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sta21  (Selleck Chemicals)


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    Selleck Chemicals sta21
    Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of <t>STAT3,</t> pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by <t>STA21</t> (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.
    Sta21, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sta21/Ochromycinone/pm36453419-98-13-18
    Average 91 stars, based on 2 article reviews
    sta21 - by Bioz Stars, 2026-10
    91/100 stars

    Images

    1) Product Images from "Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury."

    Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury.

    Journal: Neural regeneration research

    doi: 10.4103/1673-5374.357912

    Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of STAT3, pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by STA21 (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.
    Figure Legend Snippet: Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of STAT3, pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by STA21 (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.

    Techniques Used: In Vitro, Staining, Western Blot, Incubation, Immunofluorescence

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    Article Title: Cationic amphiphilic antihistamines inhibit STAT3 via Ca 2+ -dependent lysosomal H + efflux
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    Protease Inhibitor:

    Article Title: Cationic amphiphilic antihistamines inhibit STAT3 via Ca 2+ -dependent lysosomal H + efflux
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-STAT3 Cell Signaling Technology Cat# 4904, RRID:AB_331269 HRP-anti-a-Tubulin Abcam Cat# ab40742, RRID:AB_880625 Rabbit anti-CCND1 Millipore Cat# 06–137, RRID:AB_2070695 Rabbit anti-P-STAT3(S727) Abcam Cat# ab86430, RRID:AB_10713219 Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-Galectin 1 Abcam Cat# ab25138, RRID:AB_2136615 Rabbit anti-BAX Cell Signaling Technology Cat# 2772, RRID:AB_10695870 Donkey anti-rabbit IgG, Alexa Fluor 594 Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637 Donkey anti-rabbit IgG, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792 Anti-rabbit IgG from goat Vector Laboratories Cat# PI-1000, RRID:AB_2336198 Chemicals, peptides, and recombinant proteins Terfenadine Sigma Aldrich Cat# T9652 Niclosamide Sigma Aldrich Cat# N3510 Forskolin Sigma Aldrich Cat# 93049 Penfluridol Sigma Aldrich Cat# P3371 Astemizole Sigma Aldrich Cat# A2861 EIPA Sigma Aldrich Cat# A3085 Ebastine Cayman Chemical Cat# T9652 WP1066 Merk Cat# N3510 STA-21 Selleckchem Cat# S7951 Stattic Abcam Cat# Ab120952 Hoechst 33,342 Sigma Aldrich Cat# B2261 Propidium iodide solution Sigma Aldrich Cat# P4864 Dextran, Fluorescein, 70,000 MW, Anionic Thermo Fisher Scientific Cat# D-1823 Dextran, Tetramethylrhodamine, 70,000 MW Thermo Fisher Scientific Cat# D-1818 BCECF-AM Thermo Fisher Scientific Cat# B1170 cOmplete Protease inhibitor Cocktail Merk Cat# 11697498001 Critical commercial assays TurboFectin 8.0 Origene Cat# TF81001 Lipofectamine 3000 Thermo Fisher Scientific Cat# L3000008 Lipofectamine RNAmax Thermo Fisher Scientific Cat# 13778075 NE-PERTM Nuclear Extraction Kit Thermo Fisher Scientific Cat# 78833 Quant-XTM One-Step qRT-PCR SYBR Kit Takara Bio Cat# 638317 Pierce Gaussia Luciferase Glow Assay Kit Thermo Fisher Scientific Cat# 16160 Fluo 4, AM Thermo Fisher Scientific Cat# F-14201 Venor GeM Classic PCR kit MB Minerva biolabs Cat# 11-1025G (Continued on next page) Cell Reports 42, 112137, February 28, 2023 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: Cell lines HeLa cervix carcinoma cell ECACC Cat# 93021013, RRID:CVCL_0030 HeLa NC4, STAT3 KO1, STAT3 KO11 Liu et al.12 N/A PANC-1 pancreatic tumor cell Creative Bioarray CSC-C8212L A549 non-small cell lung carcinoma cell ATCC Cat# CCL-185, RRID:CVCL_0023 A549 triple cell Dmitry Malkov lab N/A MCF7 breast cancer cell, TNF-sensitive subclone S1 Jaattela et al.,62 N/A Experimental models: Organisms/strains BALB/c nude mouse Beijing Vital River Laboratory Animal Technology Co., Ltd N/A Oligonucleotides Primers for qPCR: See Table S1 This paper N/A Primers for cloning: See Table S1 This paper N/A siRNA targeting STAT3, P2RX4, ADCY1, TMEM165: See Table S1 This paper N/A Recombinant DNA pC1SypHer3S Addgene Cat# 108118 Flamindo2 Addgene Cat# 73938 pLentiCMVIE-IRES-BlastR Addgene Cat# 119863 Software and algorithms Fiji Schneider et al.63 ImageJ.net/Fiji GraphPad Prism 8.0 GraphPad Prism https://www.graphpad.com/scientific-software/prism/ Image Studio lite Li-COR https://www.licor.com/bio/image-studio-lite/ MetaXpress software Molecular Device https://www.moleculardevices.com/products/ cellular-imaging-systems/acquisition-andanalysis-software/metaxpress#gref Other MicroliteTM1 Luminescence Microtiter 96-well plate VWR Scientific Cat# 62403-124 pHrodo Green-AM Thermo Fisher Scientific Cat# P35373 Live Cell Imaging Solution Thermo Fisher Scientific Cat# A14291DJ Article ll OPEN ACCESS

    Extraction:

    Article Title: Cationic amphiphilic antihistamines inhibit STAT3 via Ca 2+ -dependent lysosomal H + efflux
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-STAT3 Cell Signaling Technology Cat# 4904, RRID:AB_331269 HRP-anti-a-Tubulin Abcam Cat# ab40742, RRID:AB_880625 Rabbit anti-CCND1 Millipore Cat# 06–137, RRID:AB_2070695 Rabbit anti-P-STAT3(S727) Abcam Cat# ab86430, RRID:AB_10713219 Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-Galectin 1 Abcam Cat# ab25138, RRID:AB_2136615 Rabbit anti-BAX Cell Signaling Technology Cat# 2772, RRID:AB_10695870 Donkey anti-rabbit IgG, Alexa Fluor 594 Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637 Donkey anti-rabbit IgG, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792 Anti-rabbit IgG from goat Vector Laboratories Cat# PI-1000, RRID:AB_2336198 Chemicals, peptides, and recombinant proteins Terfenadine Sigma Aldrich Cat# T9652 Niclosamide Sigma Aldrich Cat# N3510 Forskolin Sigma Aldrich Cat# 93049 Penfluridol Sigma Aldrich Cat# P3371 Astemizole Sigma Aldrich Cat# A2861 EIPA Sigma Aldrich Cat# A3085 Ebastine Cayman Chemical Cat# T9652 WP1066 Merk Cat# N3510 STA-21 Selleckchem Cat# S7951 Stattic Abcam Cat# Ab120952 Hoechst 33,342 Sigma Aldrich Cat# B2261 Propidium iodide solution Sigma Aldrich Cat# P4864 Dextran, Fluorescein, 70,000 MW, Anionic Thermo Fisher Scientific Cat# D-1823 Dextran, Tetramethylrhodamine, 70,000 MW Thermo Fisher Scientific Cat# D-1818 BCECF-AM Thermo Fisher Scientific Cat# B1170 cOmplete Protease inhibitor Cocktail Merk Cat# 11697498001 Critical commercial assays TurboFectin 8.0 Origene Cat# TF81001 Lipofectamine 3000 Thermo Fisher Scientific Cat# L3000008 Lipofectamine RNAmax Thermo Fisher Scientific Cat# 13778075 NE-PERTM Nuclear Extraction Kit Thermo Fisher Scientific Cat# 78833 Quant-XTM One-Step qRT-PCR SYBR Kit Takara Bio Cat# 638317 Pierce Gaussia Luciferase Glow Assay Kit Thermo Fisher Scientific Cat# 16160 Fluo 4, AM Thermo Fisher Scientific Cat# F-14201 Venor GeM Classic PCR kit MB Minerva biolabs Cat# 11-1025G (Continued on next page) Cell Reports 42, 112137, February 28, 2023 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: Cell lines HeLa cervix carcinoma cell ECACC Cat# 93021013, RRID:CVCL_0030 HeLa NC4, STAT3 KO1, STAT3 KO11 Liu et al.12 N/A PANC-1 pancreatic tumor cell Creative Bioarray CSC-C8212L A549 non-small cell lung carcinoma cell ATCC Cat# CCL-185, RRID:CVCL_0023 A549 triple cell Dmitry Malkov lab N/A MCF7 breast cancer cell, TNF-sensitive subclone S1 Jaattela et al.,62 N/A Experimental models: Organisms/strains BALB/c nude mouse Beijing Vital River Laboratory Animal Technology Co., Ltd N/A Oligonucleotides Primers for qPCR: See Table S1 This paper N/A Primers for cloning: See Table S1 This paper N/A siRNA targeting STAT3, P2RX4, ADCY1, TMEM165: See Table S1 This paper N/A Recombinant DNA pC1SypHer3S Addgene Cat# 108118 Flamindo2 Addgene Cat# 73938 pLentiCMVIE-IRES-BlastR Addgene Cat# 119863 Software and algorithms Fiji Schneider et al.63 ImageJ.net/Fiji GraphPad Prism 8.0 GraphPad Prism https://www.graphpad.com/scientific-software/prism/ Image Studio lite Li-COR https://www.licor.com/bio/image-studio-lite/ MetaXpress software Molecular Device https://www.moleculardevices.com/products/ cellular-imaging-systems/acquisition-andanalysis-software/metaxpress#gref Other MicroliteTM1 Luminescence Microtiter 96-well plate VWR Scientific Cat# 62403-124 pHrodo Green-AM Thermo Fisher Scientific Cat# P35373 Live Cell Imaging Solution Thermo Fisher Scientific Cat# A14291DJ Article ll OPEN ACCESS

    Quantitative RT-PCR:

    Article Title: Cationic amphiphilic antihistamines inhibit STAT3 via Ca 2+ -dependent lysosomal H + efflux
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-STAT3 Cell Signaling Technology Cat# 4904, RRID:AB_331269 HRP-anti-a-Tubulin Abcam Cat# ab40742, RRID:AB_880625 Rabbit anti-CCND1 Millipore Cat# 06–137, RRID:AB_2070695 Rabbit anti-P-STAT3(S727) Abcam Cat# ab86430, RRID:AB_10713219 Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-Galectin 1 Abcam Cat# ab25138, RRID:AB_2136615 Rabbit anti-BAX Cell Signaling Technology Cat# 2772, RRID:AB_10695870 Donkey anti-rabbit IgG, Alexa Fluor 594 Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637 Donkey anti-rabbit IgG, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792 Anti-rabbit IgG from goat Vector Laboratories Cat# PI-1000, RRID:AB_2336198 Chemicals, peptides, and recombinant proteins Terfenadine Sigma Aldrich Cat# T9652 Niclosamide Sigma Aldrich Cat# N3510 Forskolin Sigma Aldrich Cat# 93049 Penfluridol Sigma Aldrich Cat# P3371 Astemizole Sigma Aldrich Cat# A2861 EIPA Sigma Aldrich Cat# A3085 Ebastine Cayman Chemical Cat# T9652 WP1066 Merk Cat# N3510 STA-21 Selleckchem Cat# S7951 Stattic Abcam Cat# Ab120952 Hoechst 33,342 Sigma Aldrich Cat# B2261 Propidium iodide solution Sigma Aldrich Cat# P4864 Dextran, Fluorescein, 70,000 MW, Anionic Thermo Fisher Scientific Cat# D-1823 Dextran, Tetramethylrhodamine, 70,000 MW Thermo Fisher Scientific Cat# D-1818 BCECF-AM Thermo Fisher Scientific Cat# B1170 cOmplete Protease inhibitor Cocktail Merk Cat# 11697498001 Critical commercial assays TurboFectin 8.0 Origene Cat# TF81001 Lipofectamine 3000 Thermo Fisher Scientific Cat# L3000008 Lipofectamine RNAmax Thermo Fisher Scientific Cat# 13778075 NE-PERTM Nuclear Extraction Kit Thermo Fisher Scientific Cat# 78833 Quant-XTM One-Step qRT-PCR SYBR Kit Takara Bio Cat# 638317 Pierce Gaussia Luciferase Glow Assay Kit Thermo Fisher Scientific Cat# 16160 Fluo 4, AM Thermo Fisher Scientific Cat# F-14201 Venor GeM Classic PCR kit MB Minerva biolabs Cat# 11-1025G (Continued on next page) Cell Reports 42, 112137, February 28, 2023 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: Cell lines HeLa cervix carcinoma cell ECACC Cat# 93021013, RRID:CVCL_0030 HeLa NC4, STAT3 KO1, STAT3 KO11 Liu et al.12 N/A PANC-1 pancreatic tumor cell Creative Bioarray CSC-C8212L A549 non-small cell lung carcinoma cell ATCC Cat# CCL-185, RRID:CVCL_0023 A549 triple cell Dmitry Malkov lab N/A MCF7 breast cancer cell, TNF-sensitive subclone S1 Jaattela et al.,62 N/A Experimental models: Organisms/strains BALB/c nude mouse Beijing Vital River Laboratory Animal Technology Co., Ltd N/A Oligonucleotides Primers for qPCR: See Table S1 This paper N/A Primers for cloning: See Table S1 This paper N/A siRNA targeting STAT3, P2RX4, ADCY1, TMEM165: See Table S1 This paper N/A Recombinant DNA pC1SypHer3S Addgene Cat# 108118 Flamindo2 Addgene Cat# 73938 pLentiCMVIE-IRES-BlastR Addgene Cat# 119863 Software and algorithms Fiji Schneider et al.63 ImageJ.net/Fiji GraphPad Prism 8.0 GraphPad Prism https://www.graphpad.com/scientific-software/prism/ Image Studio lite Li-COR https://www.licor.com/bio/image-studio-lite/ MetaXpress software Molecular Device https://www.moleculardevices.com/products/ cellular-imaging-systems/acquisition-andanalysis-software/metaxpress#gref Other MicroliteTM1 Luminescence Microtiter 96-well plate VWR Scientific Cat# 62403-124 pHrodo Green-AM Thermo Fisher Scientific Cat# P35373 Live Cell Imaging Solution Thermo Fisher Scientific Cat# A14291DJ Article ll OPEN ACCESS

    Luciferase:

    Article Title: Cationic amphiphilic antihistamines inhibit STAT3 via Ca 2+ -dependent lysosomal H + efflux
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-STAT3 Cell Signaling Technology Cat# 4904, RRID:AB_331269 HRP-anti-a-Tubulin Abcam Cat# ab40742, RRID:AB_880625 Rabbit anti-CCND1 Millipore Cat# 06–137, RRID:AB_2070695 Rabbit anti-P-STAT3(S727) Abcam Cat# ab86430, RRID:AB_10713219 Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-Galectin 1 Abcam Cat# ab25138, RRID:AB_2136615 Rabbit anti-BAX Cell Signaling Technology Cat# 2772, RRID:AB_10695870 Donkey anti-rabbit IgG, Alexa Fluor 594 Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637 Donkey anti-rabbit IgG, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792 Anti-rabbit IgG from goat Vector Laboratories Cat# PI-1000, RRID:AB_2336198 Chemicals, peptides, and recombinant proteins Terfenadine Sigma Aldrich Cat# T9652 Niclosamide Sigma Aldrich Cat# N3510 Forskolin Sigma Aldrich Cat# 93049 Penfluridol Sigma Aldrich Cat# P3371 Astemizole Sigma Aldrich Cat# A2861 EIPA Sigma Aldrich Cat# A3085 Ebastine Cayman Chemical Cat# T9652 WP1066 Merk Cat# N3510 STA-21 Selleckchem Cat# S7951 Stattic Abcam Cat# Ab120952 Hoechst 33,342 Sigma Aldrich Cat# B2261 Propidium iodide solution Sigma Aldrich Cat# P4864 Dextran, Fluorescein, 70,000 MW, Anionic Thermo Fisher Scientific Cat# D-1823 Dextran, Tetramethylrhodamine, 70,000 MW Thermo Fisher Scientific Cat# D-1818 BCECF-AM Thermo Fisher Scientific Cat# B1170 cOmplete Protease inhibitor Cocktail Merk Cat# 11697498001 Critical commercial assays TurboFectin 8.0 Origene Cat# TF81001 Lipofectamine 3000 Thermo Fisher Scientific Cat# L3000008 Lipofectamine RNAmax Thermo Fisher Scientific Cat# 13778075 NE-PERTM Nuclear Extraction Kit Thermo Fisher Scientific Cat# 78833 Quant-XTM One-Step qRT-PCR SYBR Kit Takara Bio Cat# 638317 Pierce Gaussia Luciferase Glow Assay Kit Thermo Fisher Scientific Cat# 16160 Fluo 4, AM Thermo Fisher Scientific Cat# F-14201 Venor GeM Classic PCR kit MB Minerva biolabs Cat# 11-1025G (Continued on next page) Cell Reports 42, 112137, February 28, 2023 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: Cell lines HeLa cervix carcinoma cell ECACC Cat# 93021013, RRID:CVCL_0030 HeLa NC4, STAT3 KO1, STAT3 KO11 Liu et al.12 N/A PANC-1 pancreatic tumor cell Creative Bioarray CSC-C8212L A549 non-small cell lung carcinoma cell ATCC Cat# CCL-185, RRID:CVCL_0023 A549 triple cell Dmitry Malkov lab N/A MCF7 breast cancer cell, TNF-sensitive subclone S1 Jaattela et al.,62 N/A Experimental models: Organisms/strains BALB/c nude mouse Beijing Vital River Laboratory Animal Technology Co., Ltd N/A Oligonucleotides Primers for qPCR: See Table S1 This paper N/A Primers for cloning: See Table S1 This paper N/A siRNA targeting STAT3, P2RX4, ADCY1, TMEM165: See Table S1 This paper N/A Recombinant DNA pC1SypHer3S Addgene Cat# 108118 Flamindo2 Addgene Cat# 73938 pLentiCMVIE-IRES-BlastR Addgene Cat# 119863 Software and algorithms Fiji Schneider et al.63 ImageJ.net/Fiji GraphPad Prism 8.0 GraphPad Prism https://www.graphpad.com/scientific-software/prism/ Image Studio lite Li-COR https://www.licor.com/bio/image-studio-lite/ MetaXpress software Molecular Device https://www.moleculardevices.com/products/ cellular-imaging-systems/acquisition-andanalysis-software/metaxpress#gref Other MicroliteTM1 Luminescence Microtiter 96-well plate VWR Scientific Cat# 62403-124 pHrodo Green-AM Thermo Fisher Scientific Cat# P35373 Live Cell Imaging Solution Thermo Fisher Scientific Cat# A14291DJ Article ll OPEN ACCESS

    Polymerase Chain Reaction:

    Article Title: Cationic amphiphilic antihistamines inhibit STAT3 via Ca 2+ -dependent lysosomal H + efflux
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-STAT3 Cell Signaling Technology Cat# 4904, RRID:AB_331269 HRP-anti-a-Tubulin Abcam Cat# ab40742, RRID:AB_880625 Rabbit anti-CCND1 Millipore Cat# 06–137, RRID:AB_2070695 Rabbit anti-P-STAT3(S727) Abcam Cat# ab86430, RRID:AB_10713219 Rabbit anti-P-STAT3(Y705) Cell Signaling Technology Cat# 9145, RRID:AB_2491009 Rabbit anti-Galectin 1 Abcam Cat# ab25138, RRID:AB_2136615 Rabbit anti-BAX Cell Signaling Technology Cat# 2772, RRID:AB_10695870 Donkey anti-rabbit IgG, Alexa Fluor 594 Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637 Donkey anti-rabbit IgG, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792 Anti-rabbit IgG from goat Vector Laboratories Cat# PI-1000, RRID:AB_2336198 Chemicals, peptides, and recombinant proteins Terfenadine Sigma Aldrich Cat# T9652 Niclosamide Sigma Aldrich Cat# N3510 Forskolin Sigma Aldrich Cat# 93049 Penfluridol Sigma Aldrich Cat# P3371 Astemizole Sigma Aldrich Cat# A2861 EIPA Sigma Aldrich Cat# A3085 Ebastine Cayman Chemical Cat# T9652 WP1066 Merk Cat# N3510 STA-21 Selleckchem Cat# S7951 Stattic Abcam Cat# Ab120952 Hoechst 33,342 Sigma Aldrich Cat# B2261 Propidium iodide solution Sigma Aldrich Cat# P4864 Dextran, Fluorescein, 70,000 MW, Anionic Thermo Fisher Scientific Cat# D-1823 Dextran, Tetramethylrhodamine, 70,000 MW Thermo Fisher Scientific Cat# D-1818 BCECF-AM Thermo Fisher Scientific Cat# B1170 cOmplete Protease inhibitor Cocktail Merk Cat# 11697498001 Critical commercial assays TurboFectin 8.0 Origene Cat# TF81001 Lipofectamine 3000 Thermo Fisher Scientific Cat# L3000008 Lipofectamine RNAmax Thermo Fisher Scientific Cat# 13778075 NE-PERTM Nuclear Extraction Kit Thermo Fisher Scientific Cat# 78833 Quant-XTM One-Step qRT-PCR SYBR Kit Takara Bio Cat# 638317 Pierce Gaussia Luciferase Glow Assay Kit Thermo Fisher Scientific Cat# 16160 Fluo 4, AM Thermo Fisher Scientific Cat# F-14201 Venor GeM Classic PCR kit MB Minerva biolabs Cat# 11-1025G (Continued on next page) Cell Reports 42, 112137, February 28, 2023 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: Cell lines HeLa cervix carcinoma cell ECACC Cat# 93021013, RRID:CVCL_0030 HeLa NC4, STAT3 KO1, STAT3 KO11 Liu et al.12 N/A PANC-1 pancreatic tumor cell Creative Bioarray CSC-C8212L A549 non-small cell lung carcinoma cell ATCC Cat# CCL-185, RRID:CVCL_0023 A549 triple cell Dmitry Malkov lab N/A MCF7 breast cancer cell, TNF-sensitive subclone S1 Jaattela et al.,62 N/A Experimental models: Organisms/strains BALB/c nude mouse Beijing Vital River Laboratory Animal Technology Co., Ltd N/A Oligonucleotides Primers for qPCR: See Table S1 This paper N/A Primers for cloning: See Table S1 This paper N/A siRNA targeting STAT3, P2RX4, ADCY1, TMEM165: See Table S1 This paper N/A Recombinant DNA pC1SypHer3S Addgene Cat# 108118 Flamindo2 Addgene Cat# 73938 pLentiCMVIE-IRES-BlastR Addgene Cat# 119863 Software and algorithms Fiji Schneider et al.63 ImageJ.net/Fiji GraphPad Prism 8.0 GraphPad Prism https://www.graphpad.com/scientific-software/prism/ Image Studio lite Li-COR https://www.licor.com/bio/image-studio-lite/ MetaXpress software Molecular Device https://www.moleculardevices.com/products/ cellular-imaging-systems/acquisition-andanalysis-software/metaxpress#gref Other MicroliteTM1 Luminescence Microtiter 96-well plate VWR Scientific Cat# 62403-124 pHrodo Green-AM Thermo Fisher Scientific Cat# P35373 Live Cell Imaging Solution Thermo Fisher Scientific Cat# A14291DJ Article ll OPEN ACCESS



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    Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of <t>STAT3,</t> pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by <t>STA21</t> (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.
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    Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of <t>STAT3,</t> pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by <t>STA21</t> (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.
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    Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of <t>STAT3,</t> pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by <t>STA21</t> (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.
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    Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of STAT3, pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by STA21 (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.

    Journal: Neural regeneration research

    Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury.

    doi: 10.4103/1673-5374.357912

    Figure Lengend Snippet: Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of STAT3, pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by STA21 (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.

    Article Snippet: To assess the role of STAT3 signaling, astrocytes were pretreated with 10 μM STA21 (an inhibitor of STAT3, Selleck, Shanghai, China, Cat# S7951) for 72 hours before coculture.

    Techniques: In Vitro, Staining, Western Blot, Incubation, Immunofluorescence