Journal: Frontiers in Immunology

Article Title: m6A eraser FTO modulates autophagy by targeting SQSTM1/P62 in the prevention of canagliflozin against renal fibrosis

doi: 10.3389/fimmu.2022.1094556

Figure Lengend Snippet: Cana causes STAT6 degradation through inducing autophagy. (A) HK2 cells were treated with the indicated doses of Cana for 48 h serum-free medium culture and cell viability was determined using MTT assay. (n = 4, * p < 0.05, Cana 0 μM vs. other doses of Cana). (B) HK2 cells were treated with Cana (0–20 μM) for 24 h, and then cell lysates were harvested and subjected to immunoblot analyses with the indicated antibodies. (C) To test its protein stability, the half-life of STAT6 was determined. HK2 cells were left untreated or treated with Cana (20 μM) for 24 h, and CHX (50 μM) was added at different time points. The intensity of STAT6 and GAPDH bands was quantified and plotted against the time after CHX addition. (D) HK2 cells were treated with Cana (0–20 μM) for 24 h and subjected to immunofluorescence staining of LC3. (E) HK2 cells were transfected with a tandem mRFP-GFP-LC3 construct for 24 h and then either left untreated or separately treated with rapamycin (1 μM), BafA1 (100 nM), or Cana (40 μM) for 24 h. Representative micrographs of the indicated cells are shown. (F) The mRNA levels of STAT6, Arg-1, PPARα, and CPT-1α in HK2 cells treated with Cana (0–20 μM) for 24 h were measured by qPCR. (* p < 0.05, Cana 0 μM vs . other doses of Cana). (G–I) HK2 cells were transfected with vector or plasmid of STAT6 overexpression. Followed by 24 h serum-free medium culture, cells were treated with TGF-β1 (5 ng/ml) for another 24 h and cotreated with or without Cana (20 μM). (G) Immunoblot analysis was conducted with the indicated antibodies. (H) Immunoblot analyses were employed to confirm the efficiency of overexpressing STAT6. Representative microphages showed the Oil Red O staining in HK2 cells with the indicated treatments. (I) TG content was determined enzymatically. (n = 4, * p < 0.05, TGF-β1 vs. TGF-β1 with other treatments).

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc-166920), TGF-β1 (sc-146), α-SMA (sc-53142), FN (sc-18827), LC3I/II (sc-398822), SQSTM1 (sc-28359), CPT-1α (sc-393070), PPARα (sc-398394), and GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: MTT Assay, Western Blot, Immunofluorescence, Staining, Transfection, Construct, Plasmid Preparation, Over Expression

Journal: Frontiers in Immunology

Article Title: m6A eraser FTO modulates autophagy by targeting SQSTM1/P62 in the prevention of canagliflozin against renal fibrosis

doi: 10.3389/fimmu.2022.1094556

Figure Lengend Snippet: Atg7 deficiency vanished the protection of Cana in tubulointerstitial fibrosis. (A) HK2 cells were treated with Cana (0–20 μM) for 24 h. The protein expression of STAT6 and p-STAT6 in nucleus and cytoplasm was separated and determined by immunoblot analyses. (B) HK2 cells were pretreated with MG132 (10 μM) or BafA1 (100 nM) for 4 h and cotreated with or without Cana after being cultured with serum-free medium for 24 h. The cell lysates were subjected to immunoblot analyses with the indicated antibodies. (C–E) WT and Atg7 cKO mice received intragastric administration of saline or Cana (20 mg/kg) after UUO operation. (C) Representative images of H&E, Sirius Red, and IHC staining of kidney sections from the indicated group of mice. Quantification of the relative collagen proportional area from each group of mice was performed. (D) TG content in kidney tissue was measured by a commercial kit. (E) Immunoblot analyses for the protein levels of p-STAT6, STAT6, Arg-1, FN, α-SMA, PPARα, CPT-1α, and GAPDH in kidney from the indicated group with quantification on the right panel (n = 8, * p < 0.05, UUO vs . UUO + Cana).

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc-166920), TGF-β1 (sc-146), α-SMA (sc-53142), FN (sc-18827), LC3I/II (sc-398822), SQSTM1 (sc-28359), CPT-1α (sc-393070), PPARα (sc-398394), and GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Cell Culture, Saline, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: m6A eraser FTO modulates autophagy by targeting SQSTM1/P62 in the prevention of canagliflozin against renal fibrosis

doi: 10.3389/fimmu.2022.1094556

Figure Lengend Snippet: FTO was downregulated by Cana to enhance the stability of SQSTM1 mRNA through installing m6A modification of its mRNA. m6A levels of HK2 cells treated with or without Cana (20 μM) were detected by using the EpiQuik™ m6A RNA methylation quantification kit (A) and RNA dot blot analyses (B) . Methylation blue (MB) staining served as a loading control. (C) RNA dot blot analyses of m6A levels in mice from the indicated group. (D) HK2 cells treated with or without Cana (20 μM) were harvested and subjected to qPCR to determined STAT6, Arg-1, SQSTM1, FTO, ALKBH5, METTL14, METTL3, and WTAP mRNA levels. (n = 4, * p < 0.05, Ctrl vs. Cana). (E) HK2 cells treated with Cana (0–20 μM) for 24 h were subjected to detected the protein levels of FTO by immunoblot analyses. (F) Mice received intragastric administration of saline or Cana (20 mg/kg) after UUO treatment. The expression of FTO was determined through IHC staining. (G) HK2 cells were pretreated with MG132 (10 μM) or BafA1 (100 nM) for 4 h and cultured with serum-free medium for 24 h. Then, the cells were left and treated with or without Cana (20 μM) for another 24 h. Immunoblot analyses was performed to detect the protein level of FTO. (H–J, L) HK2 cells were transfected with or without plasmid of FTO overexpression 48 h. Then, cells were treated with or without Cana (20 μM) for another 24 h. (H) The levels of LC3 in HK2 cells from the indicated group were detected through immunofluorescence staining. (I) Immunoblot analysis was performed to determine the protein levels of STAT6, FTO, SQSTM1, LC3I/II, and GAPDH. (J) qPCR analyses of SQSTM1 mRNA stability in HK2 cells from the indicated group (* p < 0.05). (K) RNA immunoprecipitation (RIP)-qPCR analyses revealed the SQSTM1 mRNA level enriched by the FTO antibody. The agarose gel electrophoresis analyses of qPCR products are shown on the panel (* p < 0.05). (L) MeRIP-qPCR assay indicated the m6A modification level of HK2 cells from the indicated group (* p < 0.05). (M) HK2 cells transfected with or without SQSTM1 siRNA for 24 h and then treated with Cana (20 μM) along with TGF-β1 (5 ng/ml) for another 48 h. The efficiency of knockdown SQSTM1 in HK2 cells was confirmed by immunoblot analyses. Representative microphages showed the Oil Red O staining in HK2 cells with indicated treatments.

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc-166920), TGF-β1 (sc-146), α-SMA (sc-53142), FN (sc-18827), LC3I/II (sc-398822), SQSTM1 (sc-28359), CPT-1α (sc-393070), PPARα (sc-398394), and GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques: Modification, Methylation, Dot Blot, Staining, Control, Western Blot, Saline, Expressing, Immunohistochemistry, Cell Culture, Transfection, Plasmid Preparation, Over Expression, Immunofluorescence, RNA Immunoprecipitation, Agarose Gel Electrophoresis, Knockdown

Journal: Frontiers in Immunology

Article Title: m6A eraser FTO modulates autophagy by targeting SQSTM1/P62 in the prevention of canagliflozin against renal fibrosis

doi: 10.3389/fimmu.2022.1094556

Figure Lengend Snippet: Proposed model for the therapeutic action of Cana against renal fibrosis. Cana attenuates renal tubular cells’ FAO disorder and renal fibrosis by SQSTM1/autophagy-mediated STAT6 degradation in an m6A-dependent manner.

Article Snippet: The following antibodies were used: STAT6 (sc-374021), p-STAT6 (sc-136019), Arg-1 (sc-166920), TGF-β1 (sc-146), α-SMA (sc-53142), FN (sc-18827), LC3I/II (sc-398822), SQSTM1 (sc-28359), CPT-1α (sc-393070), PPARα (sc-398394), and GAPDH (sc-32233) from Santa Cruz Biotechnology.

Techniques:

Journal: Immunity

Article Title: Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function

doi: 10.1016/j.immuni.2020.07.006

Figure Lengend Snippet:

Article Snippet: anti-pSTAT6 149 Sm-conjugated Clone 18 , Fluidigm , Cat No.3149004A.

Techniques: Recombinant, Virus, Molecular Cloning, Blocking Assay, Conjugation Assay, Staining, Western Blot, Lysis, Extraction, Mutagenesis, Isolation, Reverse Transcription, Luminex, Transfection, Amplification, Software, Variant Assay

Journal: Cell reports

Article Title: Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome

doi: 10.1016/j.celrep.2019.10.038

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-human phospho-STAT6 (Tyr641) (clone 18) , Fluidigm , Cat#3168012A; RRID:AB_2811103.

Techniques: Recombinant, Mass Cytometry, Software

Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet: KLF4 is associated with sleep deprivation-induced ADRB2 modulation of macrophage prompting on proliferation, migration, and invasion of LLC-LUC cells (A) Differentially expressed Top20 genes in metastatic lung tissues of NC group and sleep deprivation group. (B) The expression of top 5 upregulated genes in tumor tissues was analyzed by qRT-PCR. (C) KLF4 expression was detected by ADRB2-specific agonist ISO and ADRB2 antagonist ICI118,551 in macrophages treated with IL-4 in vitro . (D) The effect of ADRB2 agonist on CD206 expression in IL-4-induced KLF4-knockdown macrophages. (E) EdU assays showed the proliferation of tumor cells in the coculture system with 30% CM. CM derived from IL-4 and IL-4 + ISO induced supernatant of macrophages with KLF4 low and high expression. (F) The migration ability of tumor cells was determined by wound healing assays in the indicated coculture medium. (G) Transwell assays demonstrated invasion of tumor cells in the indicated coculture system. (H) Representative IHC images of p-JAK1 and p-STAT6 in xenograft tumors. (I) The phosphorylation of JAK1 and STAT6 expression in RAW264.7 treated with ADRB2 agonist or antagonist via Western blotting. (J) Immunoblotting of KLF4 in RAW264.7 treated with different conditions. Data represented as mean ± SD. Statistical analysis was performed using Student’s t test or Mann-Whitney U test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The paraffin sections of mice tumor tissues were subjected to baking, dewaxing, dehydration, elimination of endogenous peroxidase activity, tissue pressure-resistant antigen repair, 5% BSA blocking, dropping of rabbit polyclonal antibody against KLF4 (Proteintech), rabbit polyclonal antibody against CD31 (Proteintech), rabbit polyclonal antibody against p-JAK1 (ImmunoWay), rabbit polyclonal antibody against p-STAT6 (ImmunoWay), PBS washing at 4°C overnight, incubation with secondary antibody at 37°C for 35 min, PBS washing, and staining.

Techniques: Migration, Expressing, Quantitative RT-PCR, In Vitro, Knockdown, Derivative Assay, Phospho-proteomics, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet: NE level is elevated in NSCLC patients with sleep deprivation and is associated with a poor prognosis (A) The concentration of NE in serum of the NSCLC patients, determined by ELISA assay. (B) Representative IHC images and quantitative analysis of ADRB2 in the NSCLC patients. (C) Representative IHC images and quantitative analysis of KLF4 in the NSCLC patients. (D) Investigation into the association between ADRB2 expression and immune cell infiltration in patients diagnosed with LUAD. (E) Analysis of the correlation between KLF4 and immune cell infiltration in LUAD patients. (F–H) Correlation analysis between ADRB2, KLF4, JAK1, and STAT6 expression. (I and J) Correlation analysis between KLF4, JAK1, and STAT6 expression. (K and L) Correlation analysis between JAK1, STAT6, and pro-tumoral macrophages. Data represented as mean ± SD. Statistical analysis was evaluated using Student’s t test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: The paraffin sections of mice tumor tissues were subjected to baking, dewaxing, dehydration, elimination of endogenous peroxidase activity, tissue pressure-resistant antigen repair, 5% BSA blocking, dropping of rabbit polyclonal antibody against KLF4 (Proteintech), rabbit polyclonal antibody against CD31 (Proteintech), rabbit polyclonal antibody against p-JAK1 (ImmunoWay), rabbit polyclonal antibody against p-STAT6 (ImmunoWay), PBS washing at 4°C overnight, incubation with secondary antibody at 37°C for 35 min, PBS washing, and staining.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet:

Article Snippet: The paraffin sections of mice tumor tissues were subjected to baking, dewaxing, dehydration, elimination of endogenous peroxidase activity, tissue pressure-resistant antigen repair, 5% BSA blocking, dropping of rabbit polyclonal antibody against KLF4 (Proteintech), rabbit polyclonal antibody against CD31 (Proteintech), rabbit polyclonal antibody against p-JAK1 (ImmunoWay), rabbit polyclonal antibody against p-STAT6 (ImmunoWay), PBS washing at 4°C overnight, incubation with secondary antibody at 37°C for 35 min, PBS washing, and staining.

Techniques: Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Phagocytosis Assay, Sequencing, Software

Journal: Cell Death and Differentiation

Article Title: PP2Acα promotes macrophage accumulation and activation to exacerbate tubular cell death and kidney fibrosis through activating Rap1 and TNFα production

doi: 10.1038/s41418-021-00780-5

Figure Lengend Snippet: a , b Western blot assay ( a ) and semi-quantitative analysis ( b ) showing that deletion of PP2Acα enhanced LPS-stimulated Stat6 phosphorylation in BMDMs. * p < 0.05, n = 3. Data are presented as means ± SEM. c , d Western blot assay ( c ) and quantitative analysis ( d ) showing that overexpression of PP2Acα could inhibit Stat6 phosphorylation caused by PP2Acα ablation. * p < 0.05, n = 3. # p < 0.05, n = 3. Data are presented as means ± SEM. e Real-time qRT-PCR analysis showing the mRNA abundance of Tnfα in BMDMs. * p < 0.05, n = 6. # p < 0.05, n = 6. Data are presented as means ± SEM. f ELISA assay showing the protein abundance of TNFα in the cultural media of AS1517499-treated BMDMs. * p < 0.05, n = 3. # p < 0.05, n = 3. $ p < 0.05, n = 3. Data are presented as means ± SEM. g , h Representative PI staining images ( g ) and quantitative analysis ( h ) showing that the conditioned media of AS1517499-treated macrophages caused more tubular cell death. Scale bar, 100 μm. * p < 0.05, n = 3. # p < 0.05, n = 3. $ p < 0.05, n = 3. Data are presented as means ± SEM. i Real-time qRT-PCR analysis showing the mRNA abundance of Stat6 in scramble siRNA and Stat6 siRNA-transfected BMDMs. * p < 0.05, n = 3. Data are presented as means ± SEM. j ELISA analysis showing the protein abundance of TNFα in the cultural media of Stat6 siRNA-transfected BMDMs. * p < 0.05, n = 3. # p < 0.05, n = 3. $ p < 0.05, n = 3. Data are presented as means ± SEM. k , l Representative PI staining images ( k ) and quantitative analysis ( l ) showing that the conditioned media of Stat6 siRNA-transfected macrophages caused more tubular cell death. Scale bar, 100 μm. * p < 0.05, n = 3. # p < 0.05, n = 3. $ p < 0.05, n = 3. Data are presented as means ± SEM. m Representative immune staining images showing the induction of p-Stat6 (T645) in F4/80-positive macrophages within MФ-PP2Acα −/− fibrotic kidneys. White arrows indicate double-staining-positive cells. Scale bar, 10 μm. n , o Quantitative analysis for p-Stat6 (T645) and F4/80 double-positive cells in IRI ( n ) and UUO ( o ) kidney tissues. * p < 0.05, n = 3. Data are presented as means ± SEM. p , q Western blot analyses ( p ) and quantitative determination ( q ) showing that deletion of PP2Acα could induce the phosphorylation of Stat6 in macrophages sorted from MФ-PP2Acα −/− IRI kidneys. * p < 0.05, n = 3. Data are presented as means ± SEM. r , s Western blot analyses ( r ) and quantitative determination ( s ) showing that deletion of PP2Acα could induce the phosphorylation of Stat6 in macrophages sorted from MФ-PP2Acα −/− UUO kidneys. * p < 0.05, n = 3. Data are presented as means ± SEM.

Article Snippet: The primary antibodies were anti-PP2Ac (cat: 2038, Cell Signaling Technology, Boston, MA, USA, 1:1000), anti-PP2Acα (cat: ab106262, Abcam, Cambridge, UK, 1:1000), anti-PP2Acβ (cat: ab168371, Abcam, 1:1000), anti-methyl-PP2Ac (L309) (cat: ab66597, Abcam, 1:1000), anti-Itgb2 (cat: ab119830, Abcam, 1:1000), anti-Epac1 (cat: ab124162, Abcam, 1:1000), anti-FN (cat: F3648, Sigma-Aldrich, 1:10000), anti-Stat6 (cat: ab32520, Abcam, 1:1000), anti-p-Stat6 (T645) (cat: BS4186, Bioworld Technology, Nanjing, China, 1:1000), anti-Rap1a/b (cat: 4938, Cell Signaling Technology, 1:1000), anti-tubulin (cat: sc53646, Santa Cruz Biotechnology, 1:10000), and anti-GAPDH (cat: FL-335, Santa Cruz Biotechnology, 1:5000).

Techniques: Western Blot, Over Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Transfection, Double Staining