Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: ( A ) HER2 (diamonds), TNBC (circles), and luminal (squares and triangles) breast cancer lines were seeded into 96-well plates and treated with increasing doses of MAL3-101 for 72 hr. Viability is expressed as the average of three or more independent experiments, ± SEM. ( B ) MAL3-101-sensitive (MCF7 and MDA MB 231, denoted in blue) and resistant (MDA MB 453 and MDA MB 361, denoted in black) cells were treated with 12 µM MAL3-101 for the indicated times, and lysates were prepared and immunoblotted for cleaved caspase-3, caspase-7, and caspase-8. β-actin serves as a loading control. ( C ) The corresponding fold-increase of the indicated apoptotic markers relative to the DMSO control are plotted, ± SEM (n≥3 for cleaved caspase-3, n=3 for cleaved caspase-7, and n≥4 for cleaved caspase-8). Black asterisks correspond to statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and the red asterisk represents statistical significance between MCF7 (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle) cells; * denotes p<0.05, ** denotes p<0.005. Figure 1—source data 1. Source data for cell viability assay and apoptotic marker accumulation in .

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay, Marker

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101, a specific Hsp70 inhibitor. The indicated breast cancer lines were seeded into 96-well plates and treated with increasing doses of the indicated compounds for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for an undetermined value. MAL3-101 sensitivities of Hsp70 inhibitor resistant cells are in bold.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: The cell numbers and autophagy or proteasome inhibitor concentrations used for the cell viability assay in combination with increasing doses of MAL3-101 are shown. The concentrations of bortezomib, CQ, and bafilomycin to induce no greater than 30% of cell death in each line after 72 hr treatment are shown. ND stands for undetermined value.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101 in the presence of either autophagy or proteasome inhibitors. Cells were seeded into 96-well plates and treated with increasing doses of MAL3-101 in the presence or absence of subcritical doses of bortezomib (proteasome inhibitor), or CQ or bafilomycin (autophagy inhibitors) for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for undetermined value. MAL3-101 resistant cells are highlighted in yellow.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Plasma levels of Foxp3. Peripheral blood mononuclear cells were collected from each group, and the proportions of CD4 + CD25 + Foxp3 + T reg /CD4 + T-cells were analyzed by flow cytometry, and quantified using FlowJo 7.6.1 software. (A) Histogram. Data are presented as the mean ± standard deviation. *P<0.01, vs. the AS group. (B) Negative group, (C) AS group, (D) atorvastatin group and (E) IL-35 group. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35; APC, allophycocyanin; PE, phycoerythrin.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Clinical Proteomics, Flow Cytometry, Software, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Levels of Foxp3 in atherosclerotic lesions. The deposition of Foxp3 in arteries from various groups was detected by immunohistochemistry (magnification, ×400). Positive Foxp3 was shown as brown nuclei. In the (A) negative and (B) AS groups, the deposition of Foxp3 in the lesions was minimal. Conversely, Foxp3 deposition was increased in the lesions of the (C) atorvastatin and (D) IL-35 groups, as compared with the AS group. (E) This was shown to be significant following quantification. There was no significant difference in the levels of Foxp3 between the atorvastatin and IL-35 groups. *P<0.01, vs. the negative control. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Immunohistochemistry, Negative Control

Journal: International Journal of Nanomedicine

Article Title: A Mansonone Derivative Coupled with Monoclonal Antibody 4D5-Modified Chitosan Inhibit AKR1C3 to Treat Castration-Resistant Prostate Cancer

doi: 10.2147/IJN.S241324

Figure Lengend Snippet: Affinity of CS-4D5 and CS-4D5/6e for 22Rv1 and LNCaP cells. ( A ) Western blot analysis relative expression of HER2 receptor in 22RV1 and LNCaP cells. Data are expressed as the mean SEM (n = 3). * P < 0.05 vs α-tubulin. ( B ) Immunofluorescence double co-localization assay was used to compare the affinity of CS-4D5 and CS-4D5/6e in 22RV1 and LNCAP cells. Red fluorescence (Alexa Fluor 555) represents the location of the HER2 receptor, whereas green fluorescence (Alexa Fluor 488) represents the location of CS-4D5 and CS-4D5/6e. Abbreviations: HER2, human epidermal growth factor receptor 2; CS, chemically chitosan; 4D5, single chain antibody fragment 4D5; 6e, a derivative of mansonone F; DAPI, 4ʹ,6-diamidino-2-phenylindole; SEM, standard error of mean.

Article Snippet: Cell membranes were probed with primary antibodies overnight against AR (rabbit monoclonal antibody (mAb); 5153S; Cell Signaling Technology), PSA (rabbit mAb; 5877S; Cell Signaling Technology), AKR1C3 (rabbit mAb; ab203834; Abcam, Cambridge, UK), HER2 (mouse mAb; bsm-33051M; Bioss) or α-tubulin (rabbit mAb; A01080; Abbkine, Wuhan, China) followed by incubation with anti-mouse (AA75181; Bioworld Technology, Saint Louis Park, MN, USA) or anti-rabbit (AA01191; Bioworld Technology) horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) for 1 h, followed by chemiluminescence detection.

Techniques: Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice.

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: Fig. 3 TWEAK regulated the Th17/Treg cell ratio in AC mice. (A) The effect of TWEAK knockdown on Th17 and Treg cell ratio in spleen of AC mice was observed by flow cytometry. (B-C) WB and IHC were employed to evaluate the expression levels of FoxP3 and RORγt in mice conjunctival tissue. (D) The effect of TWEAK knockdown on the levels of Th17 and Treg cytokines in mice spleen were evaluated by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con + AAV-shNC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. AC + AAV-shNC; ns, no significant difference

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Knockdown, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice.

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: Fig. 6 Inhibition of Nrf2/HO-1 signaling pathway affected Th17/Treg cell ratio in AC mice with TWEAK knockdown. (A) The effect of Nrf2 inhibitor on Th17/Treg cell ratio in AC mice with TWEAK knockdown was assessed by flow cytometry. (B-C) WB and IHC assays were employed to evaluate the expres sion of FoxP3 and RORγt in conjunctival tissue of AC mice. (D) The levels of Th17 and Treg cytokines in mice spleen were detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. AC + AAV-shNC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. AC + AAV-shTWEAK

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Inhibition, Knockdown, Flow Cytometry, Enzyme-linked Immunosorbent Assay