Journal: Frontiers in Pharmacology

Article Title: KS18, a Mcl-1 inhibitor, improves the effectiveness of bortezomib and overcomes resistance in refractory multiple myeloma by triggering intrinsic apoptosis

doi: 10.3389/fphar.2024.1436786

Figure Lengend Snippet: List of antibodies. The following antibodies were used in this study.

Article Snippet: This mixture was then combined with 20 mg of affinity-purified rabbit polyclonal anti-STAT3 antibody (Cell signaling, #9139) or normal rabbit IgG (Abcam, ab46540) as a control.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: KS18, a Mcl-1 inhibitor, improves the effectiveness of bortezomib and overcomes resistance in refractory multiple myeloma by triggering intrinsic apoptosis

doi: 10.3389/fphar.2024.1436786

Figure Lengend Snippet: KS18 inhibits Mcl-1 at both transcriptional and post-translational levels. (A) , U266 MM cells were treated with or without the indicated doses of KS18 for 24 h (B) , KS18 therapy suppresses the activation of the STAT3-Mcl-1 promoter. The ChIP experiment was conducted as outlined in the Materials and Methods section. GraphPad Prism was utilized for graphical representations and statistical analysis. All data were shown as mean ± standard error of measurement. (C–E) , 5 μM of KS18 was applied to U266 MM cells at various time points. For section (A, C, D, E) , subsequent to incubation, the cells were lysed and analyzed via immunoblotting as outlined in the Materials and Methods section, utilizing the specified antibodies. The GAPDH antibody functioned as a loading control. Cells treated with vehicles served as the control. **** P ≤ 0.0001.

Article Snippet: This mixture was then combined with 20 mg of affinity-purified rabbit polyclonal anti-STAT3 antibody (Cell signaling, #9139) or normal rabbit IgG (Abcam, ab46540) as a control.

Techniques: Activation Assay, Incubation, Western Blot, Control

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

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.

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

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]).

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

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.

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

Journal: Frontiers in Microbiology

Article Title: Autoinducer-2 promotes Pseudomonas aeruginosa PAO1 acute lung infection via the IL-17A pathway

doi: 10.3389/fmicb.2022.948646

Figure Lengend Snippet: pSTAT3 and STAT3 expression in lungs from mice infected with P. aeruginosa . (A) Representative immunoblot graphs with pSTAT3, STAT3, and GAPDH expression from mice infected with P. aeruginosa . (B) Significantly higher intensity ratios of pSTAT3/GAPDH in lungs of AI-2+ P.a group than those in P.a group. One of at least three independent experiments with three mice in each group was shown. Results are shown as mean ± SD. * P < 0.05. PBS, phosphate-buffered saline group; AI-2, Autoinducer-2 group; P.a, P. aeruginosa PAO1 group; AI-2+ P.a , AI-2+ P. aeruginosa PAO1 group.

Article Snippet: The membranes were subsequently incubated with primary antibodies, including rabbit anti-mouse STAT3 polyclonal antibodies (1:1,000, CST, USA), rabbit anti-mouse p-STAT3 polyclonal antibodies (1:1,000, CST, USA), and rabbit anti-mouse GAPDH polyclonal antibodies (1:5,000, Proteintech, China) at 4°C overnight.

Techniques: Expressing, Infection, Western Blot, Saline

Journal: Frontiers in Microbiology

Article Title: Autoinducer-2 promotes Pseudomonas aeruginosa PAO1 acute lung infection via the IL-17A pathway

doi: 10.3389/fmicb.2022.948646

Figure Lengend Snippet: pSTAT3 and STAT3 expression in lungs from mice infected with P. aeruginosa . (A) Representative immunoblot graphs with pSTAT3, STAT3, and GAPDH expression from mice infected with P. aeruginosa . (B) Significantly higher intensity ratios of pSTAT3/GAPDH in lungs of AI-2+ P.a group than those in P.a group. One of at least three independent experiments with three mice in each group was shown. Results are shown as mean ± SD. * P < 0.05. PBS, phosphate-buffered saline group; AI-2, Autoinducer-2 group; P.a, P. aeruginosa PAO1 group; AI-2+ P.a , AI-2+ P. aeruginosa PAO1 group.

Article Snippet: The membranes were subsequently incubated with primary antibodies, including rabbit anti-mouse STAT3 polyclonal antibodies (1:1,000, CST, USA), rabbit anti-mouse p-STAT3 polyclonal antibodies (1:1,000, CST, USA), and rabbit anti-mouse GAPDH polyclonal antibodies (1:5,000, Proteintech, China) at 4°C overnight.

Techniques: Expressing, Infection, Western Blot, Saline