , Rabbit , Proteintech , 20886-AP , 1:1000 (WB).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Membrane, Fluorescence