Journal: iScience

Article Title: Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

doi: 10.1016/j.isci.2022.105779

Figure Lengend Snippet:

Article Snippet: 3xFlag-HaloTag-EZH2 HDR - pDY053 , Youmans et al. , Addgene: 171108.

Techniques: Virus, Recombinant, Western Blot, Software, Modification, DNA Extraction

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI and STAT3 are upregulated both in HCC tissues and cells. ( A , B ) SRI and STAT3 are highly expressed in HCC tissues (T) compared with normal liver tissues (N) in the GEO database (GDS4882). ( C ) Image available from Proteinatlas database showed high expression of SRI and STAT3 in tumor (T) compared with normal liver tissues (N). The expression of proteins were determined by the brown area. Scale bars = 200 μm. ( D – H ) Western blot showed SRI and STAT3 proteins were overexpressed in clinical HCC tissues and in Huh-7/HepG2/Hep3B cells. The red and blue spots represented the expression of different proteins in the T and N groups, respectively. ( I ) From the TGGA database, SRI was found to be positively correlated with STAT3 in LIHC ( p < 0.001). ( J , K ) The expressions of SRI and STAT3 are positively correlated in HCC tissues and cells lines ( p < 0.05). * p < 0.05, ** p < 0.01.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI and STAT3 are interacting proteins. ( A ) SRI and STAT3 are interacting proteins in the STRING database. ( B ) SRI and STAT3 are interacting proteins confirmed by co-immunoprecipitation assays in Huh-7/HepG2 cells. ( C , D ) SRI (shown in red) co-localized with STAT3 (shown in green) was visualized by cellular immunofluorescence in Huh-7/HepG2 cells. DAPI (shown in blue) was used for nuclear staining. Scale bars = 200 μm.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Immunoprecipitation, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI interacts with STAT3, inhibits apoptosis and activates the NF-κB signaling pathway in vitro and in vivo. ( A – D ) SRI downexpression reduced the fluorescence intensity of TMRE (shown in red) and enhanced the apoptosis sensitivities by Hoechst 33342 (shown in blue) staining assay. The results of SRI overexpression were consisted with SRI downexpression. Scale bars = 200 μm. ( E – J ) Western blot detected the expression of STAT3, p65, p-p65, p-IκB and apoptosis-related proteins when SRI overexpression or downexpression were found in HCC cell and tumor xenografts. ( K , L ) Representative IHC images of STAT3, p65, p-p65, p-IκB and apoptosis-related proteins when SRI overexpression or downexpression were found in tumor xenografts. The expression of proteins were determined by the brown area. Scale bars = 50 μm. * p < 0.05, ** p < 0.01.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: In Vitro, In Vivo, Fluorescence, Staining, Over Expression, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI and STAT3 interaction is crucial for anti-apoptosis. ( A – D ) Stattic reduced the fluorescence intensity of TMRE (shown in red) and enhanced the sensitivities to SRI induced apoptosis by Hoechst 33342 (shown in blue) staining assay. Scale bars = 200 μm. ( E – H ) The expression of SRI, p65, p-p65, p-IκB and apoptosis-related proteins were detected by Western blot in Stattic group. * p < 0.05, ** p < 0.01.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Fluorescence, Staining, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI inhibits cells apoptosis through the NF-κB signaling pathway. ( A , B ) Tumor volume and weight of orthotopic xenograft models derived from upSRI cells treated with Stattic. Symbols of different colors represented the weight of tumors in different groups. ( C , D ) Representative IHC images of STAT3, p65, p-p65, p-IκB and apoptosis-related proteins in Stattic treatment. Scale bars = 50 μm. ( C , E ) Representative IHC images of p65 and apoptosis-related proteins after treatment with AL inhibitor. The expression of proteins were determined by the brown area. ( F – H ) AL inhibits the fluorescence intensity of TMRE (shown in red) and reduced the sensitivities to SRI-induced apoptosis by Hoechst 33342 (shown in blue) staining assay. Scale bars = 200 μm. ( I – L ) Apoptosis-related proteins were detected after treated with AL inhibitor by Western blot. * p < 0.05, ** p < 0.01.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Derivative Assay, Expressing, Fluorescence, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: A schematic illustration of SRI regulating apoptosis in HCC. SRI overexpression inhibits the apoptosis of HCC cells by interacting with STAT3 via NF-κB pathway, whereas opposing effects were observed for knockdown of SRI. Stattic, an inhibitor of STAT3, promotes apoptosis of HCC cells via NF-κB pathway. Avicularin, the inhibitor of NF-κB, which promotes apoptosis of HCC cells. ↑ : represent upregulation or promotion. ↓ : represent downregulation or inhibition.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Over Expression, Knockdown, Inhibition

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: