Review

polyclonal rabbit anti phospho stat3 s727 cell signaling technology (Cell Signaling Technology Inc)

Name: polyclonal rabbit anti phospho stat3 s727 cell signaling technology
Brand: Cell Signaling Technology Inc
Rating: 96 (225 reviews)

Cell Signaling Technology Inc is a verified supplier

Cell Signaling Technology Inc manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Cell Signaling Technology Inc polyclonal rabbit anti phospho stat3 s727 cell signaling technology
Polyclonal Rabbit Anti Phospho Stat3 S727 Cell Signaling Technology, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 225 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/polyclonal rabbit anti phospho stat3 s727 cell signaling technology/product/Cell Signaling Technology Inc
Average 96 stars, based on 225 article reviews

polyclonal rabbit anti phospho stat3 s727 cell signaling technology - by Bioz Stars, 2026-03

96/100 stars

Images

Image Search Results

$Figure 1. Mitochondrial STAT3 forms a complex with LRPPRC and SLIRP (A) Schematic representation of IP-MS workﬂow. (B) Size-exclusion chromatography of STAT3-containing complexes from isolated mitochondria. Molecular weight markers were separated in previous runs, and retention time corresponding to each indicated size is indicated. (C) Venn diagram showing the overlap between STAT3-interacting proteins identiﬁed from A549 cytosolic, mitochondrial, and nuclear fractions and the mito- chondrial proteome (Mitocarta 2.0). (D) Log10 p values of Gene Ontology analysis of mitochondrial STAT3-interacting proteins. Analysis performed with the ClueGO application for Cytoscape15,16$

Journal: Cell reports

Article Title: A STAT3 protein complex required for mitochondrial mRNA stability and cancer.

doi: 10.1016/j.celrep.2023.113033

Figure Lengend Snippet: Figure 1. Mitochondrial STAT3 forms a complex with LRPPRC and SLIRP (A) Schematic representation of IP-MS workﬂow. (B) Size-exclusion chromatography of STAT3-containing complexes from isolated mitochondria. Molecular weight markers were separated in previous runs, and retention time corresponding to each indicated size is indicated. (C) Venn diagram showing the overlap between STAT3-interacting proteins identiﬁed from A549 cytosolic, mitochondrial, and nuclear fractions and the mito- chondrial proteome (Mitocarta 2.0). (D) Log10 p values of Gene Ontology analysis of mitochondrial STAT3-interacting proteins. Analysis performed with the ClueGO application for Cytoscape15,16

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-LRPPRC Abcam 205022 Mouse monoclonal anti-STAT3 Cell Signaling Technology 9139; RRID:AB_331757 Rabbit polyclonal anti-SLIRP Abcam 51523; RRID:AB_2066704 Rabbit polyclonal anti-VDAC Cell Signaling Techology 4661; RRID:AB_10557420 Rabbit polyclonal anti-MRPL37 Proteintech 15190-1-AP; RRID:AB_2146040 Rabbit polyclonal anti-MRPS35 Proteintech 16457-1-AP; RRID:AB_2146521 Rabbit monoclonal anti-BiP (C50B12) Cell Signaling Techology 3177; RRID:AB_2119845 Mouse monoclonal anti-Lamin A/C (4C11) Cell Signaling Techology 4777; RRID:AB_10545756 Mouse monoclonal anti-FLAG (M2) Merck F1804; RRID:AB_262044 Donkey anti-mouse IRDye 680LT LiCOR 926–68022 Donkey anti-rabbit IRDye 800CW LiCOR 926–32213 Bacterial and virus strains E. Coli strain Top10 Thermo Fisher C404010 E. Coli strain Stbl3 Thermo Fisher C737303 Biological samples Human LUAD patient samples and adjacent normal tissue W.A Cooper and M.R.J Kohonen-Corish Cooper et al.34 Chemicals, peptides, and recombinant proteins Celluspot cellulose array Intavis 93.010 Dynabeads Protein A Thermo Fisher 10001D Anti-FLAG M2 magnetic beads Merck M8823 Strep-Tactin XT Sepharose resin Merck GE29401324 Dynabeads MyOne Streptavidin T1 Thermo Fisher 65602 Critical commercial assays Transcriptor High Fidelity cDNA systhesis kit Merck 5091284001 Click-iT Nascent RNA Capture Kit Thermo Fisher C10365 Expre35S35S Protein Labeling Mix Perkin Elmer NEG072002MC MitoStress Test Kit Agilent 103010–100 Duolink In Situ Green Kit (Mouse/Rabbit) Merck DUO92014 Deposited data Mass spectrometry data This paper ProteomeXchange accession: PXD044281 mPAT Sequencing data This paper FigShare https://doi.org/10.26180/23849112 Experimental models: Cell lines

Techniques: Protein-Protein interactions, Size-exclusion Chromatography, Isolation, Molecular Weight

$Figure 2. STAT3 interacts with LRPPRC and SLIRP independent of RNA (A–C) Immunoprecipitation of mitochondrial fractions from A549 WT or STAT3/ cells with antibodies against (A) STAT3, (B) LRPPRC, or (C) SLIRP and western blots probed with indicated antibodies. (D) Proximity ligation assay using antibodies against either LRPPRC and STAT3 or SLIRP and STAT3 (green), counterstained with DAPI (blue) and MitoTracker (red). Scale bar: 20 mm. (E and F) Mitochondrial fractions from (E) SLIRP knockout or (F) LRPPRC knockdown cells were immunoprecipitated with LRPPRC or SLIRP antibodies, respectively, and western blots probed with the indicated antibodies. (G) LRPPRC immunoprecipitation from the mitochondrial fractions of matched A549 wild-type (WT) and A549 r0 cells. See related content in Figures S2, S4 and S5.$

Journal: Cell reports

Article Title: A STAT3 protein complex required for mitochondrial mRNA stability and cancer.

doi: 10.1016/j.celrep.2023.113033

Figure Lengend Snippet: Figure 2. STAT3 interacts with LRPPRC and SLIRP independent of RNA (A–C) Immunoprecipitation of mitochondrial fractions from A549 WT or STAT3/ cells with antibodies against (A) STAT3, (B) LRPPRC, or (C) SLIRP and western blots probed with indicated antibodies. (D) Proximity ligation assay using antibodies against either LRPPRC and STAT3 or SLIRP and STAT3 (green), counterstained with DAPI (blue) and MitoTracker (red). Scale bar: 20 mm. (E and F) Mitochondrial fractions from (E) SLIRP knockout or (F) LRPPRC knockdown cells were immunoprecipitated with LRPPRC or SLIRP antibodies, respectively, and western blots probed with the indicated antibodies. (G) LRPPRC immunoprecipitation from the mitochondrial fractions of matched A549 wild-type (WT) and A549 r0 cells. See related content in Figures S2, S4 and S5.

Techniques: Immunoprecipitation, Western Blot, Proximity Ligation Assay, Knock-Out, Knockdown

$Figure 3. STAT3 is required for mature mito- chondrial RNA stability and transport to the mitochondrial ribosome for efﬁcient mito- chondrial translation (A and B) Total RNA was isolated from the mito- chondria of A549 STAT3 WT or STAT3/ cells and cDNA synthesized with (A) oligo(d-T) primers or (B) random hexamers. The abundance of each mtRNA species was determined by quantitative real-time PCR and plotted relative to that observed in WT cells. The red dotted line represents equiva- lent expression between genotypes. (C) Nascent mitochondrial RNA was labeled with EU for 1 h prior to isolation and changes in the rate of mitochondrial transcription in the absence of STAT3 expression determined by qRT-PCR. (D) STAT3 loss decreases mitochondrial RNA sta- bility. EU pulse-chase in A549 STAT3 WT or STAT3/ cells and the abundance of each mtRNA species determined by qRT-PCR, normalized to the expression of the nuclear housekeeping gene b2M and expressed relative to WT. Data represent the mean ± SD from at least three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). See related content in Figures S6 and S7. A549 STAT3 WT or STAT3/ cells were pulsed with EU for 1 h, and mitochondrial ribosome fractions were isolated. (E) Equivalent abundance of mitochondrial ribo- somes in STAT3 WT and STAT3/ cells was conﬁrmed by western blot. (F) RNA was biotinylated and isolated from mito- chondrial ribosomal fractions and abundance of each mtRNA species at the mitochondrial ribosome determined by qRT-PCR and expressed relative to WT cells. All experiments were performed at least 3 times and plotted as the mean ± SD. Student’s t test was used to determine signiﬁcance. *p < 0.05, **p < 0.01, ***p < 0.001. (G and H) A549 STAT3 WT or STAT3/ cells (G) or 35S labeling of mitochondrially encoded proteins (H) in Ras-transformed MEFs (STAT3 WT, STAT3/, or STAT3/ reconstituted with mi- tochondrially restricted STAT3 [MTS]).$

Journal: Cell reports

Article Title: A STAT3 protein complex required for mitochondrial mRNA stability and cancer.

doi: 10.1016/j.celrep.2023.113033

Figure Lengend Snippet: Figure 3. STAT3 is required for mature mito- chondrial RNA stability and transport to the mitochondrial ribosome for efﬁcient mito- chondrial translation (A and B) Total RNA was isolated from the mito- chondria of A549 STAT3 WT or STAT3/ cells and cDNA synthesized with (A) oligo(d-T) primers or (B) random hexamers. The abundance of each mtRNA species was determined by quantitative real-time PCR and plotted relative to that observed in WT cells. The red dotted line represents equiva- lent expression between genotypes. (C) Nascent mitochondrial RNA was labeled with EU for 1 h prior to isolation and changes in the rate of mitochondrial transcription in the absence of STAT3 expression determined by qRT-PCR. (D) STAT3 loss decreases mitochondrial RNA sta- bility. EU pulse-chase in A549 STAT3 WT or STAT3/ cells and the abundance of each mtRNA species determined by qRT-PCR, normalized to the expression of the nuclear housekeeping gene b2M and expressed relative to WT. Data represent the mean ± SD from at least three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). See related content in Figures S6 and S7. A549 STAT3 WT or STAT3/ cells were pulsed with EU for 1 h, and mitochondrial ribosome fractions were isolated. (E) Equivalent abundance of mitochondrial ribo- somes in STAT3 WT and STAT3/ cells was conﬁrmed by western blot. (F) RNA was biotinylated and isolated from mito- chondrial ribosomal fractions and abundance of each mtRNA species at the mitochondrial ribosome determined by qRT-PCR and expressed relative to WT cells. All experiments were performed at least 3 times and plotted as the mean ± SD. Student’s t test was used to determine signiﬁcance. *p < 0.05, **p < 0.01, ***p < 0.001. (G and H) A549 STAT3 WT or STAT3/ cells (G) or 35S labeling of mitochondrially encoded proteins (H) in Ras-transformed MEFs (STAT3 WT, STAT3/, or STAT3/ reconstituted with mi- tochondrially restricted STAT3 [MTS]).

Techniques: Isolation, Synthesized, Real-time Polymerase Chain Reaction, Expressing, Labeling, Quantitative RT-PCR, Pulse Chase, Western Blot, Transformation Assay

Figure 4. STAT3 interaction with the LRPPRC-SLIRP complex is required for tumor growth (A) Schematic overview of STAT3 domains. C-C, coiled coil; DBD, DNA-binding domain; TAD, transcriptional activation domain. (B) Spotted cellulose arrays were incubated with lysates from STAT3/ (left panel) or SLIRP/ (right panel) to enable LRPPRC complex to bind to STAT3 peptides. Complex interaction was detected by LRPPRC antibody. Border spots are for orientation. (C) Spotted array analysis was performed three times and spot intensity calculated. Graph represents mean spot intensity ± SD for three independent experi- ments. The location of the ﬁrst amino acid of each peptide sequence is shown on the x axis, dashed lines show the border of each STAT3 domain, and the bar color follows the schematic in (A). (D) STAT3 was immunoprecipitated from mitochondria isolated from the indicated cell lines and the interaction between LRPPRC and STAT3 determined by western blot. Data are representative of at least three independent experiments.

Journal: Cell reports

Article Title: A STAT3 protein complex required for mitochondrial mRNA stability and cancer.

doi: 10.1016/j.celrep.2023.113033

Figure Lengend Snippet: Figure 4. STAT3 interaction with the LRPPRC-SLIRP complex is required for tumor growth (A) Schematic overview of STAT3 domains. C-C, coiled coil; DBD, DNA-binding domain; TAD, transcriptional activation domain. (B) Spotted cellulose arrays were incubated with lysates from STAT3/ (left panel) or SLIRP/ (right panel) to enable LRPPRC complex to bind to STAT3 peptides. Complex interaction was detected by LRPPRC antibody. Border spots are for orientation. (C) Spotted array analysis was performed three times and spot intensity calculated. Graph represents mean spot intensity ± SD for three independent experi- ments. The location of the ﬁrst amino acid of each peptide sequence is shown on the x axis, dashed lines show the border of each STAT3 domain, and the bar color follows the schematic in (A). (D) STAT3 was immunoprecipitated from mitochondria isolated from the indicated cell lines and the interaction between LRPPRC and STAT3 determined by western blot. Data are representative of at least three independent experiments.

Techniques: Binding Assay, Activation Assay, Incubation, Sequencing, Immunoprecipitation, Isolation, Western Blot

Figure 3 Rg1 inhibits the phosphorylation of STAT3 induced by LPS in BV-2 microglia. (A) The target-PPI network of Rg1. As depicted, 17 crucial targets of Rg1 were identified. They are represented by the blue rounded square. In addition, the yellow ellipse represents Rg1. The red edge denotes the relationship between Rg1 and targets, while the the gray edge represents the PPI among Rg1’s targets. The network contains 18 nodes that are interconnected and associated with 41 edges. (B) Western blotting results showing phosphorylation of STAT3 in LPS- activated BV-2 microglial cells with or without Rg1 (20 and 60μM) pretreatment. Bar graph shows the gray value analysis based on immunoblot images. ***p < 0.001 compared with the control group, ###p < 0.001 compared with the LPS group. (C) Immunofluorescence images showing STAT3 expression (red) in lectin-positive microglia (green, arrows) in BV-2 microglia. DAPI-blue. Scale bars=20μm. (D) Note the enhanced expression of NLRP3 in activated microglia, which is noticeably attenuated by Rg1 treatment. *p < 0.05 compared with the control group, #p < 0.05 compared with the LPS group. (E) Immunofluorescence images showing STAT3 expression (red) in lectin-positive microglia (green, arrows) in the corpus callosum of postnatal brain. DAPI-blue. Scale bars=50μm. (F) Note enhanced expression of NLRP3 in activated microglia, which is obviously attenuated by Rg1 treatment. **p < 0.01 compared with the control group, #p < 0.05 compared with the LPS group.

Journal: Journal of Inflammation Research

Article Title: Ginsenoside Rg1 Inhibits Microglia Pyroptosis Induced by Lipopolysaccharide Through Regulating STAT3 Signaling

doi: 10.2147/jir.s326888

Figure Lengend Snippet: Figure 3 Rg1 inhibits the phosphorylation of STAT3 induced by LPS in BV-2 microglia. (A) The target-PPI network of Rg1. As depicted, 17 crucial targets of Rg1 were identified. They are represented by the blue rounded square. In addition, the yellow ellipse represents Rg1. The red edge denotes the relationship between Rg1 and targets, while the the gray edge represents the PPI among Rg1’s targets. The network contains 18 nodes that are interconnected and associated with 41 edges. (B) Western blotting results showing phosphorylation of STAT3 in LPS- activated BV-2 microglial cells with or without Rg1 (20 and 60μM) pretreatment. Bar graph shows the gray value analysis based on immunoblot images. ***p < 0.001 compared with the control group, ###p < 0.001 compared with the LPS group. (C) Immunofluorescence images showing STAT3 expression (red) in lectin-positive microglia (green, arrows) in BV-2 microglia. DAPI-blue. Scale bars=20μm. (D) Note the enhanced expression of NLRP3 in activated microglia, which is noticeably attenuated by Rg1 treatment. *p < 0.05 compared with the control group, #p < 0.05 compared with the LPS group. (E) Immunofluorescence images showing STAT3 expression (red) in lectin-positive microglia (green, arrows) in the corpus callosum of postnatal brain. DAPI-blue. Scale bars=50μm. (F) Note enhanced expression of NLRP3 in activated microglia, which is obviously attenuated by Rg1 treatment. **p < 0.01 compared with the control group, #p < 0.05 compared with the LPS group.

Article Snippet: The rats in Rg1 Table 1 Antibodies Used for Immunofluorescence and Western Blotting Antibody Host Source Catalog Number Dilution for Staining Dilution for Western Blot NLRP3 Rabbit polyclonal Abclonal, China WH110563 1/200 1/1000 Cleaved-caspase-1 Rabbit polyclonal Cell Signaling Technology 4199s 1/1000 ASC Rabbit polyclonal Cell Signaling Technology 67824s 1/1000 β-Actin Mouse monoclonal Proteintech 66009-1-Ig 1/4000 STAT3 Mouse monoclonal Cell Signaling Technology 9139s 1/1000 p-STAT3 Rabbit polyclonal Cell Signaling Technology 9145s 1/100 1/1000 GSDMD Rabbit polyclonal Proteintech 20770-1-AP 1/2000 IL-1β Rabbit polyclonal Cell Signaling Technology 31202 1/1000 AIM Rabbit polyclonal Affinity DF3514 1/1000 Lectin Lycopersicon esculentum Sigma-Aldrich, USA L0401 1/200 DAPI Sigma-Aldrich, USA F6057 Mature-IL-1β Rabbit polyclonal Cell Signaling Technology 83186 1/1000 Pro-caspase-1 Rabbit polyclonal Cell Signaling Technology 24232 1/1000 GSDMD-N Rabbit polyclonal Cell Signaling Technology 36425 1/1000 Goat anti-rabbit IgG H&L (Alexa Fluor® 647) preadsorbed Goat anti-rabbit Abcam Ab150083 1/500 Horseradish peroxidase conjugated secondary antibody Goat anti-rabbit Affinity s0001 1/2000 Horseradish peroxidase conjugated secondary antibody Goat anti-mouse Affinity s0002 1/2000 https://doi.org/10.2147/JIR.S326888 DovePress Journal of Inflammation Research 2021:14 6622 R E T R C T E D Powered by TCPDF (www.tcpdf.org) treatment group were given Rg1 100mg/kg treatment 1h before LPS (1mg/kg) injection administrated intraperitoneally.

Techniques: Phospho-proteomics, Western Blot, Control, Immunofluorescence, Expressing

Figure 4 Rg1 inhibits pyroptosis through STAT3 signaling. (A) Conserved sequences at binding sites of STAT3. (B) STAT3 binding sites on AIM2 promoters predicted by JASPAR. (C) The binding relationship between STAT3 and AIM2 promoter verified by ChIP-qPCR experiment. *p < 0.05 compared with IgG negative control. (D) Western blotting analysis showing NLRP3, ASC, AIM2, cleaved-caspase-1, pro-caspase-1, IL-1β, mature-IL-1β, GSDMD and GSDMD-N protein expression in BV-2 cells with or without Rg1 pretreatment exposed to LPS or LPS with IL-6 stimulation, and with or without stattic treatment induced by LPS. (E–M) Bar graph shows gray value analysis based on immunoblot images. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control, #p < 0.05 and ###p < 0.001 compared with LPS treatment group, ∆p < 0.05 and ∆∆∆p < 0.001 compared with LPS+Rg1 treatment group. (N) Flow cytometry shows the percentage of dead cells over total cells in each stage. The effect of Rg1 (60μM) in down-regulating the rate of apoptotic cells induced by LPS was abrogated by IL-6. Compared with LPS (2μg/mL) treatment group, the rate of apoptotic cells was markedly decreased in stattic and LPS co-incubated group. (O) Bar graph shows the statistical results of the rate of dead cells in each group. **p < 0.01 compared with control, ##p < 0.01 compared with LPS treatment group, ∆∆p < 0.01 compared with LPS+Rg1 treatment group.

Journal: Journal of Inflammation Research

Article Title: Ginsenoside Rg1 Inhibits Microglia Pyroptosis Induced by Lipopolysaccharide Through Regulating STAT3 Signaling

doi: 10.2147/jir.s326888

Figure Lengend Snippet: Figure 4 Rg1 inhibits pyroptosis through STAT3 signaling. (A) Conserved sequences at binding sites of STAT3. (B) STAT3 binding sites on AIM2 promoters predicted by JASPAR. (C) The binding relationship between STAT3 and AIM2 promoter verified by ChIP-qPCR experiment. *p < 0.05 compared with IgG negative control. (D) Western blotting analysis showing NLRP3, ASC, AIM2, cleaved-caspase-1, pro-caspase-1, IL-1β, mature-IL-1β, GSDMD and GSDMD-N protein expression in BV-2 cells with or without Rg1 pretreatment exposed to LPS or LPS with IL-6 stimulation, and with or without stattic treatment induced by LPS. (E–M) Bar graph shows gray value analysis based on immunoblot images. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control, #p < 0.05 and ###p < 0.001 compared with LPS treatment group, ∆p < 0.05 and ∆∆∆p < 0.001 compared with LPS+Rg1 treatment group. (N) Flow cytometry shows the percentage of dead cells over total cells in each stage. The effect of Rg1 (60μM) in down-regulating the rate of apoptotic cells induced by LPS was abrogated by IL-6. Compared with LPS (2μg/mL) treatment group, the rate of apoptotic cells was markedly decreased in stattic and LPS co-incubated group. (O) Bar graph shows the statistical results of the rate of dead cells in each group. **p < 0.01 compared with control, ##p < 0.01 compared with LPS treatment group, ∆∆p < 0.01 compared with LPS+Rg1 treatment group.

Techniques: Binding Assay, ChIP-qPCR, Negative Control, Western Blot, Expressing, Control, Flow Cytometry, Incubation

Figure 5 A schematic diagram depicting the proposed signaling mechanism via which Rg1 can exert its neuroprotection effect on lipopolysaccharide (LPS)-induced microglia pyroptosis. Following LPS stimulation, phosphorylated STAT3 (Tyr705) translocates into the nucleus and binds to the promoters of related genes such as AIM2 inflammasome. AIM2 transcription is initiated and forms inflammasome complexes with ASC and pro-caspase-1. Activated caspase-1 mediates the matura tion of IL-1β, and the cleaved N-terminal of GSDMD triggers pyroptosis. However, phosphorylation of STAT3 enhanced the expression of NLRP3. NLRP3, ASC and pro-caspase-1 are assembled into inflammasome complexes and mediate microglia pyroptosis. Rg1 disrupts the pathway and protects against LPS-induced microglia pyroptosis and inflammatory response through inhibition of STAT3 signaling.

Journal: Journal of Inflammation Research

Article Title: Ginsenoside Rg1 Inhibits Microglia Pyroptosis Induced by Lipopolysaccharide Through Regulating STAT3 Signaling

doi: 10.2147/jir.s326888

Figure Lengend Snippet: Figure 5 A schematic diagram depicting the proposed signaling mechanism via which Rg1 can exert its neuroprotection effect on lipopolysaccharide (LPS)-induced microglia pyroptosis. Following LPS stimulation, phosphorylated STAT3 (Tyr705) translocates into the nucleus and binds to the promoters of related genes such as AIM2 inflammasome. AIM2 transcription is initiated and forms inflammasome complexes with ASC and pro-caspase-1. Activated caspase-1 mediates the matura tion of IL-1β, and the cleaved N-terminal of GSDMD triggers pyroptosis. However, phosphorylation of STAT3 enhanced the expression of NLRP3. NLRP3, ASC and pro-caspase-1 are assembled into inflammasome complexes and mediate microglia pyroptosis. Rg1 disrupts the pathway and protects against LPS-induced microglia pyroptosis and inflammatory response through inhibition of STAT3 signaling.

Techniques: Phospho-proteomics, Expressing, Inhibition

(A) FAO assay comparing levels of FAO in DMSO and 1µM AZD1480-treated BBM2 and BBM3 tumorspheres. Shown are mean ± SD (n=3). (B) Real-time PCR showing the effects of STAT3 siRNA on lipid metabolic genes in BBM2 tumorspheres. Shown are mean ± SD (n=3). (C) Comparison of FAO rates in CSC MDA-MB-468, BBM2 tumorspheres, and MCF-10A cells treated with control siRNA vs. STAT3 siRNA. Shown are mean ± SD (n=3). (D) Relative ATP and ADP/ATP ratio in MCF-10A cells after control or STAT3 siRNA treatment. Shown are mean ± SD (n=4). (E) BODIPY 493/503 staining showing effect of STAT3 siRNA on lipid droplets in CSC MDA-MB-468 cells. The fluorescence intensity was quantified and shown on the right side. (F) Cell survival assay of CSC MDA-MB-468 cells or tumorsphere formation of BBM2 tumor cells after indicated treatments. Shown are mean ± SD (n=4). (G) Tumorsphere formation of BBM2 after indicated treatments of 1µM AZD1480 or/and 5µM Bezafibrate. Shown are mean ± SD (n=3).*p<0.05, **p<0.005, and ***p<0.0005

Journal: Cell metabolism

Article Title: JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance

doi: 10.1016/j.cmet.2017.11.001

Figure Lengend Snippet: (A) FAO assay comparing levels of FAO in DMSO and 1µM AZD1480-treated BBM2 and BBM3 tumorspheres. Shown are mean ± SD (n=3). (B) Real-time PCR showing the effects of STAT3 siRNA on lipid metabolic genes in BBM2 tumorspheres. Shown are mean ± SD (n=3). (C) Comparison of FAO rates in CSC MDA-MB-468, BBM2 tumorspheres, and MCF-10A cells treated with control siRNA vs. STAT3 siRNA. Shown are mean ± SD (n=3). (D) Relative ATP and ADP/ATP ratio in MCF-10A cells after control or STAT3 siRNA treatment. Shown are mean ± SD (n=4). (E) BODIPY 493/503 staining showing effect of STAT3 siRNA on lipid droplets in CSC MDA-MB-468 cells. The fluorescence intensity was quantified and shown on the right side. (F) Cell survival assay of CSC MDA-MB-468 cells or tumorsphere formation of BBM2 tumor cells after indicated treatments. Shown are mean ± SD (n=4). (G) Tumorsphere formation of BBM2 after indicated treatments of 1µM AZD1480 or/and 5µM Bezafibrate. Shown are mean ± SD (n=3).*p<0.05, **p<0.005, and ***p<0.0005

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-CD44 Cell Signaling Technology Cat# 3570S; clone 156-3C11 Mouse monoclonal anti-LEPR Sigma Aldrich Cat# SAB1402837-100UG; clone 2H5 Rabbit polyclonal anti-CPT1B Abcam Cat# ab104662 Rabbit polyclonal anti-STAT3 Santa Cruz Biotech Cat# SC-482 Rabbit polyclonal anti-pY705-STAT3 Cell Signaling Technology Cat#9145S Rabbit polyclonal anti-GAPDH Santa Cruz Biotech Cat#sc-25778 Rabbit polyclonal anti-phospho-Jak2 (Tyr1007/1008) Cell Signaling Technology Cat# 3771S RNA polymerase Sigma Aldrich Cat#05-952 Normal rabbit IgG Santa Cruz Biotech Cat# SC-2027 Leptin neutralizing antibody: Mouse monoclonal anti-LEPR R&D Systems, Inc. Cat# AF497 Bacterial and Virus Strains N/A Biological Samples Breast cancer patient specimens City of Hope National Cancer Center N/A Chemicals, Peptides, and Recombinant Proteins Recombinant LEPR BD Pharmingen Cat# 564376 Recombinant CCR2 BD Pharmingen Cat# 561744 Recombinant CXCR1 BD Pharmingen Cat# 555939 Recombinant CXCR2 BD Pharmingen Cat# 551126 [ 3 H]-palmitic acid PerkinElmer Inc Cat# NET043001MC [ 3 H]-Acetyl Coenzyme A PerkinElmer Inc Cat# NET290050UC Acetyl-coA Sigma Aldrich Cat# A2056 Myristic acid Sigma Aldrich Cat# M3128 paclitaxel Sigma Aldrich Cat# T7402 Etomoxir Sigma Aldrich Cat# 236020 Accutase Millipore Cat# SCR005 Critical Commercial Assays Imprint Chromatin-Immunoprecipitation kit Sigma Aldrich Cat# CHP1-96RXN Adipokine array R&D Systems Cat# ARY024 ChIPAb+ RNA Pol II - ChIP Validated Antibody and Primer Set Sigma Aldrich Cat#17-620 RNeasy Plus Mini Kit Qiagen Cat#74134 iScript cDNA Synthesis Kit Bio-Rad Cat#1708890 iQ SYBR Green Supermix Bio-Rad Cat#1708880 Deposited Data N/A Experimental Models: Cell Lines MCF10A ATCC CRL-10317 MCF-7 ATCC HTB-22 MDA-MB-468 ATCC HTB-132 MDA-MB-231-Parental This paper N/A MDA-MB-231-Resistant This paper N/A BBM2 Neman and Jandial, 2004 N/A BBM3 Neman and Jandial, 2004 N/A Hs578T ATCC HTB-126 MDA-MB-436 ATCC HTB-130 HCC1500 ATCC CRL-2329 BT20 ATCC HTB-19 Preadipocyte Zen-Bio BR-F Experimental Models: Organisms/Strains Mouse: FVB/N-Tg(MMTV-PyVT)634Mul/J The Jackson Laboratory Cat# 002374 Oligonucleotides Stat3 siRNA (h) Santa Cruz Biotech Cat# sc-29493 Please see supplemental table 1 for primer sequence Recombinant DNA pRC-CMV-STAT3C This paper N/A pGL 3.1 Promega Cat# E1751 pGL3.1-CPT1B This paper N/A Software and Algorithms Oncomine Rhodes and Chinnaiyan, 2004. https://www.oncomine.org/ Open in a separate window Key Resources Table Inhibition of FAO preferentially eliminates BCSCs JAK/STAT3 activates FAO through transcription of CPT1B Adipocyte-derived leptin is critical for JAK/STAT3-FAO in BCSCs Targeting FAO/leptin inhibits BCSCs, chemoresistance and breast tumor growth

Techniques: Real-time Polymerase Chain Reaction, Staining, Fluorescence, Clonogenic Cell Survival Assay

(A) Real time PCR measuring CPT1A, CPT1B, and CPT1C mRNA levels in BBM2 tumorspheres and CSC MDA-MB-468 cells transfected with control or STAT3 siRNA. Shown are mean ± SD (n=3). (B) ChIP assay showing STAT3 binding to the CPT1B promoter in BBM2 tumorspheres. The results were normalized to input DNA. Shown are mean ± SD (n=3). (C) CPT1B promoterluciferase assay was measured in MDA-MB-468 and −231 breast cancer cells 24 hours after control or STAT3 siRNA transfection. Constitutively activated STAT3 (STAT3C) was used as a positive control. The luciferase activity was then normalized with control siRNA transfection. (D) Immunofluorescent images of CPT1B levels in CSC MDA-MB-468 cells transfected with control or STAT3 siRNA. White bar presents 10 µm. The fluorescence intensity was quantified and shown on the right side. (E) Western blotting measuring CPT1B and STAT3 protein levels in BBM2 tumorspheres transfected with control siRNA or STAT3 siRNA. β-actin was detected as a loading control. (F) Measurement of relative radioactivity (per µg protein) showing effects of STAT3 siRNA on CPT1 enzyme activity in CSC MDA-MB-468 cells and BBM2 tumorspheres. Shown are mean ± SD (n=3). (G) Tumorsphere formation of BBM2 tumorspheres treated with 1 uM AZD1480 or 5 µM myristic acid (MA). Shown are mean ± SD (n=3).*p<0.05, **p<0.005, and ***p<0.0005

Journal: Cell metabolism

Article Title: JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance

doi: 10.1016/j.cmet.2017.11.001

Figure Lengend Snippet: (A) Real time PCR measuring CPT1A, CPT1B, and CPT1C mRNA levels in BBM2 tumorspheres and CSC MDA-MB-468 cells transfected with control or STAT3 siRNA. Shown are mean ± SD (n=3). (B) ChIP assay showing STAT3 binding to the CPT1B promoter in BBM2 tumorspheres. The results were normalized to input DNA. Shown are mean ± SD (n=3). (C) CPT1B promoterluciferase assay was measured in MDA-MB-468 and −231 breast cancer cells 24 hours after control or STAT3 siRNA transfection. Constitutively activated STAT3 (STAT3C) was used as a positive control. The luciferase activity was then normalized with control siRNA transfection. (D) Immunofluorescent images of CPT1B levels in CSC MDA-MB-468 cells transfected with control or STAT3 siRNA. White bar presents 10 µm. The fluorescence intensity was quantified and shown on the right side. (E) Western blotting measuring CPT1B and STAT3 protein levels in BBM2 tumorspheres transfected with control siRNA or STAT3 siRNA. β-actin was detected as a loading control. (F) Measurement of relative radioactivity (per µg protein) showing effects of STAT3 siRNA on CPT1 enzyme activity in CSC MDA-MB-468 cells and BBM2 tumorspheres. Shown are mean ± SD (n=3). (G) Tumorsphere formation of BBM2 tumorspheres treated with 1 uM AZD1480 or 5 µM myristic acid (MA). Shown are mean ± SD (n=3).*p<0.05, **p<0.005, and ***p<0.0005

Techniques: Real-time Polymerase Chain Reaction, Transfection, Binding Assay, Positive Control, Luciferase, Activity Assay, Fluorescence, Western Blot, Radioactivity

(A) Western blot showing increased phosphorylation of STAT3 at Y705 (pSTAT3) in BBM2 tumorspheres after co-culture with human breast adipocytes or pre-adipocyte. BBM2 culture alone was used as control. (B) Adipokine array identifying adipokines in supernatant from human breast adipocytes. Right, table listing the most highly secreted adipokines. (C) Real-time PCR measuring LEPR mRNA levels in BBM2 and BBM3 tumorspheres (CSC) vs. adherent cells (NCSC). LEPR expression of CSC was then normalized with NCSC. Shown are mean ± SD (n=3). (D) Western blot showing increases in phosphorylation of JAK2 (pJAK2) and STAT3 (pSTAT3) in BBM2 tumorspheres upon leptin stimulation with the indicated conditions. (E) Tumorsphere formation of BBM2 cells treated with leptin at the indicated concentrations. Shown are mean ± SD (n=3). (F) Real-time PCR comparing expression of CPT1B and ACADM in BBM2 tumorspheres cultured with or without human breast adipocytes, treated with leptin neutralizing antibody (α-Leptin) or 1µM AZD1480. Shown are mean ± SD (n=3). (G) Tumor weights of tumors collected from multiple sites in MMTV-PyMT mice treated with PBS or α-Leptin (250 µg/kg). Shown are mean ± SD (n=4). (H) Flow cytometric analyses of single-cell suspensions prepared from tumors collected from MMTV-PyMT mice treated with PBS or α-Leptin (250 µg/kg). Shown are mean ± SD (n=4). (I) Expression of Leptin receptor and CPT1B in primary and resistant TNBC tumors. FFPE tissue sections from TNBC patients were stained by immunofluorescence for Leptin receptor (red) and CPT1B (green); nuclei were counterstained with Hoechst (blue).*p<0.05, **p<0.005, and ***p<0.0005.

Journal: Cell metabolism

Article Title: JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance

doi: 10.1016/j.cmet.2017.11.001

Figure Lengend Snippet: (A) Western blot showing increased phosphorylation of STAT3 at Y705 (pSTAT3) in BBM2 tumorspheres after co-culture with human breast adipocytes or pre-adipocyte. BBM2 culture alone was used as control. (B) Adipokine array identifying adipokines in supernatant from human breast adipocytes. Right, table listing the most highly secreted adipokines. (C) Real-time PCR measuring LEPR mRNA levels in BBM2 and BBM3 tumorspheres (CSC) vs. adherent cells (NCSC). LEPR expression of CSC was then normalized with NCSC. Shown are mean ± SD (n=3). (D) Western blot showing increases in phosphorylation of JAK2 (pJAK2) and STAT3 (pSTAT3) in BBM2 tumorspheres upon leptin stimulation with the indicated conditions. (E) Tumorsphere formation of BBM2 cells treated with leptin at the indicated concentrations. Shown are mean ± SD (n=3). (F) Real-time PCR comparing expression of CPT1B and ACADM in BBM2 tumorspheres cultured with or without human breast adipocytes, treated with leptin neutralizing antibody (α-Leptin) or 1µM AZD1480. Shown are mean ± SD (n=3). (G) Tumor weights of tumors collected from multiple sites in MMTV-PyMT mice treated with PBS or α-Leptin (250 µg/kg). Shown are mean ± SD (n=4). (H) Flow cytometric analyses of single-cell suspensions prepared from tumors collected from MMTV-PyMT mice treated with PBS or α-Leptin (250 µg/kg). Shown are mean ± SD (n=4). (I) Expression of Leptin receptor and CPT1B in primary and resistant TNBC tumors. FFPE tissue sections from TNBC patients were stained by immunofluorescence for Leptin receptor (red) and CPT1B (green); nuclei were counterstained with Hoechst (blue).*p<0.05, **p<0.005, and ***p<0.0005.

Techniques: Western Blot, Co-Culture Assay, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Staining, Immunofluorescence

(A, B) Oncomine analyses of the Finak§ breast cancer data set comparing relative CPT1B mRNA expression in normal breast tissues and breast carcinoma tissues and tumors from patients with recurrence and no recurrence within 5 years. (C) Oncomine analysis of Gluck§ breast cancer dataset of CPT1B mRNA expression in breast tumors from patients with no, partial, near complete, and complete response to paclitaxeltaxol chemotherapy. (D) Real time PCR to assess CPT1B and STAT3 gene expression in ex vivo cultured breast cancer biopsies collected before (primary) and after chemotherapy (resistant). (E) Real time PCR measuring CPT1B and ACADM gene expression in 231-P (parental) and 231-R (resistant) cells. Shown are mean ± SD (n=3). The results were normalized with GAPDH expression. (F) Real time PCR comparing gene expression of MSI1 and OCT4 in 231-P and 231-R cells. Shown are mean ± SD (n=3). The results were normalized with GAPDH expression. (G) Limiting dilution assay comparing tumorsphere forming ability of 231-P and 231-R cells (1 in 13.56 to 1 in 2.24 cells, p=1.26×10−16 with Χ2, n=18). (H) Immunofluorescent images comparing protein levels of CPT1B and pSTAT3 in 231-P and 231-R cells. Right, quantification of relative fluorescence intensities of CPT1B and STAT3. Shown are mean ± SD (n≥5). (I) Comparison of FAO rates between 231-P and -R cells. Shown are mean ± SD (n=3). (J) Expression of CD44 and CPT1B in primary and resistant TNBC tumors. FFPE tissue sections from TNBC patients were stained by immunofluorescence for CD44 (red) and CPT1B (green); nuclei were counterstained with Hoechst (blue). §Databases are named after the first author of originally published data. *p<0.05, **p<0.005, and ***p<0.0005.

Journal: Cell metabolism

Article Title: JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance

doi: 10.1016/j.cmet.2017.11.001

Figure Lengend Snippet: (A, B) Oncomine analyses of the Finak§ breast cancer data set comparing relative CPT1B mRNA expression in normal breast tissues and breast carcinoma tissues and tumors from patients with recurrence and no recurrence within 5 years. (C) Oncomine analysis of Gluck§ breast cancer dataset of CPT1B mRNA expression in breast tumors from patients with no, partial, near complete, and complete response to paclitaxeltaxol chemotherapy. (D) Real time PCR to assess CPT1B and STAT3 gene expression in ex vivo cultured breast cancer biopsies collected before (primary) and after chemotherapy (resistant). (E) Real time PCR measuring CPT1B and ACADM gene expression in 231-P (parental) and 231-R (resistant) cells. Shown are mean ± SD (n=3). The results were normalized with GAPDH expression. (F) Real time PCR comparing gene expression of MSI1 and OCT4 in 231-P and 231-R cells. Shown are mean ± SD (n=3). The results were normalized with GAPDH expression. (G) Limiting dilution assay comparing tumorsphere forming ability of 231-P and 231-R cells (1 in 13.56 to 1 in 2.24 cells, p=1.26×10−16 with Χ2, n=18). (H) Immunofluorescent images comparing protein levels of CPT1B and pSTAT3 in 231-P and 231-R cells. Right, quantification of relative fluorescence intensities of CPT1B and STAT3. Shown are mean ± SD (n≥5). (I) Comparison of FAO rates between 231-P and -R cells. Shown are mean ± SD (n=3). (J) Expression of CD44 and CPT1B in primary and resistant TNBC tumors. FFPE tissue sections from TNBC patients were stained by immunofluorescence for CD44 (red) and CPT1B (green); nuclei were counterstained with Hoechst (blue). §Databases are named after the first author of originally published data. *p<0.05, **p<0.005, and ***p<0.0005.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Ex Vivo, Cell Culture, Limiting Dilution Assay, Fluorescence, Staining, Immunofluorescence

(A) Heatmap depicting abundances of detected FAO- and carnitine-shuttle pathway-related metabolites in chemoresistant 231-R and parental 231-P TNBC cell lines. Values were autoscaled; clustering was conducted using Euclidean Distance and Ward’s method. Fold-change in glycerophosphoethanolamine abundance (B) and carnitine/acetylcarnitine levels (C) following 2, 4 or 6 hours of paclitaxel challenge in 231-R and -P TNBC cell lines relative to respective time-matched vehicle (DMSO) controls. P-value represents differences in the area under the curve between 231-R and -P cells. * p<0.05. (D) Cell survival assay showing perhexilline sensitizes 231-R cells to paclitaxel. Shown are mean ± SD (n=4). (E) Schematic of how JAK/STAT3 activates FAO in BCSCs through intrinsic and extrinsic pathways.

Journal: Cell metabolism

Article Title: JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance

doi: 10.1016/j.cmet.2017.11.001

Figure Lengend Snippet: (A) Heatmap depicting abundances of detected FAO- and carnitine-shuttle pathway-related metabolites in chemoresistant 231-R and parental 231-P TNBC cell lines. Values were autoscaled; clustering was conducted using Euclidean Distance and Ward’s method. Fold-change in glycerophosphoethanolamine abundance (B) and carnitine/acetylcarnitine levels (C) following 2, 4 or 6 hours of paclitaxel challenge in 231-R and -P TNBC cell lines relative to respective time-matched vehicle (DMSO) controls. P-value represents differences in the area under the curve between 231-R and -P cells. * p<0.05. (D) Cell survival assay showing perhexilline sensitizes 231-R cells to paclitaxel. Shown are mean ± SD (n=4). (E) Schematic of how JAK/STAT3 activates FAO in BCSCs through intrinsic and extrinsic pathways.

Techniques: Clonogenic Cell Survival Assay

Journal: Cell metabolism

Article Title: JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance

doi: 10.1016/j.cmet.2017.11.001

Figure Lengend Snippet: Key Resources Table

Techniques: Recombinant, Chromatin Immunoprecipitation, SYBR Green Assay, Sequencing, Software

Review

Structured Review

Images

Similar Products

Image Search Results