Journal: Acta Pharmaceutica Sinica. B

Article Title: Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation

doi: 10.1016/j.apsb.2021.03.011

Figure Lengend Snippet: Primers used in qPCR, DNA methylation and histone methylation.

Article Snippet: Lonicerin (C 27 H 30 O 15 , MW: 594.5, >98% purity) was purchased from Chengdu Push Biotech (Chengdu, China); 5-ASA was purchased from Ipsen Pharma (Houdan, France); Myeloperoxidase (MPO) Kit was purchased from Jiancheng Biotech (Nanjing, China); lipopolysaccharide (LPS), phorbol myristate acetate (PMA), muramyldipeptide (MDP), adenosine triphosphate (ATP), nigericin, monosodium urate crystals (MSU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA); DSS was purchased from MP Biomedicals (Solon, OH, USA); recombinant mouse macrophage colony stimulating factor (rmM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA); NLRP3, ASC, IL-1 β , pro-caspase 1, cleaved caspase-1, cleaved IL-1 β , enhancer of zeste homolog 2 (EZH2), LC3B, P62, autophagy-related gene 5 (ATG5) and ATG7 primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); trimethylation of lysine 27 on histone 3 (H3K27me3) antibody was purchased from Abcam (Cambridge, UK, USA); F4/80 antibody was purchased from Wuhan Servicebio Biotech (Wuhan, China); Annexin V-FITC/PI Apoptosis Detection Kit was purchased from ThermoFisher Scientific (Waltham, MA, USA); phosphatase inhibitor cocktail, bafilomycin A1 (BafA1) and chloroquine (CQ) were purchased from Selleck Chemicals (Houston, TX, USA); RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA); phenylmethanesulfonyl fluoride (PMSF), RIPA lysis buffer, Chromatin Immunoprecipitation (ChIP) Assay Kit, proteinase K and NP-40 lysis buffer were purchased from Beyotime Biotechnology (Shanghai, China); Genomic DNA Mimi Preparation Kit, QIAquick PCR Purification Kit and the EZ DNA Methylation-Gold Kit were purchased from Tiangen Biotech Co., Ltd. (Beijing, China); mouse and human IL-1 β , IL-18 ELISA Kit were purchased from Dakewe Biotech (Shenzhen, China); Trizol reagent, HiScript™QRTSuperMix and AceQ™qPCR SYBR® Green Master Mix were purchased from Vazyme Biotech (Nanjing, China); Enhanced Chemiluminescent Plus Reagent Kit was purchased from Yesen Biotech (Shanghai, China); rhodamine-conjugated goat anti-rat IgG (H + L) secondary antibody, β -actin and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Bioworld Technology (St Louis Park, MN, USA); the Entranster™- in vivo transfection regent was purchased from Engreen Biosystem Co., Ltd. (Beijing, China).

Techniques: DNA Methylation Assay, Methylation, Sequencing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation

doi: 10.1016/j.apsb.2021.03.011

Figure Lengend Snippet: Lonicerin promotes autophagy in macrophages. (A)–(E) The differentiated THP-1 cells and BMDMs were pre-treated with LPS (100 ng/mL) for 3 h, and then cultured with lonicerin (3, 10, and 30 μmol/L) for 6 h. The protein expressions of LC3B-II/I (A) and P62 (B) were detected by using Western blotting assay; the LC3B dot formation was detected by immunofluorescence assay, and the images were taken at 1000 × magnification (scale bar: 10 μm) (C); the formation of autophagosome was detected by using transmission electron microscopy (scale bar: 2 μm; Red arrow, the autophagosome) (D); the levels of ATG5 and ATG7 were detected by using qPCR and Western blotting assays, respectively (E). (F) The C57BL/6 mice were subjected to DSS-induced colitis model, lonicerin (3, 10, and 30 mg/kg) and 5-ASA (200 mg/kg) were orally administrated daily for consecutive 10 days. At the end of the experiment, the mice were sacrificed, the colons were collected and the protein expression of ATG5 in colons was detected by using the Western blotting assay. Data are expressed as mean ± SEM of at least three independent experiments or six mice in each group. ∗P < 0.05 and ∗∗ P < 0.01 vs . LPS group or DSS group.

Article Snippet: Lonicerin (C 27 H 30 O 15 , MW: 594.5, >98% purity) was purchased from Chengdu Push Biotech (Chengdu, China); 5-ASA was purchased from Ipsen Pharma (Houdan, France); Myeloperoxidase (MPO) Kit was purchased from Jiancheng Biotech (Nanjing, China); lipopolysaccharide (LPS), phorbol myristate acetate (PMA), muramyldipeptide (MDP), adenosine triphosphate (ATP), nigericin, monosodium urate crystals (MSU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA); DSS was purchased from MP Biomedicals (Solon, OH, USA); recombinant mouse macrophage colony stimulating factor (rmM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA); NLRP3, ASC, IL-1 β , pro-caspase 1, cleaved caspase-1, cleaved IL-1 β , enhancer of zeste homolog 2 (EZH2), LC3B, P62, autophagy-related gene 5 (ATG5) and ATG7 primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); trimethylation of lysine 27 on histone 3 (H3K27me3) antibody was purchased from Abcam (Cambridge, UK, USA); F4/80 antibody was purchased from Wuhan Servicebio Biotech (Wuhan, China); Annexin V-FITC/PI Apoptosis Detection Kit was purchased from ThermoFisher Scientific (Waltham, MA, USA); phosphatase inhibitor cocktail, bafilomycin A1 (BafA1) and chloroquine (CQ) were purchased from Selleck Chemicals (Houston, TX, USA); RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA); phenylmethanesulfonyl fluoride (PMSF), RIPA lysis buffer, Chromatin Immunoprecipitation (ChIP) Assay Kit, proteinase K and NP-40 lysis buffer were purchased from Beyotime Biotechnology (Shanghai, China); Genomic DNA Mimi Preparation Kit, QIAquick PCR Purification Kit and the EZ DNA Methylation-Gold Kit were purchased from Tiangen Biotech Co., Ltd. (Beijing, China); mouse and human IL-1 β , IL-18 ELISA Kit were purchased from Dakewe Biotech (Shenzhen, China); Trizol reagent, HiScript™QRTSuperMix and AceQ™qPCR SYBR® Green Master Mix were purchased from Vazyme Biotech (Nanjing, China); Enhanced Chemiluminescent Plus Reagent Kit was purchased from Yesen Biotech (Shanghai, China); rhodamine-conjugated goat anti-rat IgG (H + L) secondary antibody, β -actin and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Bioworld Technology (St Louis Park, MN, USA); the Entranster™- in vivo transfection regent was purchased from Engreen Biosystem Co., Ltd. (Beijing, China).

Techniques: Cell Culture, Western Blot, Immunofluorescence, Transmission Assay, Electron Microscopy, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation

doi: 10.1016/j.apsb.2021.03.011

Figure Lengend Snippet: Lonicerin inactivates NLRP3 inflammasome via autophagy. The differentiated THP-1 cells and BMDMs were transfected with ATG5 shRNA and pre-treated with LPS (100 ng/mL) for 3 h, and then cultured with lonicerin (30 μmol/L) for 6 h, followed by 1 h incubation with ATP (5 mmol/L). (A) The assembly of NLRP3 inflammasome was detected by using co-immunoprecipitation assay. (B) The protein expressions of cleaved caspase-1 and cleaved IL-1 β were detected by using Western blotting assay. (C) The production of IL-1 β and IL-18 was detected by using ELISA. Data are expressed as mean ± SEM of at least three independent experiments. ## P < 0.01 vs . normal group; ∗∗ P < 0.01 vs . LPS + ATP group; $$ P < 0.01 vs . lonicerin (30 μmol/L)+scramble shRNA group.

Article Snippet: Lonicerin (C 27 H 30 O 15 , MW: 594.5, >98% purity) was purchased from Chengdu Push Biotech (Chengdu, China); 5-ASA was purchased from Ipsen Pharma (Houdan, France); Myeloperoxidase (MPO) Kit was purchased from Jiancheng Biotech (Nanjing, China); lipopolysaccharide (LPS), phorbol myristate acetate (PMA), muramyldipeptide (MDP), adenosine triphosphate (ATP), nigericin, monosodium urate crystals (MSU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA); DSS was purchased from MP Biomedicals (Solon, OH, USA); recombinant mouse macrophage colony stimulating factor (rmM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA); NLRP3, ASC, IL-1 β , pro-caspase 1, cleaved caspase-1, cleaved IL-1 β , enhancer of zeste homolog 2 (EZH2), LC3B, P62, autophagy-related gene 5 (ATG5) and ATG7 primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); trimethylation of lysine 27 on histone 3 (H3K27me3) antibody was purchased from Abcam (Cambridge, UK, USA); F4/80 antibody was purchased from Wuhan Servicebio Biotech (Wuhan, China); Annexin V-FITC/PI Apoptosis Detection Kit was purchased from ThermoFisher Scientific (Waltham, MA, USA); phosphatase inhibitor cocktail, bafilomycin A1 (BafA1) and chloroquine (CQ) were purchased from Selleck Chemicals (Houston, TX, USA); RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA); phenylmethanesulfonyl fluoride (PMSF), RIPA lysis buffer, Chromatin Immunoprecipitation (ChIP) Assay Kit, proteinase K and NP-40 lysis buffer were purchased from Beyotime Biotechnology (Shanghai, China); Genomic DNA Mimi Preparation Kit, QIAquick PCR Purification Kit and the EZ DNA Methylation-Gold Kit were purchased from Tiangen Biotech Co., Ltd. (Beijing, China); mouse and human IL-1 β , IL-18 ELISA Kit were purchased from Dakewe Biotech (Shenzhen, China); Trizol reagent, HiScript™QRTSuperMix and AceQ™qPCR SYBR® Green Master Mix were purchased from Vazyme Biotech (Nanjing, China); Enhanced Chemiluminescent Plus Reagent Kit was purchased from Yesen Biotech (Shanghai, China); rhodamine-conjugated goat anti-rat IgG (H + L) secondary antibody, β -actin and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Bioworld Technology (St Louis Park, MN, USA); the Entranster™- in vivo transfection regent was purchased from Engreen Biosystem Co., Ltd. (Beijing, China).

Techniques: Transfection, shRNA, Cell Culture, Incubation, Co-Immunoprecipitation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation

doi: 10.1016/j.apsb.2021.03.011

Figure Lengend Snippet: Lonicerin directly binds to the EZH2. (A)–(C) The differentiated THP-1 cells and BMDMs were treated with LPS (100 ng/mL) for 3 h, and then cultured with lonicerin (3, 10, and 30 μmol/L) for 6 h. The DNA methylation on Atg5 and Atg7 promoter was detected by using the methylation-specific PCR assay, M stands for methylated and U stands for unmethylated (A); the enrichment of H3K27me3 in Atg5 and Atg7 promoter was analyzed by using the chromatin immunoprecipitation assay (B); the protein expression of EZH2 was detected by using Western blotting assay (C). (D) The differentiated THP-1 cells and BMDMs were incubated with DMSO or lonicerin (30 μmol/L) for 1 h, and the whole cell lysis was suffered from CETSA assay to detect the stabilization of EZH2. (E) The interaction between lonicerin and EZH2 was detected by using molecular docking. (F) The enzyme catalytic activity of EZH2 in vitro was detected by using the EZH2 Chemiluminescent Assay Kit. (G) The differentiated THP-1 cells were transfected with Tyr111, His129, Tyr658, Arg685 mutation plasmids and then treated with DMSO or lonicerin (30 μmol/L) for 1 h. The interaction between lonicerin and EZH2 was detected by using CETSA assay. Data are expressed as mean ± SEM of at least three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01 vs . LPS group.

Article Snippet: Lonicerin (C 27 H 30 O 15 , MW: 594.5, >98% purity) was purchased from Chengdu Push Biotech (Chengdu, China); 5-ASA was purchased from Ipsen Pharma (Houdan, France); Myeloperoxidase (MPO) Kit was purchased from Jiancheng Biotech (Nanjing, China); lipopolysaccharide (LPS), phorbol myristate acetate (PMA), muramyldipeptide (MDP), adenosine triphosphate (ATP), nigericin, monosodium urate crystals (MSU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA); DSS was purchased from MP Biomedicals (Solon, OH, USA); recombinant mouse macrophage colony stimulating factor (rmM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA); NLRP3, ASC, IL-1 β , pro-caspase 1, cleaved caspase-1, cleaved IL-1 β , enhancer of zeste homolog 2 (EZH2), LC3B, P62, autophagy-related gene 5 (ATG5) and ATG7 primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); trimethylation of lysine 27 on histone 3 (H3K27me3) antibody was purchased from Abcam (Cambridge, UK, USA); F4/80 antibody was purchased from Wuhan Servicebio Biotech (Wuhan, China); Annexin V-FITC/PI Apoptosis Detection Kit was purchased from ThermoFisher Scientific (Waltham, MA, USA); phosphatase inhibitor cocktail, bafilomycin A1 (BafA1) and chloroquine (CQ) were purchased from Selleck Chemicals (Houston, TX, USA); RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA); phenylmethanesulfonyl fluoride (PMSF), RIPA lysis buffer, Chromatin Immunoprecipitation (ChIP) Assay Kit, proteinase K and NP-40 lysis buffer were purchased from Beyotime Biotechnology (Shanghai, China); Genomic DNA Mimi Preparation Kit, QIAquick PCR Purification Kit and the EZ DNA Methylation-Gold Kit were purchased from Tiangen Biotech Co., Ltd. (Beijing, China); mouse and human IL-1 β , IL-18 ELISA Kit were purchased from Dakewe Biotech (Shenzhen, China); Trizol reagent, HiScript™QRTSuperMix and AceQ™qPCR SYBR® Green Master Mix were purchased from Vazyme Biotech (Nanjing, China); Enhanced Chemiluminescent Plus Reagent Kit was purchased from Yesen Biotech (Shanghai, China); rhodamine-conjugated goat anti-rat IgG (H + L) secondary antibody, β -actin and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Bioworld Technology (St Louis Park, MN, USA); the Entranster™- in vivo transfection regent was purchased from Engreen Biosystem Co., Ltd. (Beijing, China).

Techniques: Cell Culture, DNA Methylation Assay, Methylation, Chromatin Immunoprecipitation, Expressing, Western Blot, Incubation, Lysis, Activity Assay, In Vitro, Transfection, Mutagenesis

Journal: Acta Pharmaceutica Sinica. B

Article Title: Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation

doi: 10.1016/j.apsb.2021.03.011

Figure Lengend Snippet: Lonicerin significantly induces the expression of ATG5 and inhibits the activation of NLRP3 inflammasome via targeting EZH2. (A and B) The differentiated THP-1 cells were transfected with Tyr111, His129, Tyr658, Arg685 mutation plasmids and pre-treated with LPS (100 ng/mL) for 3 h, and then cultured with lonicerin (30 μmol/L) for 6 h. The protein expressions of ATG5, LC3B-II/I were detected by using Western blotting assay (A); the mRNA expression of ATG5 was detected by using qPCR assay (B). (C) and (D) The differentiated THP-1 cells were transfected with Tyr111, His129, Tyr658, Arg685 mutation plasmids and pre-treated with LPS (100 ng/mL) for 3 h, and then cultured with lonicerin (30 μmol/L) for 6 h, followed by 1 h incubation with ATP (5 mmol/L). The protein expressions of cleaved caspase-1 and cleaved IL-1 β were detected by using Western blotting assay (C); the production of IL-1 β was detected by using ELISA (D). Data are expressed as mean ± SEM of at least three independent experiments. ## P < 0.01 vs . normal group; ∗∗ P < 0.01 vs . LPS + ATP group; $ P < 0.05, $$ P < 0.01 vs . lonicerin (30 μmol/L)+vector group.

Article Snippet: Lonicerin (C 27 H 30 O 15 , MW: 594.5, >98% purity) was purchased from Chengdu Push Biotech (Chengdu, China); 5-ASA was purchased from Ipsen Pharma (Houdan, France); Myeloperoxidase (MPO) Kit was purchased from Jiancheng Biotech (Nanjing, China); lipopolysaccharide (LPS), phorbol myristate acetate (PMA), muramyldipeptide (MDP), adenosine triphosphate (ATP), nigericin, monosodium urate crystals (MSU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA); DSS was purchased from MP Biomedicals (Solon, OH, USA); recombinant mouse macrophage colony stimulating factor (rmM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA); NLRP3, ASC, IL-1 β , pro-caspase 1, cleaved caspase-1, cleaved IL-1 β , enhancer of zeste homolog 2 (EZH2), LC3B, P62, autophagy-related gene 5 (ATG5) and ATG7 primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); trimethylation of lysine 27 on histone 3 (H3K27me3) antibody was purchased from Abcam (Cambridge, UK, USA); F4/80 antibody was purchased from Wuhan Servicebio Biotech (Wuhan, China); Annexin V-FITC/PI Apoptosis Detection Kit was purchased from ThermoFisher Scientific (Waltham, MA, USA); phosphatase inhibitor cocktail, bafilomycin A1 (BafA1) and chloroquine (CQ) were purchased from Selleck Chemicals (Houston, TX, USA); RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA); phenylmethanesulfonyl fluoride (PMSF), RIPA lysis buffer, Chromatin Immunoprecipitation (ChIP) Assay Kit, proteinase K and NP-40 lysis buffer were purchased from Beyotime Biotechnology (Shanghai, China); Genomic DNA Mimi Preparation Kit, QIAquick PCR Purification Kit and the EZ DNA Methylation-Gold Kit were purchased from Tiangen Biotech Co., Ltd. (Beijing, China); mouse and human IL-1 β , IL-18 ELISA Kit were purchased from Dakewe Biotech (Shenzhen, China); Trizol reagent, HiScript™QRTSuperMix and AceQ™qPCR SYBR® Green Master Mix were purchased from Vazyme Biotech (Nanjing, China); Enhanced Chemiluminescent Plus Reagent Kit was purchased from Yesen Biotech (Shanghai, China); rhodamine-conjugated goat anti-rat IgG (H + L) secondary antibody, β -actin and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Bioworld Technology (St Louis Park, MN, USA); the Entranster™- in vivo transfection regent was purchased from Engreen Biosystem Co., Ltd. (Beijing, China).

Techniques: Expressing, Activation Assay, Transfection, Mutagenesis, Cell Culture, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation

doi: 10.1016/j.apsb.2021.03.011

Figure Lengend Snippet: Lonicerin attenuates colitis through regulating the EZH2/ATG5-autophagy/NLRP3 inflammasome activation. The C57BL/6 mice were subjected to the DSS-induced colitis. The lonicerin (30 mg/kg) were orally administrated daily for consecutive 10 days. The scramble plasmid and EZH2 plasmid was mixed with equal volume Entranster in vivo transfection reagent, and intracolonically (i.c.) administered daily for consecutive 10 days. (A) The body weight change, (B) the disease activity index (DAI), (C) the colon length, (D) the spleen index, (E) the myeloperoxidase (MPO) activity in colons was measured. (F) The pathological changes of colons were detected by using H&E staining, and the images were taken at 200 × magnification (scale bar: 50 μm). (G) The infiltration of macrophages in colons was detected by using immunofluorescence assay, and the images were taken at 200 × magnification (Scale bar: 50 μm). (H) and (I) The protein expressions of ATG5, LC3B–I/II and cleaved-caspase-1 in colons were detected by using Western blotting assay. Data are expressed as mean ± SEM of six mice in each group; ## P < 0.01 vs. normal group; ∗∗ P < 0.01 vs. DSS group. $$ P < 0.01 vs . lonicerin (30 mg/kg)+scramble plasmid group.

Article Snippet: Lonicerin (C 27 H 30 O 15 , MW: 594.5, >98% purity) was purchased from Chengdu Push Biotech (Chengdu, China); 5-ASA was purchased from Ipsen Pharma (Houdan, France); Myeloperoxidase (MPO) Kit was purchased from Jiancheng Biotech (Nanjing, China); lipopolysaccharide (LPS), phorbol myristate acetate (PMA), muramyldipeptide (MDP), adenosine triphosphate (ATP), nigericin, monosodium urate crystals (MSU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA); DSS was purchased from MP Biomedicals (Solon, OH, USA); recombinant mouse macrophage colony stimulating factor (rmM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA); NLRP3, ASC, IL-1 β , pro-caspase 1, cleaved caspase-1, cleaved IL-1 β , enhancer of zeste homolog 2 (EZH2), LC3B, P62, autophagy-related gene 5 (ATG5) and ATG7 primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); trimethylation of lysine 27 on histone 3 (H3K27me3) antibody was purchased from Abcam (Cambridge, UK, USA); F4/80 antibody was purchased from Wuhan Servicebio Biotech (Wuhan, China); Annexin V-FITC/PI Apoptosis Detection Kit was purchased from ThermoFisher Scientific (Waltham, MA, USA); phosphatase inhibitor cocktail, bafilomycin A1 (BafA1) and chloroquine (CQ) were purchased from Selleck Chemicals (Houston, TX, USA); RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA); phenylmethanesulfonyl fluoride (PMSF), RIPA lysis buffer, Chromatin Immunoprecipitation (ChIP) Assay Kit, proteinase K and NP-40 lysis buffer were purchased from Beyotime Biotechnology (Shanghai, China); Genomic DNA Mimi Preparation Kit, QIAquick PCR Purification Kit and the EZ DNA Methylation-Gold Kit were purchased from Tiangen Biotech Co., Ltd. (Beijing, China); mouse and human IL-1 β , IL-18 ELISA Kit were purchased from Dakewe Biotech (Shenzhen, China); Trizol reagent, HiScript™QRTSuperMix and AceQ™qPCR SYBR® Green Master Mix were purchased from Vazyme Biotech (Nanjing, China); Enhanced Chemiluminescent Plus Reagent Kit was purchased from Yesen Biotech (Shanghai, China); rhodamine-conjugated goat anti-rat IgG (H + L) secondary antibody, β -actin and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Bioworld Technology (St Louis Park, MN, USA); the Entranster™- in vivo transfection regent was purchased from Engreen Biosystem Co., Ltd. (Beijing, China).

Techniques: Activation Assay, Plasmid Preparation, In Vivo, Transfection, Activity Assay, Staining, Immunofluorescence, Western Blot

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A) Schematic representation of Rab11a-regulated recycling in mock- and IAV-infected cells. In mock-infected cells, Rab11a endosomes are involved in recycling material from several organelles to the plasma membrane. Upon IAV infection, progeny vRNPs bind to Rab11a endosomes and start concentrating at ERES (steps 1–2) to form liquid viral inclusions by a mechanism ill-defined (step 3). The ER likely facilitates formation of viral inclusions to promote assembly of the 8-vRNP genome (step 3). How assembled genomes reach the plasma membrane is unknown (step 4). (B) Cells (GFP-Rab11a WT low and DN low ) were infected or mock-infected with PR8 virus for 12 h at an MOI of 3. The levels of Tf-Alexa647-fluorescent conjugates were quantified inside cells and at the cell surface by flow cytometry upon 5, 10, and 15 min of incubation at 37°C. Results were plotted as the percentage (%) of recycled Tf as a function of time of incubation. Values were normalized to the mock condition at 15 min of incubation. Three pooled independent experiments are shown. Statistical analysis was done by two-way ANOVA, followed by a Sidak’s multiple comparisons test (** p < 0.01). (C) The levels of Tf were quantified inside cells and at the cell surface by flow cytometry at 15 min of incubation at 37°C. Results were plotted as the percentage (%) of recycled Tf as a function of cell type. Four pooled independent experiments are shown. Statistical analysis was done by one-way ANOVA, followed by a Tukey’s multiple comparisons test (** p < 0.01, *** p < 0.001). (D) Cells (GFP-Rab11a WT low , green) were simultaneously transfected with a plasmid encoding mCherry tagged to the ER (magenta) and infected or mock-infected with PR8 virus for 12 h at an MOI of 10. Cells were imaged under time-lapse conditions at 12 h postinfection. Representative cells are shown on the left. The respective individual frames with single moving particles are shown in the small panels on the right. The yellow arrowheads highlight fusion/fission events of viral inclusions (green), as well as their interaction with the ER (magenta). Bar = 10 μm. Images were extracted from and Videos. (E) A linescan was drawn as indicated to assess Rab11a dynamics associated with the ER. The fluorescence intensity of ER tubules (magenta) and Rab11a endosomes or viral inclusions (green) at indicated times was plotted against the distance (in μm). Representative analysis was performed using images from ( D ). Experiments were performed twice. For each condition, at least 10 cells were analyzed. All the values of individual and pooled experiments are provided in File. ER, endoplasmic reticulum; ERES, ER exit site; IAV, influenza A virus; MOI, multiplicity of infection; Tf, transferrin; vRNP, viral ribonucleoprotein.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Clinical Proteomics, Membrane, Virus, Flow Cytometry, Incubation, Transfection, Plasmid Preparation, Fluorescence

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A) Schematic representation of a liquid viral inclusion according to 2D light and electron microscopy analysis. (B) Schematic representation of how 4 sequential tomograms (of 120 nm each) were acquired and stitched together (approximately 480 nm total thickness). (C, D) Cells (GFP-Rab11a WT low ) were infected or mock-infected with PR8 virus for 12 h at an MOI of 3. Cells were processed by high-pressure freezing/freeze substitution and imaged by ET-TEM. Representative cells are shown with 3 individual sections (including section height in nm) and the 3D cumulative model. Bar = 500 nm. Images were extracted from , , , and . Abbreviations: pm, plasma membrane (gray); er, endoplasmic reticulum (blue); v, budding virions (pink); m, mitochondria (purple); smv, single-membrane vesicle (light green); dmv, double-membrane vesicle (yellow); *, ER dilation (dark green). (E) Photomontages of single sections covering an entire plane of the cell were acquired by TEM at several times postinfection (4–16 h), as exemplified here at 16 h postinfection. The photomontages were used to score the number of single (smv) and double (dmv) membrane vesicles in ( F ) and ( G ). Bar = 2 μm. (F, G) The number of single (smv) and double (dmv) membrane vesicles was manually scored and plotted as a function of time of infection. Statistical analysis was done by Kruskal–Wallis test (* p < 0.05). On average, 10 cells were analyzed per condition ( C - G ). Experiments were performed twice. All the values of individual and pooled experiments are provided in File. ER, endoplasmic reticulum; ET, electron tomography; MOI, multiplicity of infection; TEM, transmission electron microscopy; 2D, 2-dimensional; 3D, 3-dimensional.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Electron Microscopy, Infection, Virus, Clinical Proteomics, Membrane, Tomography, Transmission Assay

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: Cells (A549) were treated with siRNA non-targeting (siNT) or targeting ULK1 (siULK1), ULK2 (siULK2), TBC1D14 (siTBC1D14), ATG2A (siATG2A), or ATG9A (siATG9A) for 48 h and then infected or mock-infected (M) with PR8 virus for 8 h, at an MOI of 3. (A) Viral production was determined at 8 h postinfection by plaque assay and plotted as PFU per mL ± SEM. Data were pooled from 3–6 independent experiments. Statistical analysis was done by one-way ANOVA, followed by a Dunnett’s multiple comparisons test (*** p < 0.001). (B) The mRNA level of ULK1/2 and TBC1D14 before infection was quantified by real-time RT-qPCR and plotted as the relative expression to GAPDH mRNA level ± SEM. Expression was normalized to siNT from mock-infected cells. Data are a pool from 3 independent experiments. Statistical analysis was done by Student t test (*** p < 0.01). (C) Protein levels of ATG2A and ATG9A before infection were determined by western blotting and plotted as the relative expression to actin protein levels ± SEM. Expression was normalized to siNT from mock-infected cells. Data are a pool from 3–6 independent experiments. Statistical analysis was done by Student t test (*** p < 0.01). The original uncropped blots can be found in Images. (D) Localisation of Rab11a (gray), tubulin (green), and viral NP (magenta) proteins at 8 h postinfection was determined by immunofluorescence using antibody staining. Viral inclusions/vRNPs/Rab11a are highlighted by white boxes. Cell periphery and nuclei (blue, Hoechst staining) are delineated by yellow and white dashed lines, respectively. Bar = 10 μm. The figure panels for the corresponding mock-infected cells can be found in . (E) A schematic representation of shape classification based on circularity versus roundness is shown. (F) The roundness and circularity of viral inclusions/vRNPs, marked by NP staining, were determined at 8 h postinfection using the Shape Descriptor tool (Image J, NIH) and plotted against each other for siNT and siATG9A-treated cells. The maximum value of roundness and circularity (1) corresponds to a circular structure, whereas the minimum value represents a linear structure (0). More than 80 cells, pooled from 3 independent experiments, were analyzed per condition. Statistical analysis was done by Mann–Whitney test (*** p < 0.001). A similar analysis done for the other autophagy factors is shown in . The frequency distribution of roundness and circularity of viral inclusions/vRNPs is shown in . (G-I) Protein levels of LC3-I and LC3-II were quantified by western blotting and plotted as the relative expression to actin protein levels ± SEM. Expression was normalized to siNT from mock-infected (M) cells. Data are a pool from 6 independent experiments. Statistical analysis was done by one-way ANOVA, followed by a Tukey’s multiple comparisons test (no statistical significance detected). All the values of individual and pooled experiments are provided in File, and the original uncropped blots can be found in Images. IAV, influenza A virus; MOI, multiplicity of infection; NP, nucleoprotein; PFU, plaque-forming unit; RT-qPCR, quantitative reverse transcription PCR; SEM, standard error of the mean; vRNP, viral ribonucleoprotein.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Virus, Plaque Assay, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, MANN-WHITNEY, Reverse Transcription

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A–C) Cells (A549) were infected or mock-infected with PR8 virus, at an MOI of 3, for the indicated times. (A) The localization of host proteins ATG9A (green) and GM130 (gray) and viral protein NP (magenta) was determined by immunofluorescence using antibodies against these proteins. Mock-infected cells were collected at the same time as the 14 h-infected cells. Nuclei (blue, Hoechst staining) and cell periphery are delimited by white and yellow dashed lines, respectively. Bar = 10 μm. (B) Colocalization between ATG9A and GM130 in the images acquired in ( A ) was determined using the Colocalization Threshold analysis tool (FIJI/Image J, NIH) and plotted as the Pearson R value. Approximately 30 cells, from a single experiment, were analyzed per experimental condition. Red bar represents the median of values. Statistical analysis was done by Kruskal–Wallis test (** p > 0.01; *** p > 0.001). (C) The levels of ATG9A, actin, and viral NP protein in cell lysates at the indicated time points were determined by western blotting. ATG9A band intensity was quantified using FIJI (ImageJ, NIH) and normalized to actin levels. Original blots can be found in Images. Experiments ( A – C ) were performed twice. (D–F) Cells (A549) were transfected with a plasmid encoding GFP-ATG9A for 24 h and then infected or mock-infected with PR8 virus, at an MOI of 10, for 8 h. The localization of endogenous host proteins (GM130 –Golgi, Calnexin–ER, or Rab11a –recycling endosome) and viral protein NP was determined by immunofluorescence using antibodies against these proteins. Nuclei (blue or gray, Hoechst staining) and cell periphery are delimited by white and yellow dashed lines, respectively. Yellow arrowheads highlight areas of contact between viral inclusions and overexpressed GFP-ATG9A protein. Red arrowheads highlight areas of colocalization between GFP-ATG9A and the Golgi marker GM130. Bar = 10 μm. Control cells expressing GFP alone can be found in . Experiments ( D – F ) were performed twice. All the values of individual and pooled experiments are provided in File. ER, endoplasmic reticulum; GFP, green fluorescent protein; IAV, influenza A virus; MOI, multiplicity of infection; NP, nucleoprotein; TGN, trans -Golgi network.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Virus, Immunofluorescence, Staining, Western Blot, Transfection, Plasmid Preparation, Marker, Control, Expressing

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A–C) Cells (GFP-Rab11a WT low or GFP-Rab11a DN low ) were treated with siRNA non-targeting (siNT) or targeting ATG9A (siATG9A) for 48 h and then infected or mock-infected with PR8 virus for 10 h, at an MOI of 3. (A) Viral production was determined by plaque assay and plotted as PFU per milliliter (mL) ± SEM. Data represent 6 replicates from a single experiment. Two independent experiments were performed. Statistical analysis was done by one-way ANOVA, followed by a Kruskal–Wallis test (* p < 0.05; *** p < 0.001). (B) The protein level of ATG9A, lamin B, GFP, and Rab11a before infection were quantified by western blotting. The levels of ATG9A were plotted as the relative expression to lamin B level ± SEM. Expression was normalized to siNT from mock-infected cells. The data are a pool from 3 independent experiments. Statistical analysis was done by unpaired t test between siNT vs. siATG9A conditions of each condition (Rab11a WT vs. DN mock; *** p < 0.01). (C) Localisation of Rab11a (magenta) and PDI (gray) at 10 h postinfection was determined by immunofluorescence using antibody staining. Viral inclusions/Rab11a are highlighted by white boxes. Cell periphery and nuclei (blue, Hoechst staining) are delineated by yellow and white dashed lines, respectively. Mock-infected cells can be found in . Bar = 10 μm. (D–F) Cells (A549) were treated with siRNA non-targeting (siNT) or targeting ATG9A (siATG9A) for 48 h and then infected or mock-infected with PR8 virus for 8 h, at an MOI of 3. (D) The localisation of host Rab11a (green) and viral NP (magenta) proteins at 8 h postinfection was determined by immunofluorescence using antibody staining. Viral inclusions/vRNPs are highlighted by white boxes. Cell periphery and nuclei (blue, Hoechst staining) are delineated by yellow and white dashed lines, respectively. Bar = 10 μm. Experiments were performed twice. (E) Colocalization between Rab11a and NP in the images acquired in ( D ) was determined using the Colocalization Threshold analysis tool (Image J, NIH) and plotted as the Pearson R value. At least 20 cells, pooled from 2 independent experiments, were analyzed per experimental condition. Red bar represents the median of values. Statistical analysis was done by Mann–Whitney test (n.s., not significant). (F) The roundness and circularity of Rab11a structures in the images acquired in ( D ) were determined using the Shape Descriptor tool (Image J, NIH) and plotted against each other. The maximum value of roundness and circularity (1) corresponds to a circular structure, whereas the minimum value represents a linear structure (0). Approximately 30 cells, from 2 independent experiments, were analyzed per condition. Statistical analysis was done by Mann–Whitney test (*** p < 0.001). The frequency distribution of roundness and circularity of structures marked by Rab11a is shown in . All the values of individual and pooled experiments are provided in File. GFP, green fluorescent protein; IAV, influenza A virus; MOI, multiplicity of infection; NP, nucleoprotein; PFU, plaque-forming unit; SEM, standard error of the mean; vRNP, viral ribonucleoprotein.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Virus, Plaque Assay, Western Blot, Expressing, Immunofluorescence, Staining, MANN-WHITNEY

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A–D) Cells (GFP-Rab11a WT low , magenta) were treated with siRNA non-targeting (siNT) or targeting ATG9A (siATG9A) for 48 h. Upon this period, cells were infected or mock-infected with PR8 virus for 8 h, at an MOI of 3, and simultaneously treated with 200 nM Sir-Tubulin dye to stain the microtubules (green) in live cells. Cells were imaged for 10 min (2 s/frame) under time-lapse conditions at 8 h postinfection. White boxes show viral inclusions/Rab11a. Individual frames with single moving particles highlighted with yellow arrows are shown in the small panels. Bar = 10 μm. Images from selected infected cells were extracted from and Videos. Images from mock-infected cells were extracted from and Videos. For each case, a linescan was drawn as indicated to assess the dynamics of Rab11a and tubulin. The fluorescence intensity of Rab11a endosomes or viral inclusions (magenta) and tubulin (green) at indicated times was plotted against the distance (in μm). Representative analysis was performed using images from ( A - D ). (E, F) Cells (GFP-Rab11a WT low ) were treated as explained above (in A–D). At 8 h postinfection, cells were treated with DMSO or 10 μg/mL of nocodazole for 2 h. Cells were imaged at 10 h postinfection. White boxes show viral inclusions/Rab11a. Bar = 10 μm. (G) Scheme illustrates how viral inclusion/Rab11a endosome deviation from a reference position (in X and Y direction) was tracked by live cell imaging. The formula used to quantify the mean squared displacement (MSD, μm 2 ) is also shown. (H) Each viral inclusion/Rab11a endosome in a cell was tracked using the TrackMate plugin (FIJI, NIH) and displacement was quantified as explained in ( G ). Data were plotted as the MSD (μm 2 ) per treatment. The red dot indicates the median in the boxplots. Statistical analysis was done by a Kruskal–Wallis test (*** p < 0.001). (I) Colocalization between microtubules (tubulin) and viral inclusions (Rab11a) in live cells was determined as the Manders’ Overlap Coefficient tM1 (thresholded; explained in Methods section). Only infected conditions are shown. Given that very small Rab11a endosomes were scattered throughout the cytosol in mock-infected controls, we could not obtain reliable correlation coefficients. This result is, however, corroborated by quantifying colocalization between microtubules (tubulin) and viral inclusions using a fluorescent virus (PA-mNeonGreen PR8), as shown in . Between 6 and 10 cells per condition were analyzed. Statistical analysis was done by a Student t test (*** p < 0.001). Experiments were performed twice. All the values of individual and pooled experiments are provided in File.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Virus, Staining, Fluorescence, Live Cell Imaging

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: We currently view liquid viral inclusions, composed of Rab11a endosomes and vRNPs, as sites dedicated to the assembly of the IAV genome [ , , ]. We have previously shown that liquid viral inclusions develop in close contact with the ERES . Here, we describe the initial events on the left panel that may lead to the formation of liquid viral inclusions on the right to facilitate the formation of IAV genomic complex. In this study, we demonstrate that IAV infection reduces the Rab11a-regulated recycling capacity of the host cell (step 1). This effect is likely a consequence of vRNP binding to Rab11a endosomes, which are then rerouted to the ERES to form viral inclusions. Such trafficking of Rab11a endosomes carrying the vRNPs to the ER is regulated by the host factor ATG9A. We identified that ATG9A is mobilized from the Golgi during IAV infection (step 2) and leads to the removal of Rab11a-vRNP complexes from microtubules when at the ER (step 3). It is thus possible that ATG9A moves to the ER to promote the linkage of viral inclusions to microtubules. In this location, vRNPs-Rab11a units may establish multiple and dynamic contacts forming liquid percolation-driven condensates. We also show (although with overexpression experiments) that ATG9A engages in multiple contacts with viral inclusions (step 4). We propose that the liquid properties of viral inclusions favor the formation of the 8-segmented IAV genome that is transported to the plasma membrane (step 5). ATG9A, autophagy related gene 9A; ER, endoplasmic reticulum; ERES, ER exit site; IAV, influenza A virus; vRNP, viral ribonucleoprotein.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Binding Assay, Over Expression, Clinical Proteomics, Membrane, Virus

Journal: bioRxiv

Article Title: The tubulin poly-glutamylase complex, TPGC, is required for phosphatidyl inositol homeostasis and cilium assembly and maintenance

doi: 10.1101/2025.03.03.641315

Figure Lengend Snippet: A ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr and immuno-stained with GT335, anti-acetylated tubulin and anti-Arl13b antibodies. ≥90 cells per sample were analyzed in three independent experiments. Error bars, S.D. ***p ≤ 0.001, **** p ≤ 0.0001. B ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr, and immuno-stained with antibodies against GT335, Arl13b, Myo-Va, Cep89, Rab8a, Rabin8, IFT88, Cep290 and RPGRIP1L. Scale bar, 1µm. C ) sgRNA control and TBC1D19 knockout cells were serum starved as in panels A and B and visualized by staining with GT335 and antibodies against CP110. Scale bar, 1µm. N ≥ 90 cells per sample were analyzed in three independent experiments. Error bars, S.D. D ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr, and immunostained with antibodies against Rab34 and Arl13b. Scale bar, 1µm. E ) sgRNA control and TBC1D19 knockout cells were serum starved as in panels A-C and immunostained with antibodies against GT335 and Arl13b. Scale bar, 1µm. F ) Primary cilia in sgRNA control and TBC1D19 knockout cells were examined by transmission electron microscopy (TEM) after 24 hr of serum starvation. Images of TBC1D19 knockout cilia from two consecutive sections are shown.

Article Snippet: Mouse anti-α-tubulin (1:10000 for WB 66031-1-Ig), rabbit anti-Arl13b (1:2000 for IF and 1:1000 for WB, 17711-1-AP), rabbit anti-Rab8a (1:50 for IF, 55296-1-AP), rabbit anti-IFT88 (1:500 for IF, 13967-1-AP), rabbit anti-INPP5E (1:1000 for WB and 1:1000 for IF, 17797-1-AP), mouse anti-β-actin (1:10000 for WB, 66009-1-Ig PT), mouse anti-GAPDH (1:10000 for WB, 60004-1-Ig), rabbit ant-CCP1 (1:1000 for WB, 14067-1-AP), Rabbit anti-TBC1D19 (1:1000 for WB, 21085-1-AP) and rabbit anti-Tulp3 (1:1000 for WB, 1:150 for IF, 13637-1-AP), RPGRIP1L (1:150 for IF, 55160-1-AP) were from Proteintech.

Techniques: Control, Knock-Out, Staining, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: The tubulin poly-glutamylase complex, TPGC, is required for phosphatidyl inositol homeostasis and cilium assembly and maintenance

doi: 10.1101/2025.03.03.641315

Figure Lengend Snippet: A ) Control (sgCTL) and TBC1D19 knockout cells were treated with non-targeting control or CCP1 siRNA for 48 hr and serum starved for 24 hr, and extracts were subjected to immunoblotting with the indicated antibodies. B ) TBC1D19 knockout cells were infected with lentivirus expressing Flag-map4m or Flag-map4m-TTLL1 for 72 hr, serum starved for 24 hr and subjected to immunoblotting with the indicated antibodies. C ) Control (sgCTL), TBC1D19 knockout cells, and TBC1D19 knockout cells stably expressing TTLL5-EYFP or TTLL6-EYFP were serum starved for 24 hr and subjected to immunoblotting with the indicated antibodies. D ) Control (sgCTL), TBC1D19 -/- , C11ORF49 -/- , and TTLL1 -/- cells were serum starved for 24 hr and subjected to immunoblotting with the indicated antibodies. E ) sgCTL, C11ORF49 -/- , and LRRC49 -/- cells were serum starved for 24 hr, immuno-stained with antibodies against acetylated tubulin and INPP5E. N ≥ 50 cilia were counted per sample in two independent experiments. Error bars, S.D. ns, not significant. F ) 293T cells were co-transfected with GFP or GFP-TBC1D19 with mCherry-Arl13b for 32 hr and serum starved for 16 hr. Lysates were subjected to immunoprecipitation with GFP-trap beads and immuno-blotted with antibodies against GFP and mCherry.

Article Snippet: Mouse anti-α-tubulin (1:10000 for WB 66031-1-Ig), rabbit anti-Arl13b (1:2000 for IF and 1:1000 for WB, 17711-1-AP), rabbit anti-Rab8a (1:50 for IF, 55296-1-AP), rabbit anti-IFT88 (1:500 for IF, 13967-1-AP), rabbit anti-INPP5E (1:1000 for WB and 1:1000 for IF, 17797-1-AP), mouse anti-β-actin (1:10000 for WB, 66009-1-Ig PT), mouse anti-GAPDH (1:10000 for WB, 60004-1-Ig), rabbit ant-CCP1 (1:1000 for WB, 14067-1-AP), Rabbit anti-TBC1D19 (1:1000 for WB, 21085-1-AP) and rabbit anti-Tulp3 (1:1000 for WB, 1:150 for IF, 13637-1-AP), RPGRIP1L (1:150 for IF, 55160-1-AP) were from Proteintech.

Techniques: Control, Knock-Out, Western Blot, Infection, Expressing, Stable Transfection, Staining, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: A) HeLa cells were transduced with ACE2 lentivirus to generate stable cell lines. Mock and ACE2 transduced cells were lysed and immunoblotted for ACE2. Tubulin was used as a loading control. (B) HeLa WT +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green) and DAPI (blue). (C) HeLa WT +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green), tetherin (red) and DAPI (blue). Uninfected cells shown with asterisk. (D) Electron micrographs showing plasma membrane associated SARS-CoV-2 virions and virus filled intracellular organelles. SARS-CoV-2 infected HeLa WT +ACE2 cells (MOI 0.5) were fixed at 24 hpi and processed for TEM. Left micrograph – plasma membrane-associated virus, middle micrograph – virus-filled tubulovesicular compartments are directed towards the plasma membrane, right micrograph – virions within DMVs. (E) Surface immunogold electron microscopy of SARS-CoV-2 infected HeLa WT +ACE2 cells. Cells were infected with SARS-CoV-2 (MOI 0.5), fixed at 24 hpi and immunogold labelled with antibodies against tetherin. (F) As (E) but labelled with antibodies against SARS-CoV-2 spike.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Transduction, Stable Transfection, Control, Infection, Staining, Clinical Proteomics, Membrane, Virus, Electron Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: (A) A549 cells were transduced with ACE2 lentivirus to generate stable cell lines. Mock and ACE2 transduced cells were lysed and immunoblotted for ACE2. Tubulin served as a loading control. (B) A549+ACE2 cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green) and DAPI (blue). Uninfected cells shown with asterisk (C) A549+ACE2 cells were treated with IFNα (1000 U/ml, 24 hours) to upregulate tetherin expression. Cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green), tetherin (red) and DAPI (blue). Uninfected cells shown with asterisk. (D) A549+ACE2 cells were treated with IFNα (1000 U/ml) and infected with SARS-CoV-2 (MOI 0.5), fixed at 24 hpi and processed for TEM. Infected cells display very few virions on their plasma membrane (left inset) but significant DMV formation (right inset). (E) Electron micrograph of the perinuclear region of A549+ACE2 mock infected cells. Zoomed area shows typical Golgi morphology. (F) Electron microscopy of the perinuclear region of SARS-CoV-2 infected A549+ACE2 cells. Cells were infected at an MOI of 0.5 and fixed at 24 hpi. Zoomed area shows membrane rearrangements and DMVs. (G) T84 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi and stained for spike (green) and tetherin (red). (H) T84 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi and processed for TEM. Tethered virions were frequently present at the plasma membrane.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Transduction, Stable Transfection, Control, Infection, Staining, Expressing, Clinical Proteomics, Membrane, Electron Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: (A) Lentiviral ACE2 was used to generate stable HeLa Bst2KO +ACE2 cells, and ACE2 expression was verified by Western blotting. GAPDH served as a loading control. (B) HeLa WT +ACE2 and HeLa Bst2KO +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi. Cells were stained for spike (green) to demonstrate infection with SARS-CoV-2, and tetherin (red). (C) High MOI viral growth curves were performed by infecting HeLa WT +ACE2 and HeLa Bst2KO +ACE2 cells with SARS-CoV-2 at MOI (5). Titres were measured by plaque assays. (D) Low MOI viral growth curves were performed by infecting HeLa WT +ACE2 and HeLa Bst2KO +ACE2 cells with SARS-CoV-2 at MOI (1). Titres were measured by plaque assays.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Expressing, Western Blot, Control, Infection, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: (A) Schematic diagram to illustrate the domain organization of SARS-CoV-1 ORF7a and SARS-CoV-2 ORF7a. SP = signal peptide. TM = transmembrane domain. Expanded region below shows regions flanking the transmembrane domain with amino acid differences (*) and deletions (-) between SARS-CoV-1 ORF7a and SARS-CoV-2 ORF7a. (B) Representative confocal immunofluorescence images of HeLa cells transiently transfected with SARS-CoV-1 ORF7a-FLAG or SARS-CoV-2 ORF7a-FLAG. SARS-CoV-1 ORF7a-FLAG predominately colocalizes with TGN46 (red), while SARS-CoV-2 ORF7a-FLAG shows additional staining outside that colocalizing with TGN46 (arrowheads). (C) Manders’ coefficients were calculated to measure the ORF7a-FLAG overlap with TGN46. n = 3 independent experiments. Means of each independent experiment are plotted. Two-tailed, unpaired t-tests were performed. *** p < 0.001. (D) SARS-CoV-2 infected HeLa WT +ACE2 cells display fragmentation of Golgi markers. HeLa WT +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi. Infected cells were identified by spike staining (green) and cells were costained with Golgi markers TGN46 (top) and ZFPL1 (below). Areas of TGN46 and ZFPL1 are enlarged (right) to highlight Golgi fragmentation in SARS-CoV-2 infected cells.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Immunofluorescence, Transfection, Staining, Two Tailed Test, Infection

Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a Schematic illustrating the SARS-COV-2 genomic landscape and the deletions/substitutions in ΔE G /ΔE G 68, main structural and accessory proteins indicated. Four overlapping fragments covering the whole SARS-CoV-2 genome were amplified by PCR (Fragments A-D, see also Supplementary Fig. ). b Complementation efficiency of Vero-E2T cells, analyzed by FFU (focus forming units) quantification after infection with ΔE G 3* (ΔE G with an additional stop codon in ORF3a) at different multiplicities of infection (MOI) or medium-only control (ctrl) three and six days post-infection ( n = 2 individual cultures), for corresponding genome copies, see Supplementary Fig. . c Passaging of 1:10 and 1:100 (after p2) dilutions of cell-free supernatant (Input = Passage 0) of wild-type SARS-CoV-2 (Muc-1, B.1), ΔE G 3* and ΔE G 68 on non-complementing Vero E6 cells (initial infection MOI = 1). Data from one representative experiment are shown; analysis was performed in duplicates. d Transmission electron microscopy analysis of recombinant wild-type SARS-CoV-2 (rCoV2) or vaccine candidates ΔE G and ΔE G 68 showing the presence of the characteristic spike protein (indicated with arrows). e Immunoblot analysis of viral protein production in Vero E6-TMPRSS2 cells infected for 24 h with rCoV2, E**fs, ΔE G 3*, ΔE G 68 or medium only (ctrl), probed with anti-NSP2, anti-N, anti-S, anti-ORF3a (full-length [fl] and truncated [tr] forms indicated with arrows), anti-ORF6, anti-ORF7a, anti-ORF8, and anti-beta-actin (β-ACT) antibodies. f Detection of N and S (magenta), F-actin (green), nuclei (blue) and ORF6 or ORF8 in Vero E6-TMPRSS2 cells infected with rCoV2, E**fs, ΔE G 3* or ΔE G 68. Scale bar is 100 nm in ( d ), 50 µm and 20 µm in ( f ) (overview and ROI images, respectively).

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), rabbit polyclonal anti-SARS-CoV Nucleocapsid protein (Rockland; 200-401-A50; RRID:AB_828403), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche ), sheep polyclonal anti-SARS-CoV-2 ORF3a , rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto ), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).

Techniques: Amplification, Infection, Control, Passaging, Transmission Assay, Electron Microscopy, Recombinant, Western Blot

Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a – c Modulation after transfection: Flow cytometry staining of THP-1 cells for HLA-A/B/C, CD80, CD275, and HLA-DR surface expression 48 h after transfection with expression plasmids for ORF6 ( a ), ORF8 ( b ), or Envelope ( c ) proteins, compared with control transfection. d – i Modulation after infection: d A549-ACE2-TMPRSS2 cells were infected with recombinant wild-type (rCoV2), E**fs, ΔE G 68, or XBB.1.5 SARS-CoV-2 virus (MOI = 0.1) for 24 h and stained for HLA-A/B/C, CD44 and CD275. e Median fluorescence intensity (MFI) of HLA-A/B/C and CD275. The same infection was conducted on HEK293T-ACE2 and their respective supernatant was then applied on THP-1 for 48 h before surface staining and analysis. f Histogram showing the expression of CD44, HLA-A/B/C, CD80, CD275, and HLA-DR on THP-1 after 48 h. g Median fluorescence intensity of CD44, HLA-A/B/C, CD80, and CD275 markers on THP-1 after 48 h incubation. h Comparison of wild-type or ΔE G 68 conditions for their expression of CD80 and HLA-A/B/C. The frequency of cells inside the gate in ( h ) is shown in ( i ). Median is shown for ( e ) and ( g ), mean and S.D. for ( i ). The gating strategy is shown in Supplementary Fig. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), rabbit polyclonal anti-SARS-CoV Nucleocapsid protein (Rockland; 200-401-A50; RRID:AB_828403), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche ), sheep polyclonal anti-SARS-CoV-2 ORF3a , rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto ), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).

Techniques: Transfection, Flow Cytometry, Staining, Expressing, Control, Infection, Recombinant, Virus, Fluorescence, Incubation, Comparison

Journal: Chinese medicine

Article Title: Combination of mangiferin and T0901317 targeting autophagy promotes cholesterol efflux from macrophage foam cell in atherosclerosis.

doi: 10.1186/s13020-023-00876-9

Figure Lengend Snippet: Fig. 3 Autophagy is implicated in lipid droplet degradation under combined treatment of T0 and MGF. Representative digital images of electron microscopy reveal autophagic vacuoles accumulating in the cytoplasm of peritoneal macrophages (A) and RAW264.7 cells (B). Scale bar, 50 μm, 1 μm, 500 nm. Expression of p-mTOR, mTOR, p-AMPK, and AMPK in peritoneal macrophages (C) and RAW264.7 cells (D) was determined by western blot with total proteins extracted from cell samples. Expression of p62, LC3, Beclin1 and ATG5 in peritoneal macrophages (E) and RAW264.7 cells (F) was determined by western blot with total proteins extracted from cell samples

Article Snippet: Primary rabbit polyclonal antibodies against α-SMA (14395), CD36 (18836), Beclin1 (11306), ATG5 (10181), and ATG7 (10088) were purchased from Proteintech Group, Inc. (Rosemont, IL, USA).

Techniques: Electron Microscopy, Expressing, Western Blot

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a – e Schematic diagrams of the brain of mouse ( a ), and the red box in the cerebral cortex shows the location where the images were taken. Immunofluorescence double labelling ( b , c , 2 double-labelled neurons are indicated as examples in ( b , c )) and quantification ( d , e , n = 6 images from 3 mice) of GDF11 (green, b ) and NeuN (red, b ) or GDF11 (green, c ) and CaMKIIα (red, c ) in the cerebral cortices of the mice aged 3 months (3 M). f Representative images of immuno-electron microscopy (Immuno-EM) of GDF11 labelled with nanogold particles (there are many GDF11 labelled black dots and only some examples are indicated with red arrows) in the cerebral cortex of the mice aged 3 M ( n = 3 mice). Nuc, nucleus; Den, dendrite. g Immunofluorescence double labelling of GDF11 (green, arrow) and GABA (red, double arrowheads) ( n = 3 mice). h Immunofluorescence double labelling of GDF11 (green) together with Olig2 (red, left), GFAP (red, middle), Iba1 (red, middle) in the cerebral cortex (Cx) and Dcx (red, right) in the dentate gyrus (DG) of the mice aged 3 M ( n = 3 mice). The GDF11 negative cells are indicated by arrows in ( h ). i Schematic diagrams of the brain of the marmoset (one aged 62 M and another aged 70 M), and the red box in the cerebral cortex shows the location of the images ( n = 2 marmosets). j – o Immunofluorescence double labelling ( j , m , n , o ) and quantification ( k , l ) of GDF11 (green) together with CaMKIIα (red, j , k , l , 2 double-labelled neurons are indicated as examples in ( j ); n = 8 images from 2 marmosets) or GABA (red, m ), Olig2 (red, n ) or GFAP (red, o ). The GDF11 negative cells are indicated by arrows in ( m , n , q ). p Schematic diagrams of the human brain. The red box in the cerebral cortex shows the location of the images. q – s Immunofluorescence double labelling ( q , male patient aged 24 years (Y) and female patient aged 23Y diagnosed with intractable epilepsy and the focus of epileptic cortices had to be removed surgically) and quantification ( r , s , n = 4 patients, male patient aged 23Y, male patient aged 52Y, female patient aged 54Y and male patient aged 60Y suffered brain injury) of GDF11 (green) together with CaMKIIα (red) in the cerebral cortex of patients and 2 double-labelled neurons are indicated by arrows in ( q ). t Immunofluorescence double labelling of GDF11 (green) together with GABA (red, left), Olig2 (red, middle), GFAP (red, middle) and Iba1 (red, right) in the cerebral cortex of patients ( n = 4 patients). The GDF11 negative cells are indicated by arrows in ( t ). Scale bars, as shown on the images, 30 μm ( b , c ), 250 nm ( f ), 10 μm ( g ), 40 μm ( j , m , n , o ), 20 μm ( h , q , t ). Data are presented as mean ± SEM. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Immunofluorescence, Immuno-Electron Microscopy

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Quantification by qPCR of the relative mRNA of GDF11 in the brain of the WT mice aged 3 M, 9 M or 36 M ( n = 3 mice/group). b Immunofluorescence double labelling of GDF11 (green) and CaMKIIα (red) in the cerebral cortices of the mice aged 3 M, 9 M and 36 M. One GDF11 + CaMKIIα + neuron is indicated by an arrow as an example per group. c Quantification of the average gray value of GDF11 in GDF11 + CaMKIIα + neurons in the cerebral cortices of the mice aged 3 M, 9 M and 36 M (3 M, n = 140; 9 M, n = 160; 36 M, n = 232 cells). d – g Representative images ( d ) and quantification ( e – g ) of the SA-β-Gal + cells in layers 4 and 5 ( d , up, and e , the dashed lines indicate the borders of layers 4 and 5, WT, n = 6; GDF11 f/f , n = 8; GDF11 cKO , n = 6), layer 6a ( d , middle, and f layer 6a is the deep layer cortex near the corpus callosum (CC), WT, n = 8; GDF11 f/f , n = 8; GDF11 cKO , n = 8) of the insular cortex (IC), and layers 2 and 3 of the piriform cortex ( d , down, and g the dashed lines indicate the borders of layers 2 and 3, WT, n = 8; GDF11 f/f , n = 10; GDF11 cKO , n = 10) of GDF11 cKO or GDF11 f/f or WT mice aged 10 M. h–j Representative images ( h ) and quantification of the SA-β-Gal + cells in the cingulate cortex of GDF11 cKO or GDF11 f/f mice aged 10 M ( i , GDF11 f/f , n = 8; GDF11 cKO , n = 6) and 17 M ( j , GDF11 f/f , n = 3; GDF11 cKO , n = 4). Examples of the SA-β-Gal + cells are indicated by double arrowheads in ( d , h ). k A schematic summary on the distribution of the SA-β-Gal + cells in the brain of GDF11 cKO or GDF11 f/f mice aged 10 M and 17 M. l Representative images of double labelling of SA-β-Gal staining (blue) and immunofluorescence of NeuN (fluorescence shown in white) in the insular cortex of GDF11 cKO or GDF11 f/f mice aged 10 M. Examples of the SA-β-Gal + NeuN + neurons are indicated by red arrowheads. m Representative images of double labelling of SA-β-Gal staining (blue) and immunohistochemical staining of CaMKIIα (brown) in the cerebral cortices of GDF11 cKO or GDF11 f/f mice aged 10 M. Examples of the SA-β-Gal + CaMKIIα + ENs are indicated by black arrows. n Survival curves of GDF11 f/f ( n = 35 mice) and GDF11 cKO mice ( n = 15 mice) which died naturally, and log-rank test P value was shown. Median survival is 25 months in GDF11 f/f mice and 22.8 months in GDF11 cKO mice. Scale bars, as shown on the images, 20 μm ( b , d up, m ), 40 μm ( d , middle and down), 50 μm ( h ) and 10 μm ( l ). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. a ( F (2, 6) = 6.672, e 0.0298; 3 M versus 36 M, P = 0.0270), c ( F (2529) = 18.77, P < 0.0001; 3 M versus 9 M, P < 0.0001; 3 M versus 36 M, P < 0.0001; 9 M versus 36 M, P = 0.5477), e ( F (2, 17) = 20.14, P < 0.0001; WT versus GDF11 f/f , P = 0.9950; GDF11 f/f , versus GDF11 cKO , P < 0.0001), f ( F (2, 21) = 4.825, P = 0.0189; WT versus GDF11 f/f , P = 0.9963; GDF11 f/f , versus GDF11 cKO , P = 0.0322) and g ( F (2, 25) = 11.61, P = 0.0003; WT versus GDF11 f/ f, P = 0.4738; GDF11 f/f , versus GDF11 cKO , P = 0.0002). One-way ANOVA with post Tukey multiple comparisons test. i ( P = 0.3427) and j ( P = 0.0280), unpaired two-tailed t test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Immunofluorescence, Staining, Fluorescence, Immunohistochemical staining, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Immunofluorescence image of NeuN (green) in Neuro-2a cells ( n = 6 fields). Scale bar, 40 μm. b PCR of the cell genomes verified successful knockout of the targeted part of exon 2 of GDF11 in Neuro-2a cells (GDF11 KO ) ( n = 3 clones of GDF11 KO cells). c Verification of GDF11 knockout by comparing the mRNA enrichment tracks of GDF11 between GDF11 KO and WT Neuro2a cells by bulk RNA-seq. d Quantification of the relative mRNA of GDF11 in the GDF11 KO and WT Neuro-2a cells by qPCR ( n = 3 biological repeats/group). e , f Western blot ( e ) and Immunofluorescence of GDF11 ( f , scale bar, 40 μm) in GDF11 KO or WT Neuro-2a cells ( n = 3 biological repeats/ group). g , h Representative images ( g ) and quantification ( h , GDF11 KO , n = 13; WT, n = 12 fields) of the SA-β-Gal + cells (blue) in GDF11 KO and WT Neuro-2a cells. All cells are indicated by black stars, and a few representative SA-β-Gal + cells are indicated by black arrows. Scale bar, 50 μm. i Quantification of SA-β-Gal + cells in 3 independent clones of GDF11 KO and WT Neuro-2a cells (GDF11 KO , n = 3; WT, n = 3 clones). j , k Representative images ( j , DAPI, blue) and quantification ( k , GDF11 KO , n = 234 cells; WT, n = 211 cells) of the nuclei of GDF11 KO and WT Neuro-2a cells. Scale bar, 3 μm. l Volcano plot of upregulated (706) and downregulated (411) genes caused by deletion of GDF11 in Neuro-2a cells and revealed by bulk-RNA-seq ( n = 3 clones). m Bulk RNA-seq gene ontology (GO) analysis reveals the top 10 enriched biological processes downregulated by GDF11 deletion in Neuro-2a cells, and the logarithm base 2 of the fold change below −1 was included. n Heatmap of downregulated (11) or upregulated (1) genes involved in “lipid metabolic process” listed in m or “lipid droplets” caused by deletion of GDF11 in Neuro-2a cells, and the logarithm base 2 of the fold change above 1 or below −1 was included. o Representative images of transmission electron microscope (TEM) show the ultrastructure features of GDF11 KO and WT Neuro-2a cells. Cell nucleus (Nuc), lipofuscin (light blue arrows), neurosecretory granules (red double arrowheads) and mitochondrion (brown arrowheads) are indicated as examples. Scale bars, 2 μm. p – r Representative TEM images ( p , lipofuscins, light blue arrows) and quantification of the number (Q, GDF11 KO , n = 20 cells; WT, n = 20 cells) or the area ( r , GDF11 KO , n = 141; WT, n = 85 lipofuscins) of lipofuscins in the GDF11 KO and WT Neuro-2a cells. Scale bars, 500 nm. s – u Representative TEM images ( s , mitochondrion, brown arrowheads; neurosecretory granules, red double arrowheads) and quantification of the number ( t , GDF11 KO , n = 10 cells; WT, n = 10 cells) or the area ( u , GDF11 KO , n = 299; WT, n = 254 mitochondria) of the mitochondria of the GDF11 KO and WT Neuro-2a cells. Scale bars, 500 nm. v Quantification of the number of neurosecretory granules (GDF11 KO , n = 8 cells; WT, n = 10 cells) of the GDF11 KO and WT Neuro-2a cells. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and “ns” indicates not significant, d ( P < 0.0001), h ( P < 0.0001), i ( P = 0.0024), k ( P = 0.0030), q ( P = 0.0002), r ( P = 0.0274), t ( P = 0.8009), u ( P < 0.0001), v ( P = 0.0047), unpaired two-tailed t test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Immunofluorescence, Knock-Out, Clone Assay, RNA Sequencing Assay, Western Blot, Transmission Assay, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Schematic diagrams (left) and representative images (right) of the cingulate gyrus 2 (Cg2), in the prefrontal cortex of GDF11 f/f mice aged 4M-5M, where bilateral focal injection of AAV9-CaMKIIα-Cre-P2A-GFP virus (KO) or AAV9-CaMKIIα-GFP virus (Ctrl) was received at age of 2–3 M and survived for two more months. b Infrared-differential interference contrast (IR-DIC) image (top) and GFP fluorescent image (bottom) of an example of GFP + EN which is undergoing whole-cell patch clamp recording ( n = 64 cells from six mice). c Representative whole-cell recordings in brain slice of a control EN (in Cg2 of GDF11 f/f mice, Ctrl, blue) and a GDF11 deleted-EN (in Cg2 of fGDF11 cKO mice, KO, red) show the firing of action potentials (AP) in response to a series of step current injections. d Examples show typical firing patterns of GFP + EN of fGDF11 cKO mice. e Pie graphs show the percentage of GFP + EN with diverse firing patterns (RS, regular spiking; IS, irregular spiking; IB, intrinsic bursting; RB, repetitive bursting) in WT or KO mice. f Left, plots of the AP frequency as a function of injected currents. Curves are color coded (Ctrl, blue, n = 31 cells from three mice; KO, red, n = 33 cells from three mice). Inset shows the beginning of the curve. Right, plots of the rheobase (Ctrl: 113 ± 16 vs. KO: 81 ± 10 pA, P = 0.049) and slope (Ctrl: 0.18 ± 0.01 vs. KO: 0.30 ± 0.03, P = 0.000) in the two groups (Ctrl, n = 31 cells from three mice; KO, n = 30 cells from three mice). g Left, representative AP waveforms (top) and phase plots (bottom) from Ctrl (blue) or KO (red) group. Right, plots of the AP threshold (Ctrl: −37.9 ± 0.8 vs. KO: −35.0 ± 0.7 mV, P = 0.014), amplitude (AMP) (Ctrl: 85.8 ± 1.6 vs. KO: 78.6 ± 2.2 mV, P = 0.010) and half-width (Ctrl: 0.79 ± 0.03 vs. KO: 0.74 ± 0.03 ms, P = 0.30) in the two groups (Ctrl, n = 29 cells from three mice; KO, n = 24 cells from three mice). h Left-top, representative membrane potential responses to negative current pulses from Ctrl (blue) or KO (red) groups. Plots of the input resistance (Ctrl: 104 ± 10 vs. KO: 214 ± 21 MΩ, P = 0.000), membrane constant (Ctrl: 14.4 ± 1.1 vs. KO: 22.1 ± 2.0 ms, P = 0.003), Sag ratio (Ctrl: 1.18 ± 0.02 vs. KO: 1.27 ± 0.03, P = 0.033), membrane capacitance (Ctrl: 147 ± 11 vs. KO: 95 ± 5 pF, P = 0.000) and RMP (Ctrl: −67.3 ± 1.0 vs. KO: −63.1 ± 0.9 mV, P = 0.004) in the two groups (Ctrl, n = 31 cells from three mice; KO, n = 33 cells from three mice). i Representative whole-cell recordings of mIPSC from the EN in GDF11 f/f mice (Ctrl, blue) and fGDF11 cKO mice (KO, red). j Left, scaled mIPSC examples in the two groups. Right, plots of rising time (Ctrl: 0.65 ± 0.04 vs. KO: 0.85 ± 0.06 ms, P = 0.005) and decay time (Ctrl: 4.44 ± 0.21 vs. KO: 4.69 ± 0.34 ms, P = 0.53) of mIPSCs in the two groups (Ctrl, n = 18 cells from four mice; KO, n = 16 cells from four mice). k , l Cumulative frequency curve of the inter-event-interval ( k ) and amplitude ( l ) of mIPSCs. Insets show the group plots of mIPSC frequency ( k , Ctrl: 34.6 ± 5.2 vs. KO: 4.0 ± 0.9 Hz, P = 0.000) and amplitude ( l , Ctrl: 24.0 ± 1.6 vs. KO: 20.5 ± 1.8 pA, P = 0.16). m – p Recordings of mEPSCs (Ctrl, n = 24 cells from four mice; KO, n = 28 cells from 4 mice) and similar plots as the mIPSCs shown above. Rising time ( n , ctrl: 0.87 ± 0.05 vs. KO: 0.81 ± 0.06 ms, P = 0.46); Decay time ( n , ctrl: 3.54 ± 0.20 vs. KO: 2.98 ± 0.24 ms, P = 0.041); Frequency ( o , Ctrl: 3.66 ± 0.84 vs. KO: 3.13 ± 0.65 Hz, p = 0.82); Amplitude ( p , Ctrl: 14.5 ± 0.8 vs. KO: 14.3 ± 0.9 pA, P = 0.33). q , r Representative traces showing IPSC ( q , left) or EPSC ( r , left) evoked by extracellular electric stimulations for the comparison of paired-pulse ratio (PPR) in GDF11 f/f mice (Ctrl, blue) and fGDF11 cKO mice (KO, red). Group plots of PPR for IPSC ( q , right, Ctrl, n = 7 cells from 3 mice: 0.98 ± 0.07 vs. KO, n = 9 cells from three mice: 1.16 ± 0.20, P = 0.92) and EPSC ( r , right, Ctrl, n = 9 cells from 3 mice: 1.38 ± 0.07 vs. KO, n = 6 cells from three mice: 1.26 ± 0.06, P = 0.24). s Track diagrams in the 3-chamber test (3CT) between the fGDF11 cKO (KO) and GDF11 f/f (Ctrl) mice aged 4–5 M. O object, S1 stranger mouse, S2 new stranger mouse. t Quantification of the exploration time in 3CT (KO, n = 13; Ctrl, n = 13 mice) on objects between the fGDF11 cKO (KO) and GDF11 f/f (Ctrl) mice aged 4–5 M. O1, object 1; O2, object 2. u Quantification of the preference index (S1-O) between the S1 and object in the KO and Ctrl groups (KO, n = 13; Ctrl, n = 13 mice). v Quantification of the preference index (S2-S1) between the S2 and S1 in the KO and Ctrl groups (KO, n = 13; Ctrl, n = 13 mice). w Schematic diagram of the novel object recognition test (NORT) between the GDF11 cKO and GDF11 f/f mice aged 10 M. Red squares indicate the familiar toy while blue triangle indicates a novel toy. x Quantification of the percentage of exploration time (GDF11 cKO , n = 9; GDF11 f/f , n = 6 mice) on the familiar or a novel toy in the GDF11 cKO and GDF11 f/f mice aged 10 M. y Quantification of the novel object discrimination index ((novel-familiar)/(novel + familiar)) between the familiar or a novel toy in the GDF11 cKO and GDF11 f/f mice aged 10 M (GDF11 cKO , n = 9; GDF11 f/f , n = 6 mice). Data are presented as mean ± SEM. Whisker boxplots in ( f , h ) represent the median and interquartile range; whiskers represent 1.5× interquartile range. * P < 0.05, ** P < 0.01 and “ns” represents not significant. f (Rheobase/Slope), h (Input resistance/Membrane constant/Sag ratio/Capacitance), j (Rising time), k , n (Decay time), o – q Mann–Whitney U test. g , h (RMP), j (Decay time), l , n (Rising time), r , u ( P = 0.0118), v ( P = 0.0128), x (GDF11 f/f : Familiar versus Novel, P = 0.0331; GDF11 cKO : Familiar versus Novel, P = 0.0188) and y ( P = 0.0254), unpaired two-tailed t test. t (Ctrl: O1 versus O2, P = 0.3210; KO: O1 versus O2, P = 0.2200), two-way ANOVA with post Sidak’s multiple comparisons test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Injection, Virus, Patch Clamp, Slice Preparation, Control, Membrane, Comparison, Whisker Assay, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Schematic diagrams of the cingulate gyrus 2 (Cg2), in the prefrontal cortex of GDF11 f/f mice aged 4–5 M, where bilateral focal injection of AAV9-CaMKIIα-Cre-P2A-GFP virus (KO) or AAV9-CaMKIIα-GFP virus (Ctrl) was received at age of 2–3 M and survived for two more months. b UMAP of the clustered 16 cell types in snRNA-seq of the Cg2 in both 3 KO mice and 3 control mice (Ctrl) aged 4–5 M. c Violin chart of the relative mRNA of GDF11 by snRNA-seq in KO-GFP + , KO-GFP - , Ctrl-GFP + or Ctrl-GFP - EN. The KO-EN were divided into KO-GFP + and KO-GFP - groups whereas “Ctrl-EN” were divided into Ctrl-GFP + and Ctrl-GFP − groups. d and e , Heatmap shows the average transcription of downregulated and upregulated ageing-related genes ( d ) and SASP-related genes ( e ) in snRNA-seq of KO-GFP + , KO-GFP − , Ctrl-GFP + or Ctrl-GFP − EN. f Confocal images (Left) and 3D-reconstruction (Right) of representative EN from Ctrl (Top) or KO (Bottom) groups. Dendrites and soma are presented in red, and axons are in blue. Scale bar, 50 μm. g , h Plots of the number of intersections of dendrites ( g ) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice) and the group data showing the number of total dendrite intersections ( h , Ctrl: 448 ± 28 vs. KO: 346 ± 36, P = 0.028). i – k Group data show the total number of apical dendrite intersections ( i , Ctrl: 238 ± 17 vs. KO: 181 ± 18, P = 0.036), the total length of apical dendrites ( j , Ctrl: 3.77 ± 0.28 vs. KO: 2.83 ± 0.34 mm, P = 0.044), and the apical branch orders against the averaged dendrite length ( k , branch order 1, Ctrl: 445 ± 28 vs. KO: 403 ± 22 μm, P = 0.26; branch order 2, Ctrl: 115 ± 3 vs. KO: 93 ± 8 μm, P = 0.017; order 3, Ctrl: 91 ± 4 vs. KO: 70 ± 6 μm, P = 0.007; branch order 4, Ctrl: 72 ± 6 vs. KO: 56 ± 7 μm, P = 0.12) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice). l – n Group data comparing the number of total basal intersections ( l , Ctrl: 207 ± 15 vs. KO: 162 ± 22, P = 0.11), total basal dendrite length ( m , Ctrl: 2.73 ± 0.18 vs. KO: 2.16 ± 0.29 mm, P = 0.11) and the basal branch orders against the averaged dendrite length ( n , branch order 1, Ctrl: 102 ± 4 vs. KO: 102 ± 8 μm, P = 0.98; branch order 2, Ctrl: 82 ± 3 vs. KO: 82 ± 9 μm, P = 0.32; order 3, Ctrl: 69 ± 8 vs. KO: 59 ± 2 μm, P = 0.25) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice). o , p Plots of the axon distance from soma against the number of intersections ( o ) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice). Group data show the number of total axon branches intersections ( p , Ctrl: 239 ± 17 vs. KO: 190 ± 28, P = 0.15). q Confocal examples of dendritic spines (red arrows indicate the big mushroom spines while yellow arrows point to small mushroom spines) in the two groups. Scale bar, 5 μm. r , s Group data show total spine density per 10 μm ( r , Ctrl: 6.28 ± 0.23 vs. KO: 1.61 ± 0.13/10 μm, P = 0.000) and mushroom spine diameter ( s , Ctrl: 0.66 ± 0.01 vs. KO: 0.80 ± 0.02 μm, P = 0.000) in two groups (Ctrl, n = 68 dendrites from 16 cells; KO, n = 70 dendrites from 16 cells). t Plots of spine density against the mushroom spine diameter in the two groups (Ctrl, n = 16 cells from three mice; KO, n = 16 cells from three mice). u A schematic summary: GDF11 deletion results in hyperexcitability of the EN as reflected by an enhancement in their firing frequency (due to increased input resistance and elevated RMP) and a decrease in mIPSC frequency. In addition, GDF11 deletion in the EN prunes and shortens their apical dendrites, reduces their dendritic mushroom spine density while enlarges its size. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. h , i , j , k , l , m , n , p , unpaired two-tailed t test; r , s , Mann–Whitney U test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Injection, Virus, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a , b SnRNA-seq GO analysis reveals the top ten enriched biological processes of upregulated ( a ) or downregulated ( b ) in the KO-GFP + EN in comparison with the KO-GFP - EN, and the EN were obtained from the Cg2 of the “KO” mice and the “Ctrl” mice aged 4–5 M. c Volcano plot shows upregulated and downregulated DEGs in the KO-GFP + EN in comparison with the Ctrl-GFP + EN. Some of the top upregulated and downregulated genes were annotated. c , d FC fold change. P value was calculated using Wilcox test and adjusted for multiple testing using Benjamini–Hochberg correction. d Volcano plot shows upregulated and downregulated DEG in the KO-GFP + EN in comparison with the KO-GFP - EN. Some of the top upregulated and downregulated genes were indicated. e UMAP visualization highlights the distribution and the transcription of Cdkn1a/p21 in the identified cell types in snRNA-seq. f Dot plot representing the frequency and average transcription of Cdkn1a/p21 in the identified cell types in snRNA-seq. g , h Relative mRNA of Cdkn1a/p21 ( g ) or p53 ( h ) among four types of EN: Ctrl-GFP - , Ctrl-GFP + , KO-GFP - and KO-GFP + by snRNA-seq. i Heatmap of upregulated (10) and downregulated (6) genes involved in “cellular senescence” caused by deletion of GDF11 in Neuro-2a cells, and the logarithm base 2 of the fold change above 1 or below −1 was included. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and “ns” indicates not significant. a , b Hypergeometric test with Benjamini and Hochberg (BH) correction. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Comparison

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a , b Genetic strategy for generation of p21 f/f mice ( a ) and CaMKIIα-Cre; GDF11 f/f ; p21 f/f mice ( b ) to selectively delete both GDF11 and p21 in CaMKIIα + neurons through Cre/Loxp system. c – g Representative images ( c ) and quantification ( d – g ) of the SA-β-Gal + cells in the cingulate cortex ( c , up, and d , n = 4 per group), layers 4 and 5 ( c , middle, and e GDF11 f/f , n = 4; GDF11 cKO , n = 3; CaMKIIα-Cre; GDF11 f/f ;p21 f/f , n = 5), layer 6a ( c middle, and f layer 6a is the deep layer cortex near the corpus callosum (CC), GDF11 f/f , n = 5; GDF11 cKO , n = 4; CaMKIIα-Cre; GDF11 f/f ;p21 f/f , n = 4) of the insular cortex (IC), and layers 2 and 3 of the piriform cortex ( c down, and g the dashed lines indicate the borders of layers 2 and 3, GDF11 f/f , n = 8; GDF11 cKO , n = 4; CaMKIIα-Cre; GDF11 f/f ;p21 f/f , n = 8) of CaMKIIα-Cre; GDF11 f/f ;p21 f/f or GDF11 cKO or GDF11 f/f mice aged 17 M. Examples of the SA-β-Gal + cells are indicated by double arrows. Scale bars, as shown on the images, 50 μm ( c , up and middle) and 20 μm ( c , middle and down). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. d ( F (2, 9) = 72.52, P < 0.0001; GDF11 f/f versus GDF11 cKO , P = 0.0006; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P = 0.0004), e ( F (2, 9) = 78.16, P < 0.0001; GDF11 f/f versus GDF11 cKO , P = 0.0020; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001), f ( F (2, 10) = 49.87, P < 0.0001; GDF11 f/f versus GDF11 cKO , P < 0.0001; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P = 0.0347) and g ( F (2, 17) = 102.8, P < 0.0001; GDF11 f/f versus GDF11 cKO , P = 0.0227; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f; p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P = 0.0001), One-way ANOVA with post Tukey multiple comparisons test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques:

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Quantification by qPCR of the relative p21 mRNA in the GDF11 KO and WT Neuro-2a cells ( n = 3 clones). b – e Immunofluorescence representative images ( b ) and quantification of the number of p21 + cells per field ( c , n = 5 fields/group), the proportion of p21 + cells ( d , n = 6 fields/group) or the average gray value of p21 per cell ( e , GDF11 KO , n = 420 cells; WT, n = 280 cells) in the GDF11 KO and WT Neuro-2a cells. Scale bar, 25 μm. Examples of the p21 + cells are indicated by double arrowheads. f – h The same snRNA-seq data were used, as described in Fig. . f Rank for regulons in the EN based on regulon specificity score (RSS). The EN were obtained from the Cg2 of the “KO” mice and the “Ctrl” mice aged 4–5 M. g Regulons activity analysis based on area under the curve (AUC) in the identified cell types in snRNA-seq of the “KO” mice and the “Ctrl” mice aged 4–5 M. The activity of regulon Smad3 (highlighted in red) is high in the EN. h Cytoscape network visualization of genes including GDF11, Cdkn1a (p21), Smad2, Smad3 (highlighted in red) and their transcription factors (TFs, yellow). i – m Representative images ( i and l ) and quantification by densitometry of western blot analysis of Smad2 ( j ), phosphorylated Smad2 (pSmad2, k ) and Smad3 ( m ) in the total protein extracted from the GDF11 KO and WT Neuro-2a cells ( n = 3 biological repeats/group). n ChIP-qPCR assessment of the enrichment of Smad2 at the promoter of Cdkn1a/p21 in the GDF11 KO and WT Neuro-2a cells ( n = 3 biological repeats/group). o A proposed working model for loss of GDF11 on cellular senescence. Loss of GDF11 upregulates pSmad2, enhances nuclear entry of Smad2/3 tricomplex and then Smad2 binds to the promoter of p21 and promotes the pro-senescence factor p21 transcription, and eventually causes cellular senescence. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and “ns” indicates not significant. a ( P = 0.0037), c ( P = 0.0033), d ( P = 0.0157), e ( P < 0.0001), j ( P = 0.6648), k ( P = 0.0040) and m ( P = 0.0299), unpaired two-tailed t test. n (IgG: WT versus GDF11 KO , P = 0.57; Smad2: WT versus GDF11 KO , P < 0.001), two-way ANOVA with Sidak’s test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Clone Assay, Immunofluorescence, Activity Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: Evidence of both in vitro (left) and in vivo (right) indicates that growth differentiation factor 11-Smad2/3-p21 pathway acts as a brake on excitatory neuronal senescence and brain ageing.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: In Vitro, In Vivo