lkb1 cst 3047 Search Results


p lkb1 ser428  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc p lkb1 ser428
a Western blots (WB) of total ERK1/2, p90RSK, <t>LKB1,</t> AMPK, mTOR, p70S6K, and S6RP in livers from 12, 25, 50, 75, and 110-week-old male mice for the respective phosphoproteins shown in Fig. . b WB of IL11 and GAPDH in visceral gonadal white adipose tissue (vWAT) and gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). c WB of p-ERK1/2, p-p90RSK, <t>p-LKB1,</t> p-AMPK, p-mTOR, p-p70S6K, p-S6RP, and their respective total proteins in gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). d WB of IL11 and GAPDH in the liver, vWAT and gastrocnemius from 12-week-old and 110-week-old male and female mice (n = 3/group). e - g Representative immunofluorescence images (scale bars, 100 µm) of EGFP expression in the livers, vWAT, and gastrocnemius, colocalized with parenchymal cell markers Adiponectin (AdipoQ) in vWAT and Four and a half LIM domains (FHL1) in gastrocnemius, endothelial cells (CD31), smooth muscle transgelin (SM22α), and pan-fibroblast marker (PDGFRα) of 10 and 110-week old Il11 - EGFP mice (representative dataset from n = 3/group).
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lkb1 p ser28  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc lkb1 p ser28
a Western blots (WB) of total ERK1/2, p90RSK, <t>LKB1,</t> AMPK, mTOR, p70S6K, and S6RP in livers from 12, 25, 50, 75, and 110-week-old male mice for the respective phosphoproteins shown in Fig. . b WB of IL11 and GAPDH in visceral gonadal white adipose tissue (vWAT) and gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). c WB of p-ERK1/2, p-p90RSK, <t>p-LKB1,</t> p-AMPK, p-mTOR, p-p70S6K, p-S6RP, and their respective total proteins in gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). d WB of IL11 and GAPDH in the liver, vWAT and gastrocnemius from 12-week-old and 110-week-old male and female mice (n = 3/group). e - g Representative immunofluorescence images (scale bars, 100 µm) of EGFP expression in the livers, vWAT, and gastrocnemius, colocalized with parenchymal cell markers Adiponectin (AdipoQ) in vWAT and Four and a half LIM domains (FHL1) in gastrocnemius, endothelial cells (CD31), smooth muscle transgelin (SM22α), and pan-fibroblast marker (PDGFRα) of 10 and 110-week old Il11 - EGFP mice (representative dataset from n = 3/group).
Lkb1 P Ser28, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phosphoerk thr202/tyr204cst-4370 antibody  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc phosphoerk thr202/tyr204cst-4370 antibody
a Western blots (WB) of total ERK1/2, p90RSK, <t>LKB1,</t> AMPK, mTOR, p70S6K, and S6RP in livers from 12, 25, 50, 75, and 110-week-old male mice for the respective phosphoproteins shown in Fig. . b WB of IL11 and GAPDH in visceral gonadal white adipose tissue (vWAT) and gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). c WB of p-ERK1/2, p-p90RSK, <t>p-LKB1,</t> p-AMPK, p-mTOR, p-p70S6K, p-S6RP, and their respective total proteins in gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). d WB of IL11 and GAPDH in the liver, vWAT and gastrocnemius from 12-week-old and 110-week-old male and female mice (n = 3/group). e - g Representative immunofluorescence images (scale bars, 100 µm) of EGFP expression in the livers, vWAT, and gastrocnemius, colocalized with parenchymal cell markers Adiponectin (AdipoQ) in vWAT and Four and a half LIM domains (FHL1) in gastrocnemius, endothelial cells (CD31), smooth muscle transgelin (SM22α), and pan-fibroblast marker (PDGFRα) of 10 and 110-week old Il11 - EGFP mice (representative dataset from n = 3/group).
Phosphoerk Thr202/Tyr204cst 4370 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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atr  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc atr
<t>LKB1</t> is required for p53/TIGAR pathway activation in Beas-2B cells upon PM2.5 exposure. ( A , B ) Beas-2B cells were transfected with <t>ATR</t> siRNA, CHK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression of TIGAR was examined at 12 h after PM2.5 exposure. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 for 12 h or a single dose of PM2.5 (100 μg/mL) for the indicated time periods and then the activation of LKB1 was detected. ( E ) Beas-2B cells were transfected with LKB1 siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). The activation of p53/TIGAR pathway was examined at 12 h after PM2.5 exposure. ( F ) Beas-2B cells stably transfected with the p53-dependent luciferase reporter were transfected with LKB1 siRNA or control siRNA and treated with PM2.5 (100 μg/mL). The induction of the p53-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).
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phospho-mdm2-ser166  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc phospho-mdm2-ser166
<t>LKB1</t> is required for p53/TIGAR pathway activation in Beas-2B cells upon PM2.5 exposure. ( A , B ) Beas-2B cells were transfected with <t>ATR</t> siRNA, CHK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression of TIGAR was examined at 12 h after PM2.5 exposure. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 for 12 h or a single dose of PM2.5 (100 μg/mL) for the indicated time periods and then the activation of LKB1 was detected. ( E ) Beas-2B cells were transfected with LKB1 siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). The activation of p53/TIGAR pathway was examined at 12 h after PM2.5 exposure. ( F ) Beas-2B cells stably transfected with the p53-dependent luciferase reporter were transfected with LKB1 siRNA or control siRNA and treated with PM2.5 (100 μg/mL). The induction of the p53-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).
Phospho Mdm2 Ser166, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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chk1  (Cell Signaling Technology Inc)
97
Cell Signaling Technology Inc chk1
LKB1 is required for p53/TIGAR pathway activation in Beas-2B cells upon PM2.5 exposure. ( A , B ) Beas-2B cells were transfected with ATR siRNA, <t>CHK1</t> siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression of TIGAR was examined at 12 h after PM2.5 exposure. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 for 12 h or a single dose of PM2.5 (100 μg/mL) for the indicated time periods and then the activation of LKB1 was detected. ( E ) Beas-2B cells were transfected with LKB1 siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). The activation of p53/TIGAR pathway was examined at 12 h after PM2.5 exposure. ( F ) Beas-2B cells stably transfected with the p53-dependent luciferase reporter were transfected with LKB1 siRNA or control siRNA and treated with PM2.5 (100 μg/mL). The induction of the p53-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).
Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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stat3  (Cell Signaling Technology Inc)
99
Cell Signaling Technology Inc stat3
TIGAR was involved in autophagy-dependent VEGF induction upon PM2.5 exposure. ( A – C ) Beas-2B cells were transfected with TIGAR, Sestrin2, DAPK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression LC3BI/II, Beclin1, p62 and VEGF was examined at 12 h after PM2.5 exposure. ( D , E ) Beas-2B cells were transfected with TIGAR siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). Then autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent ( D ) or by quantitative flow cytrometric assay ( E ) at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected as described in ( D ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( G ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). Then, the induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( H ) Beas-2B cells were transfected and treated as described in ( D ). Then the activation of <t>Src/STAT3</t> pathway was examined at 12 h after PM2.5 exposure. ( I ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).
Stat3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lc3bi ii  (Cell Signaling Technology Inc)
99
Cell Signaling Technology Inc lc3bi ii
TIGAR was involved in autophagy-dependent VEGF induction upon PM2.5 exposure. ( A – C ) Beas-2B cells were transfected with TIGAR, Sestrin2, DAPK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression LC3BI/II, Beclin1, p62 and VEGF was examined at 12 h after PM2.5 exposure. ( D , E ) Beas-2B cells were transfected with TIGAR siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). Then autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent ( D ) or by quantitative flow cytrometric assay ( E ) at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected as described in ( D ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( G ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). Then, the induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( H ) Beas-2B cells were transfected and treated as described in ( D ). Then the activation of <t>Src/STAT3</t> pathway was examined at 12 h after PM2.5 exposure. ( I ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).
Lc3bi Ii, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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sestrin2  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc sestrin2
Induction of autophagy-related transcriptional targets of p53 upon PM2.5 exposure. ( A ) Beas-2B cells were treated with different doses of PM2.5 for 12 h and then the expression levels of TIGAR, <t>Sestrin2</t> and DAPK1 were detected. ( B ) Beas-2B cells were treated with PM2.5 (100 μg/mL) for the indicated time periods and then the expression levels of TIGAR, Sestrin2 and DAPK1 were detected. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 and then cell viability was determined by PI/Annexin V staining assay ( C ) and by using Cell Counting Kit (CCK-8) ( D ) at 24 h after PM2.5 exposure. ( E ) Beas-2B cells were transfected with p53 siRNA or the control siRNA and then treated with PM2.5 (100 μg/mL). The expression levels of p53, TIGAR, Sestrin2 and DAPK1 were detected at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected and treated as described in ( E ). Then the transcription of TIGAR, Sestrin2 and DAPK1 were detected.
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p-rps6  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc p-rps6
Increased mTORC1 activity in germ cells from Lkb1 newborn cKO testis. ( a ) Western blot of testis proteins at different postnatal ages using specific antibodies against p-S6K1 (T389), <t>p-rpS6</t> (S235/6), rpS6 and β -tubulin. rpS6, and β -tubulin were used as internal controls. ( b ) Expression of PI3K-, mTOR- and SPC-related markers by western blot in P8 testes from control and Lkb1 cKO mice. ( c ) Immunohistochemistry for VASA, GATA4, p-rpS6 in P8 control testis. ( d ) Immunohistochemistry for Lin28a and p-rpS6 in P8 control testis. Serial sections were used in order to compare cellular localizations of these markers in the same tubule. Asterisks, different distribution of Lin28a and p-rpS6 in the same tubule resulting in strong positive for Lin28a and weak staining for p-rpS6. Arrows, cells expressing both Lin28a and p-rpS6. ( e ) Immunohistochemistry for Plzf and p-rpS6 in control and cKO mice. ( f ) Mean number of Plzf- or p-rpS6-positive cells in each tubule (positive cells/total tubules). The data were shown as mean±S.E.M. of at least three replicates. * P <0.05. Scale bars=50 μ m
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src  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc src
Increased mTORC1 activity in germ cells from Lkb1 newborn cKO testis. ( a ) Western blot of testis proteins at different postnatal ages using specific antibodies against p-S6K1 (T389), <t>p-rpS6</t> (S235/6), rpS6 and β -tubulin. rpS6, and β -tubulin were used as internal controls. ( b ) Expression of PI3K-, mTOR- and SPC-related markers by western blot in P8 testes from control and Lkb1 cKO mice. ( c ) Immunohistochemistry for VASA, GATA4, p-rpS6 in P8 control testis. ( d ) Immunohistochemistry for Lin28a and p-rpS6 in P8 control testis. Serial sections were used in order to compare cellular localizations of these markers in the same tubule. Asterisks, different distribution of Lin28a and p-rpS6 in the same tubule resulting in strong positive for Lin28a and weak staining for p-rpS6. Arrows, cells expressing both Lin28a and p-rpS6. ( e ) Immunohistochemistry for Plzf and p-rpS6 in control and cKO mice. ( f ) Mean number of Plzf- or p-rpS6-positive cells in each tubule (positive cells/total tubules). The data were shown as mean±S.E.M. of at least three replicates. * P <0.05. Scale bars=50 μ m
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p62  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc p62
TIGAR was involved in autophagy-dependent VEGF induction upon PM2.5 exposure. ( A – C ) Beas-2B cells were transfected with TIGAR, Sestrin2, DAPK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression LC3BI/II, Beclin1, <t>p62</t> and VEGF was examined at 12 h after PM2.5 exposure. ( D , E ) Beas-2B cells were transfected with TIGAR siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). Then autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent ( D ) or by quantitative flow cytrometric assay ( E ) at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected as described in ( D ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( G ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). Then, the induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( H ) Beas-2B cells were transfected and treated as described in ( D ). Then the activation of Src/STAT3 pathway was examined at 12 h after PM2.5 exposure. ( I ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).
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Image Search Results


a Western blots (WB) of total ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K, and S6RP in livers from 12, 25, 50, 75, and 110-week-old male mice for the respective phosphoproteins shown in Fig. . b WB of IL11 and GAPDH in visceral gonadal white adipose tissue (vWAT) and gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). c WB of p-ERK1/2, p-p90RSK, p-LKB1, p-AMPK, p-mTOR, p-p70S6K, p-S6RP, and their respective total proteins in gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). d WB of IL11 and GAPDH in the liver, vWAT and gastrocnemius from 12-week-old and 110-week-old male and female mice (n = 3/group). e - g Representative immunofluorescence images (scale bars, 100 µm) of EGFP expression in the livers, vWAT, and gastrocnemius, colocalized with parenchymal cell markers Adiponectin (AdipoQ) in vWAT and Four and a half LIM domains (FHL1) in gastrocnemius, endothelial cells (CD31), smooth muscle transgelin (SM22α), and pan-fibroblast marker (PDGFRα) of 10 and 110-week old Il11 - EGFP mice (representative dataset from n = 3/group).

Journal: Nature

Article Title: Inhibition of IL-11 signalling extends mammalian healthspan and lifespan

doi: 10.1038/s41586-024-07701-9

Figure Lengend Snippet: a Western blots (WB) of total ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K, and S6RP in livers from 12, 25, 50, 75, and 110-week-old male mice for the respective phosphoproteins shown in Fig. . b WB of IL11 and GAPDH in visceral gonadal white adipose tissue (vWAT) and gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). c WB of p-ERK1/2, p-p90RSK, p-LKB1, p-AMPK, p-mTOR, p-p70S6K, p-S6RP, and their respective total proteins in gastrocnemius from 12, 25, 50, 75, and 110-week-old male mice (n = 5/group). d WB of IL11 and GAPDH in the liver, vWAT and gastrocnemius from 12-week-old and 110-week-old male and female mice (n = 3/group). e - g Representative immunofluorescence images (scale bars, 100 µm) of EGFP expression in the livers, vWAT, and gastrocnemius, colocalized with parenchymal cell markers Adiponectin (AdipoQ) in vWAT and Four and a half LIM domains (FHL1) in gastrocnemius, endothelial cells (CD31), smooth muscle transgelin (SM22α), and pan-fibroblast marker (PDGFRα) of 10 and 110-week old Il11 - EGFP mice (representative dataset from n = 3/group).

Article Snippet: Adiponectin (AdipoQ, 21613-1-AP, Proteintech), p-AMPK Thr172 (2535, clone 40H9, CST), AMPK (5832, clone D63G4, CST), CD31 (ab222783, clone EPR17260-263, abcam), CD68 (ab125212, abcam), cyclin D1 (55506, clone E3P5S, CST), p-ERK1/2 Thr202/Tyr204 (4370, clone D13.14.4E, CST), ERK1/2 (4695, clone 137F5, CST), GAPDH (2118, clone 14C10, CST), FHL1 (10991-1-AP, Proteintech), GFP (ab290 and ab6673, abcam), p-LKB1 Ser428 (3482, clone C67A3, CST), LKB1 (3047, clone D60C5, CST), p-mTOR Ser2448 (2971, CST), mTOR (2972,CST), p-NF-κB p65 Ser536 (3033, clone 93H1, CST), NF-κB p65 (8242, clone D14E12, CST), p16 (human, ab108349, clone EPR1473, abcam), p16 (mouse, ab232402, clone EPR20418, abcam), p21 (human, ab109520, clone EPR362, abcam), p21 (mouse, ab188224, clone EPR18021, abcam), p-p70S6K Thr389 (9234, clone 108D2, CST), p70S6K (2708, clone 49D7, CST), p-p90RSK Ser380 (11989, clone D3H11, CST), p90RSK (9355, clone 32D7, CST), PDGFRα (AF1062, R&D systems), p-S6 ribosomal protein Ser235/236 (4858, clone D57.2.2E, CST), S6 ribosomal protein (2217, clone 5G10, CST), PCNA (13110, clone D3H8P, CST), PGC1α (ab191838, abcam), SLC10A1 (MBS177905, MyBioSource), SM22α (ab14106, abcam), p-STAT3 Tyr705 (4113, clone M9C6, CST), STAT3 (4904, clone 79D7, CST), UCP1 (72298, clone E9Z2V, CST), anti-rabbit horseradish peroxidase (HRP) (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor 488 (ab150077, abcam), anti-goat Alexa Fluor 488 (ab150129, abcam) and anti-rabbit Alexa Fluor 555 (ab150074, abcam).

Techniques: Western Blot, Immunofluorescence, Expressing, Marker

a WB of total proteins in livers for Fig. . WB showing the activation status of ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K, S6RP, and protein expression levels of p16, p21 and GAPDH in b vWAT and c gastrocnemius from 10 and 110-week-old male WT and Il11ra1 −/− mice (n = 5/group). d body temperatures, e indexed weights of liver, vWAT and gastrocnemius, and f the levels of liver triglycerides (TG), g serum cholesterol, and h serum triglycerides of 110-week-old male and female WT and Il11ra1 −/− mice. Relative gene expression levels of i Ccl2 , Ccl5 , Tnfα , Il1β , Il6 , j Acc , Fasn and Srebp1c in livers, and serum levels of k ALT and l AST in young and old male and female WT and Il11ra1 −/− mice. Gastrocnemius m telomere length and n mitochondria DNA (mtDNA) copy number from young and old male and female WT and Il11ra1 −/− mice. d - l Data are shown as mean ± SD. d - n young male WT, n = 8 ( i - n , except for i ( Acc and Fasn ), n = 7); young male Il11ra1 −/− , n = 7 ( i - n , except for i ( Acc and Fasn ), n = 8); old male WT, n = 11 ( e (liver), f - n ), n = 12 ( d , e (vWAT and gastrocnemius); old male Il11ra1 −/− , n = 15 ( e (liver)), n = 16 ( d , f - l ), n = 17 ( e (vWAT and gastrocnemius), i ( Ccl5 ), m - n ); young female WT, n = 7; young female Il11ra1 −/− , n = 8; old female WT, n = 14 ( e (liver and vWAT)), n = 15 ( d , e (gastrocnemius), f - n ); old female Il11ra1 −/− , n = 12 ( m - n ), n = 13 ( d - l ); two-way ANOVA with Sidak’s correction. For gel source data, see Supplementary Fig. . BW: body weight; FC: fold change.

Journal: Nature

Article Title: Inhibition of IL-11 signalling extends mammalian healthspan and lifespan

doi: 10.1038/s41586-024-07701-9

Figure Lengend Snippet: a WB of total proteins in livers for Fig. . WB showing the activation status of ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K, S6RP, and protein expression levels of p16, p21 and GAPDH in b vWAT and c gastrocnemius from 10 and 110-week-old male WT and Il11ra1 −/− mice (n = 5/group). d body temperatures, e indexed weights of liver, vWAT and gastrocnemius, and f the levels of liver triglycerides (TG), g serum cholesterol, and h serum triglycerides of 110-week-old male and female WT and Il11ra1 −/− mice. Relative gene expression levels of i Ccl2 , Ccl5 , Tnfα , Il1β , Il6 , j Acc , Fasn and Srebp1c in livers, and serum levels of k ALT and l AST in young and old male and female WT and Il11ra1 −/− mice. Gastrocnemius m telomere length and n mitochondria DNA (mtDNA) copy number from young and old male and female WT and Il11ra1 −/− mice. d - l Data are shown as mean ± SD. d - n young male WT, n = 8 ( i - n , except for i ( Acc and Fasn ), n = 7); young male Il11ra1 −/− , n = 7 ( i - n , except for i ( Acc and Fasn ), n = 8); old male WT, n = 11 ( e (liver), f - n ), n = 12 ( d , e (vWAT and gastrocnemius); old male Il11ra1 −/− , n = 15 ( e (liver)), n = 16 ( d , f - l ), n = 17 ( e (vWAT and gastrocnemius), i ( Ccl5 ), m - n ); young female WT, n = 7; young female Il11ra1 −/− , n = 8; old female WT, n = 14 ( e (liver and vWAT)), n = 15 ( d , e (gastrocnemius), f - n ); old female Il11ra1 −/− , n = 12 ( m - n ), n = 13 ( d - l ); two-way ANOVA with Sidak’s correction. For gel source data, see Supplementary Fig. . BW: body weight; FC: fold change.

Article Snippet: Adiponectin (AdipoQ, 21613-1-AP, Proteintech), p-AMPK Thr172 (2535, clone 40H9, CST), AMPK (5832, clone D63G4, CST), CD31 (ab222783, clone EPR17260-263, abcam), CD68 (ab125212, abcam), cyclin D1 (55506, clone E3P5S, CST), p-ERK1/2 Thr202/Tyr204 (4370, clone D13.14.4E, CST), ERK1/2 (4695, clone 137F5, CST), GAPDH (2118, clone 14C10, CST), FHL1 (10991-1-AP, Proteintech), GFP (ab290 and ab6673, abcam), p-LKB1 Ser428 (3482, clone C67A3, CST), LKB1 (3047, clone D60C5, CST), p-mTOR Ser2448 (2971, CST), mTOR (2972,CST), p-NF-κB p65 Ser536 (3033, clone 93H1, CST), NF-κB p65 (8242, clone D14E12, CST), p16 (human, ab108349, clone EPR1473, abcam), p16 (mouse, ab232402, clone EPR20418, abcam), p21 (human, ab109520, clone EPR362, abcam), p21 (mouse, ab188224, clone EPR18021, abcam), p-p70S6K Thr389 (9234, clone 108D2, CST), p70S6K (2708, clone 49D7, CST), p-p90RSK Ser380 (11989, clone D3H11, CST), p90RSK (9355, clone 32D7, CST), PDGFRα (AF1062, R&D systems), p-S6 ribosomal protein Ser235/236 (4858, clone D57.2.2E, CST), S6 ribosomal protein (2217, clone 5G10, CST), PCNA (13110, clone D3H8P, CST), PGC1α (ab191838, abcam), SLC10A1 (MBS177905, MyBioSource), SM22α (ab14106, abcam), p-STAT3 Tyr705 (4113, clone M9C6, CST), STAT3 (4904, clone 79D7, CST), UCP1 (72298, clone E9Z2V, CST), anti-rabbit horseradish peroxidase (HRP) (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor 488 (ab150077, abcam), anti-goat Alexa Fluor 488 (ab150129, abcam) and anti-rabbit Alexa Fluor 555 (ab150074, abcam).

Techniques: Activation Assay, Expressing

a , Signalling pathway by which IL-11 induces canonical STAT3 activation and non-canonical ERK activation, LKB1–AMPK inactivation and mTOR activation. b , Western blots of the indicated liver phosphoproteins from male mice aged 12–110 weeks ( n = 5 per group); total (phosphorylated plus unphosphorylated) proteins are shown in Extended Data Fig. . c , Heat map showing densitometry of IL-11 protein expression normalized to GAPDH (immunoblots are shown in Extended Data Fig. ) in gastrocnemius (gastroc) and vWAT from 12- to 110-week-old male mice ( n = 5 per group). d , Representative immunofluorescence images (scale bars, 100 µm) of liver EGFP and SLC10A1 expression from 10 and 110-week-old Il11 - EGFP mice and age-matched wild-type (WT) controls ( n = 3 per group). Scale bars, 100 μm. e , Western blot of liver extracts from 10- and 110-week-old male wild-type and Il11ra1 −/− mice ( n = 5 per group); total proteins are shown in Extended Data Fig. . f , g , Body weight ( f ) and percentages of fat and lean mass (male wild-type, n = 12; male Il11ra1 −/− , n = 16; female wild-type, n = 15; female Il11ra1 −/− , n = 13). h , i , Telomere length ( h ) and mtDNA copy number ( i ) in liver from young (10-week-old) and old (110-week-old) male and female wild-type and Il11ra1 −/− mice (young male wild-type, n = 8; young male Il11ra1 −/− , n = 7; old male wild-type, n = 11; old male Il11ra1 −/− , n = 17; young female wild-type, n = 7; young female Il11ra1 −/− , n = 8; old female wild-type, n = 15; old female Il11ra1 −/− , n = 13). FC, fold change. j , IL-11 and GAPDH immunoblots from the indicated organs of 12- and 105-week-old male wild-type and Il11 −/− mice (liver and soleus, n = 4 per group; vWAT and gastrocnemius, n = 6 per group). f – i , Data are mean ± s.d.; the table below each panel shows summary statistics from two-way ANOVA with Sidak’s correction. For gel source data, see Supplementary Fig. .

Journal: Nature

Article Title: Inhibition of IL-11 signalling extends mammalian healthspan and lifespan

doi: 10.1038/s41586-024-07701-9

Figure Lengend Snippet: a , Signalling pathway by which IL-11 induces canonical STAT3 activation and non-canonical ERK activation, LKB1–AMPK inactivation and mTOR activation. b , Western blots of the indicated liver phosphoproteins from male mice aged 12–110 weeks ( n = 5 per group); total (phosphorylated plus unphosphorylated) proteins are shown in Extended Data Fig. . c , Heat map showing densitometry of IL-11 protein expression normalized to GAPDH (immunoblots are shown in Extended Data Fig. ) in gastrocnemius (gastroc) and vWAT from 12- to 110-week-old male mice ( n = 5 per group). d , Representative immunofluorescence images (scale bars, 100 µm) of liver EGFP and SLC10A1 expression from 10 and 110-week-old Il11 - EGFP mice and age-matched wild-type (WT) controls ( n = 3 per group). Scale bars, 100 μm. e , Western blot of liver extracts from 10- and 110-week-old male wild-type and Il11ra1 −/− mice ( n = 5 per group); total proteins are shown in Extended Data Fig. . f , g , Body weight ( f ) and percentages of fat and lean mass (male wild-type, n = 12; male Il11ra1 −/− , n = 16; female wild-type, n = 15; female Il11ra1 −/− , n = 13). h , i , Telomere length ( h ) and mtDNA copy number ( i ) in liver from young (10-week-old) and old (110-week-old) male and female wild-type and Il11ra1 −/− mice (young male wild-type, n = 8; young male Il11ra1 −/− , n = 7; old male wild-type, n = 11; old male Il11ra1 −/− , n = 17; young female wild-type, n = 7; young female Il11ra1 −/− , n = 8; old female wild-type, n = 15; old female Il11ra1 −/− , n = 13). FC, fold change. j , IL-11 and GAPDH immunoblots from the indicated organs of 12- and 105-week-old male wild-type and Il11 −/− mice (liver and soleus, n = 4 per group; vWAT and gastrocnemius, n = 6 per group). f – i , Data are mean ± s.d.; the table below each panel shows summary statistics from two-way ANOVA with Sidak’s correction. For gel source data, see Supplementary Fig. .

Article Snippet: Adiponectin (AdipoQ, 21613-1-AP, Proteintech), p-AMPK Thr172 (2535, clone 40H9, CST), AMPK (5832, clone D63G4, CST), CD31 (ab222783, clone EPR17260-263, abcam), CD68 (ab125212, abcam), cyclin D1 (55506, clone E3P5S, CST), p-ERK1/2 Thr202/Tyr204 (4370, clone D13.14.4E, CST), ERK1/2 (4695, clone 137F5, CST), GAPDH (2118, clone 14C10, CST), FHL1 (10991-1-AP, Proteintech), GFP (ab290 and ab6673, abcam), p-LKB1 Ser428 (3482, clone C67A3, CST), LKB1 (3047, clone D60C5, CST), p-mTOR Ser2448 (2971, CST), mTOR (2972,CST), p-NF-κB p65 Ser536 (3033, clone 93H1, CST), NF-κB p65 (8242, clone D14E12, CST), p16 (human, ab108349, clone EPR1473, abcam), p16 (mouse, ab232402, clone EPR20418, abcam), p21 (human, ab109520, clone EPR362, abcam), p21 (mouse, ab188224, clone EPR18021, abcam), p-p70S6K Thr389 (9234, clone 108D2, CST), p70S6K (2708, clone 49D7, CST), p-p90RSK Ser380 (11989, clone D3H11, CST), p90RSK (9355, clone 32D7, CST), PDGFRα (AF1062, R&D systems), p-S6 ribosomal protein Ser235/236 (4858, clone D57.2.2E, CST), S6 ribosomal protein (2217, clone 5G10, CST), PCNA (13110, clone D3H8P, CST), PGC1α (ab191838, abcam), SLC10A1 (MBS177905, MyBioSource), SM22α (ab14106, abcam), p-STAT3 Tyr705 (4113, clone M9C6, CST), STAT3 (4904, clone 79D7, CST), UCP1 (72298, clone E9Z2V, CST), anti-rabbit horseradish peroxidase (HRP) (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor 488 (ab150077, abcam), anti-goat Alexa Fluor 488 (ab150129, abcam) and anti-rabbit Alexa Fluor 555 (ab150074, abcam).

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence

a - c Data for HCF at passage 4 (P4), 7, 10, and 14 that had been passaged in the presence of either IgG or anti-IL11RA (X209; 2 µg/ml) from P2. a WB of total and p-ERK1/2, p-p90RSK, p-LKB1, p-AMPK, p-mTOR, p-p70S6K, p-S6RP, p-NFκB, p-STAT3, p16, p21, PCNA, Cyclin D, and GAPDH (n = 6/group). b Immunofluorescence images (scale bars, 100 μm; representative datasets from n = 7/group) and quantification of intensity/area (n = 14/group) for p16 and p21 staining. c IL11, IL6 and IL8 levels in the supernatant based on ELISA (n = 6/group). d WB showing the expression levels of p16, p21, and GAPDH from HCFs P4 that were stimulated for 8, 24, 48, and 72 h with media collected from HCFs P14 that had been grown and passaged in the presence of either IgG or anti-IL11RA (X209; 2 µg/ml) from P2 (n = 4/group). e Telomere length (n = 6/group) and f mtDNA copy number (n = 6/group) and seahorse assay (n = 8/group) showing g mitochondrial oxygen consumption rate (OCR), h changes in OCR during basal respiration and ATP production states, and i oxidative and glycolytic energy phenotypes at baseline in HCFs P4 and P14 either untreated or in the presence of either IgG or anti-IL11RA (X209; 2 µg/ml). b , c , e - i Data are shown as mean ± SD. b , c Two-way ANOVA with Sidak’s correction, e , f , h one-way ANOVA with Tukey’s correction. For gel source data, see Supplementary Fig. .

Journal: Nature

Article Title: Inhibition of IL-11 signalling extends mammalian healthspan and lifespan

doi: 10.1038/s41586-024-07701-9

Figure Lengend Snippet: a - c Data for HCF at passage 4 (P4), 7, 10, and 14 that had been passaged in the presence of either IgG or anti-IL11RA (X209; 2 µg/ml) from P2. a WB of total and p-ERK1/2, p-p90RSK, p-LKB1, p-AMPK, p-mTOR, p-p70S6K, p-S6RP, p-NFκB, p-STAT3, p16, p21, PCNA, Cyclin D, and GAPDH (n = 6/group). b Immunofluorescence images (scale bars, 100 μm; representative datasets from n = 7/group) and quantification of intensity/area (n = 14/group) for p16 and p21 staining. c IL11, IL6 and IL8 levels in the supernatant based on ELISA (n = 6/group). d WB showing the expression levels of p16, p21, and GAPDH from HCFs P4 that were stimulated for 8, 24, 48, and 72 h with media collected from HCFs P14 that had been grown and passaged in the presence of either IgG or anti-IL11RA (X209; 2 µg/ml) from P2 (n = 4/group). e Telomere length (n = 6/group) and f mtDNA copy number (n = 6/group) and seahorse assay (n = 8/group) showing g mitochondrial oxygen consumption rate (OCR), h changes in OCR during basal respiration and ATP production states, and i oxidative and glycolytic energy phenotypes at baseline in HCFs P4 and P14 either untreated or in the presence of either IgG or anti-IL11RA (X209; 2 µg/ml). b , c , e - i Data are shown as mean ± SD. b , c Two-way ANOVA with Sidak’s correction, e , f , h one-way ANOVA with Tukey’s correction. For gel source data, see Supplementary Fig. .

Article Snippet: Adiponectin (AdipoQ, 21613-1-AP, Proteintech), p-AMPK Thr172 (2535, clone 40H9, CST), AMPK (5832, clone D63G4, CST), CD31 (ab222783, clone EPR17260-263, abcam), CD68 (ab125212, abcam), cyclin D1 (55506, clone E3P5S, CST), p-ERK1/2 Thr202/Tyr204 (4370, clone D13.14.4E, CST), ERK1/2 (4695, clone 137F5, CST), GAPDH (2118, clone 14C10, CST), FHL1 (10991-1-AP, Proteintech), GFP (ab290 and ab6673, abcam), p-LKB1 Ser428 (3482, clone C67A3, CST), LKB1 (3047, clone D60C5, CST), p-mTOR Ser2448 (2971, CST), mTOR (2972,CST), p-NF-κB p65 Ser536 (3033, clone 93H1, CST), NF-κB p65 (8242, clone D14E12, CST), p16 (human, ab108349, clone EPR1473, abcam), p16 (mouse, ab232402, clone EPR20418, abcam), p21 (human, ab109520, clone EPR362, abcam), p21 (mouse, ab188224, clone EPR18021, abcam), p-p70S6K Thr389 (9234, clone 108D2, CST), p70S6K (2708, clone 49D7, CST), p-p90RSK Ser380 (11989, clone D3H11, CST), p90RSK (9355, clone 32D7, CST), PDGFRα (AF1062, R&D systems), p-S6 ribosomal protein Ser235/236 (4858, clone D57.2.2E, CST), S6 ribosomal protein (2217, clone 5G10, CST), PCNA (13110, clone D3H8P, CST), PGC1α (ab191838, abcam), SLC10A1 (MBS177905, MyBioSource), SM22α (ab14106, abcam), p-STAT3 Tyr705 (4113, clone M9C6, CST), STAT3 (4904, clone 79D7, CST), UCP1 (72298, clone E9Z2V, CST), anti-rabbit horseradish peroxidase (HRP) (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor 488 (ab150077, abcam), anti-goat Alexa Fluor 488 (ab150129, abcam) and anti-rabbit Alexa Fluor 555 (ab150074, abcam).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Expressing

a , Representative photograph of 105-week-old female wild-type and Il11 −/− mice. b – g , Body weight ( b ), percentage of fat and lean mass (normalized to body weight), frailty score ( d ), full body grip strength ( e ), serum cholesterol and triglycerides ( f ), and GTT and ITT ( g ) of young (12-week-old) and old (105-week-old) female wild-type and Il11 −/− mice. h – j , Indexed vWAT and scWAT weight ( h ), relative vWAT mRNA expression level of Acc1 , Fasn and Srebp1c ( i ), and western blot showing activation status of ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K and S6RP and protein expression levels of p16, p21 and GAPDH ( j ). n = 6 per group. Western blots for the respective total proteins are shown in Extended Data Fig. . k , l , Telomere length and mtDNA copy number ( l ) in vWAT from young and old female wild-type and Il11 −/− mice. b – i , k – l , Data are mean ± s.d. (young wild-type, n = 8; young Il11 −/− , n = 9; old wild-type, n = 16; old Il11 −/− , n = 18; except for h (scWAT): young wild-type, n = 5; young Il11 −/− , n = 7; old wild-type and Il11 −/− , n = 16). Two-way ANOVA with Sidak’s correction ( b – f , h , i , k , l ); two-way repeated measures ANOVA with Sidak’s correction ( h ). For gel source data, see Supplementary Fig. .

Journal: Nature

Article Title: Inhibition of IL-11 signalling extends mammalian healthspan and lifespan

doi: 10.1038/s41586-024-07701-9

Figure Lengend Snippet: a , Representative photograph of 105-week-old female wild-type and Il11 −/− mice. b – g , Body weight ( b ), percentage of fat and lean mass (normalized to body weight), frailty score ( d ), full body grip strength ( e ), serum cholesterol and triglycerides ( f ), and GTT and ITT ( g ) of young (12-week-old) and old (105-week-old) female wild-type and Il11 −/− mice. h – j , Indexed vWAT and scWAT weight ( h ), relative vWAT mRNA expression level of Acc1 , Fasn and Srebp1c ( i ), and western blot showing activation status of ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K and S6RP and protein expression levels of p16, p21 and GAPDH ( j ). n = 6 per group. Western blots for the respective total proteins are shown in Extended Data Fig. . k , l , Telomere length and mtDNA copy number ( l ) in vWAT from young and old female wild-type and Il11 −/− mice. b – i , k – l , Data are mean ± s.d. (young wild-type, n = 8; young Il11 −/− , n = 9; old wild-type, n = 16; old Il11 −/− , n = 18; except for h (scWAT): young wild-type, n = 5; young Il11 −/− , n = 7; old wild-type and Il11 −/− , n = 16). Two-way ANOVA with Sidak’s correction ( b – f , h , i , k , l ); two-way repeated measures ANOVA with Sidak’s correction ( h ). For gel source data, see Supplementary Fig. .

Article Snippet: Adiponectin (AdipoQ, 21613-1-AP, Proteintech), p-AMPK Thr172 (2535, clone 40H9, CST), AMPK (5832, clone D63G4, CST), CD31 (ab222783, clone EPR17260-263, abcam), CD68 (ab125212, abcam), cyclin D1 (55506, clone E3P5S, CST), p-ERK1/2 Thr202/Tyr204 (4370, clone D13.14.4E, CST), ERK1/2 (4695, clone 137F5, CST), GAPDH (2118, clone 14C10, CST), FHL1 (10991-1-AP, Proteintech), GFP (ab290 and ab6673, abcam), p-LKB1 Ser428 (3482, clone C67A3, CST), LKB1 (3047, clone D60C5, CST), p-mTOR Ser2448 (2971, CST), mTOR (2972,CST), p-NF-κB p65 Ser536 (3033, clone 93H1, CST), NF-κB p65 (8242, clone D14E12, CST), p16 (human, ab108349, clone EPR1473, abcam), p16 (mouse, ab232402, clone EPR20418, abcam), p21 (human, ab109520, clone EPR362, abcam), p21 (mouse, ab188224, clone EPR18021, abcam), p-p70S6K Thr389 (9234, clone 108D2, CST), p70S6K (2708, clone 49D7, CST), p-p90RSK Ser380 (11989, clone D3H11, CST), p90RSK (9355, clone 32D7, CST), PDGFRα (AF1062, R&D systems), p-S6 ribosomal protein Ser235/236 (4858, clone D57.2.2E, CST), S6 ribosomal protein (2217, clone 5G10, CST), PCNA (13110, clone D3H8P, CST), PGC1α (ab191838, abcam), SLC10A1 (MBS177905, MyBioSource), SM22α (ab14106, abcam), p-STAT3 Tyr705 (4113, clone M9C6, CST), STAT3 (4904, clone 79D7, CST), UCP1 (72298, clone E9Z2V, CST), anti-rabbit horseradish peroxidase (HRP) (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor 488 (ab150077, abcam), anti-goat Alexa Fluor 488 (ab150129, abcam) and anti-rabbit Alexa Fluor 555 (ab150074, abcam).

Techniques: Expressing, Western Blot, Activation Assay

a , Schematic of anti-IL-11 (X203) therapeutic dosing experiment in old male mice for experiments shown in b – m . Mice were either aged naturally (untreated) or given either X203 or an IgG control antibody (40 mg kg −1 , every 3 weeks) starting from 75 weeks of age for a duration of 25 weeks. Created with BioRender.com. b , Body weights across time. c , d , Changes (Δ) in fat and lean mass percentage ( c ) and area under the curve (AUC) of GTT and ITT ( d ) (values at endpoint (100-week-old) − values at starting point (75-week-old)). a.u., arbitrary units. e , Frailty scores at start and endpoint. Data are shown as values recorded at starting and endpoint. f , Full body grip strength. g , RER in young (14-week-old) and IgG or X203-treated old (81-week-old) mice, 6 weeks after IgG or X203 administration was started ( n = 10 per group). h – j , Body temperatures ( h ), serum ALT ( i ) and liver triglycerides ( j ). k , l , Indexed weights of ( k ) and total collagen content (by hydroxyproline assay) in ( l ) liver, gastrocnemius and vWAT. m , Western blot showing activation status of ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K, S6RP and protein expression levels of IL-11, p16, p21 and GAPDH in vWAT ( n = 6 per group). Western blots of total protein are presented in Extended Data Fig. . b – d , f , h – l , Data are mean ± s.d. 75-week-old control: n = 10 ( f ), n = 14 ( i – l ); untreated 100-week-old: n = 6 (except for k (liver), n = 5); IgG-treated 100-week-old: n = 13; X203-treated 100-week-old: n = 12. Two-way repeated measures ANOVA with Sidak’s correction ( b ); one-way ANOVA with Tukey’s correction ( c , d (GTT), e , f , h – l ); one-way ANOVA with Kruskal–Wallis correction ( d (ITT)). For gel source data, see Supplementary Fig. .

Journal: Nature

Article Title: Inhibition of IL-11 signalling extends mammalian healthspan and lifespan

doi: 10.1038/s41586-024-07701-9

Figure Lengend Snippet: a , Schematic of anti-IL-11 (X203) therapeutic dosing experiment in old male mice for experiments shown in b – m . Mice were either aged naturally (untreated) or given either X203 or an IgG control antibody (40 mg kg −1 , every 3 weeks) starting from 75 weeks of age for a duration of 25 weeks. Created with BioRender.com. b , Body weights across time. c , d , Changes (Δ) in fat and lean mass percentage ( c ) and area under the curve (AUC) of GTT and ITT ( d ) (values at endpoint (100-week-old) − values at starting point (75-week-old)). a.u., arbitrary units. e , Frailty scores at start and endpoint. Data are shown as values recorded at starting and endpoint. f , Full body grip strength. g , RER in young (14-week-old) and IgG or X203-treated old (81-week-old) mice, 6 weeks after IgG or X203 administration was started ( n = 10 per group). h – j , Body temperatures ( h ), serum ALT ( i ) and liver triglycerides ( j ). k , l , Indexed weights of ( k ) and total collagen content (by hydroxyproline assay) in ( l ) liver, gastrocnemius and vWAT. m , Western blot showing activation status of ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K, S6RP and protein expression levels of IL-11, p16, p21 and GAPDH in vWAT ( n = 6 per group). Western blots of total protein are presented in Extended Data Fig. . b – d , f , h – l , Data are mean ± s.d. 75-week-old control: n = 10 ( f ), n = 14 ( i – l ); untreated 100-week-old: n = 6 (except for k (liver), n = 5); IgG-treated 100-week-old: n = 13; X203-treated 100-week-old: n = 12. Two-way repeated measures ANOVA with Sidak’s correction ( b ); one-way ANOVA with Tukey’s correction ( c , d (GTT), e , f , h – l ); one-way ANOVA with Kruskal–Wallis correction ( d (ITT)). For gel source data, see Supplementary Fig. .

Article Snippet: Adiponectin (AdipoQ, 21613-1-AP, Proteintech), p-AMPK Thr172 (2535, clone 40H9, CST), AMPK (5832, clone D63G4, CST), CD31 (ab222783, clone EPR17260-263, abcam), CD68 (ab125212, abcam), cyclin D1 (55506, clone E3P5S, CST), p-ERK1/2 Thr202/Tyr204 (4370, clone D13.14.4E, CST), ERK1/2 (4695, clone 137F5, CST), GAPDH (2118, clone 14C10, CST), FHL1 (10991-1-AP, Proteintech), GFP (ab290 and ab6673, abcam), p-LKB1 Ser428 (3482, clone C67A3, CST), LKB1 (3047, clone D60C5, CST), p-mTOR Ser2448 (2971, CST), mTOR (2972,CST), p-NF-κB p65 Ser536 (3033, clone 93H1, CST), NF-κB p65 (8242, clone D14E12, CST), p16 (human, ab108349, clone EPR1473, abcam), p16 (mouse, ab232402, clone EPR20418, abcam), p21 (human, ab109520, clone EPR362, abcam), p21 (mouse, ab188224, clone EPR18021, abcam), p-p70S6K Thr389 (9234, clone 108D2, CST), p70S6K (2708, clone 49D7, CST), p-p90RSK Ser380 (11989, clone D3H11, CST), p90RSK (9355, clone 32D7, CST), PDGFRα (AF1062, R&D systems), p-S6 ribosomal protein Ser235/236 (4858, clone D57.2.2E, CST), S6 ribosomal protein (2217, clone 5G10, CST), PCNA (13110, clone D3H8P, CST), PGC1α (ab191838, abcam), SLC10A1 (MBS177905, MyBioSource), SM22α (ab14106, abcam), p-STAT3 Tyr705 (4113, clone M9C6, CST), STAT3 (4904, clone 79D7, CST), UCP1 (72298, clone E9Z2V, CST), anti-rabbit horseradish peroxidase (HRP) (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor 488 (ab150077, abcam), anti-goat Alexa Fluor 488 (ab150129, abcam) and anti-rabbit Alexa Fluor 555 (ab150074, abcam).

Techniques: Control, Hydroxyproline Assay, Western Blot, Activation Assay, Expressing

LKB1 is required for p53/TIGAR pathway activation in Beas-2B cells upon PM2.5 exposure. ( A , B ) Beas-2B cells were transfected with ATR siRNA, CHK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression of TIGAR was examined at 12 h after PM2.5 exposure. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 for 12 h or a single dose of PM2.5 (100 μg/mL) for the indicated time periods and then the activation of LKB1 was detected. ( E ) Beas-2B cells were transfected with LKB1 siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). The activation of p53/TIGAR pathway was examined at 12 h after PM2.5 exposure. ( F ) Beas-2B cells stably transfected with the p53-dependent luciferase reporter were transfected with LKB1 siRNA or control siRNA and treated with PM2.5 (100 μg/mL). The induction of the p53-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Journal: Scientific Reports

Article Title: LKB1/p53/TIGAR/autophagy-dependent VEGF expression contributes to PM2.5-induced pulmonary inflammatory responses

doi: 10.1038/s41598-019-53247-6

Figure Lengend Snippet: LKB1 is required for p53/TIGAR pathway activation in Beas-2B cells upon PM2.5 exposure. ( A , B ) Beas-2B cells were transfected with ATR siRNA, CHK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression of TIGAR was examined at 12 h after PM2.5 exposure. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 for 12 h or a single dose of PM2.5 (100 μg/mL) for the indicated time periods and then the activation of LKB1 was detected. ( E ) Beas-2B cells were transfected with LKB1 siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). The activation of p53/TIGAR pathway was examined at 12 h after PM2.5 exposure. ( F ) Beas-2B cells stably transfected with the p53-dependent luciferase reporter were transfected with LKB1 siRNA or control siRNA and treated with PM2.5 (100 μg/mL). The induction of the p53-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Article Snippet: The following primary antibodies were used in this study, including Beclin1 (CST, 3495), LC3BI/II (CST, 3868), p62 (CST, 8025), p53-p-Ser15(CST, 9284), p53 (CST, 2524), Src-p-Tyr416 (CST, 6943), Src (CST, 2109), STAT3-p-Tyr705 (CST, 9145), STAT3 (CST, 9139), LKB1-p-Ser28 (CST, 3482), LKB1 (CST, 3047), ATR (CST, 2790), CHK1 (CST, 2360), VEGF(sc-65617), TIGAR (sc-166291), Sestrin2 (CST, 8487), DAPK1 (CST, 3008), VCAM-1(SC-13160), P-Selectin (SC-8419), E-Selectin (SC-71017), and β-actin (sc-69879) antibodies.

Techniques: Activation Assay, Transfection, Control, Expressing, Stable Transfection, Luciferase, Activity Assay

LKB1 is required for p53/TIGAR pathway activation in Beas-2B cells upon PM2.5 exposure. ( A , B ) Beas-2B cells were transfected with ATR siRNA, CHK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression of TIGAR was examined at 12 h after PM2.5 exposure. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 for 12 h or a single dose of PM2.5 (100 μg/mL) for the indicated time periods and then the activation of LKB1 was detected. ( E ) Beas-2B cells were transfected with LKB1 siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). The activation of p53/TIGAR pathway was examined at 12 h after PM2.5 exposure. ( F ) Beas-2B cells stably transfected with the p53-dependent luciferase reporter were transfected with LKB1 siRNA or control siRNA and treated with PM2.5 (100 μg/mL). The induction of the p53-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Journal: Scientific Reports

Article Title: LKB1/p53/TIGAR/autophagy-dependent VEGF expression contributes to PM2.5-induced pulmonary inflammatory responses

doi: 10.1038/s41598-019-53247-6

Figure Lengend Snippet: LKB1 is required for p53/TIGAR pathway activation in Beas-2B cells upon PM2.5 exposure. ( A , B ) Beas-2B cells were transfected with ATR siRNA, CHK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression of TIGAR was examined at 12 h after PM2.5 exposure. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 for 12 h or a single dose of PM2.5 (100 μg/mL) for the indicated time periods and then the activation of LKB1 was detected. ( E ) Beas-2B cells were transfected with LKB1 siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). The activation of p53/TIGAR pathway was examined at 12 h after PM2.5 exposure. ( F ) Beas-2B cells stably transfected with the p53-dependent luciferase reporter were transfected with LKB1 siRNA or control siRNA and treated with PM2.5 (100 μg/mL). The induction of the p53-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Article Snippet: The following primary antibodies were used in this study, including Beclin1 (CST, 3495), LC3BI/II (CST, 3868), p62 (CST, 8025), p53-p-Ser15(CST, 9284), p53 (CST, 2524), Src-p-Tyr416 (CST, 6943), Src (CST, 2109), STAT3-p-Tyr705 (CST, 9145), STAT3 (CST, 9139), LKB1-p-Ser28 (CST, 3482), LKB1 (CST, 3047), ATR (CST, 2790), CHK1 (CST, 2360), VEGF(sc-65617), TIGAR (sc-166291), Sestrin2 (CST, 8487), DAPK1 (CST, 3008), VCAM-1(SC-13160), P-Selectin (SC-8419), E-Selectin (SC-71017), and β-actin (sc-69879) antibodies.

Techniques: Activation Assay, Transfection, Control, Expressing, Stable Transfection, Luciferase, Activity Assay

TIGAR was involved in autophagy-dependent VEGF induction upon PM2.5 exposure. ( A – C ) Beas-2B cells were transfected with TIGAR, Sestrin2, DAPK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression LC3BI/II, Beclin1, p62 and VEGF was examined at 12 h after PM2.5 exposure. ( D , E ) Beas-2B cells were transfected with TIGAR siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). Then autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent ( D ) or by quantitative flow cytrometric assay ( E ) at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected as described in ( D ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( G ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). Then, the induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( H ) Beas-2B cells were transfected and treated as described in ( D ). Then the activation of Src/STAT3 pathway was examined at 12 h after PM2.5 exposure. ( I ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Journal: Scientific Reports

Article Title: LKB1/p53/TIGAR/autophagy-dependent VEGF expression contributes to PM2.5-induced pulmonary inflammatory responses

doi: 10.1038/s41598-019-53247-6

Figure Lengend Snippet: TIGAR was involved in autophagy-dependent VEGF induction upon PM2.5 exposure. ( A – C ) Beas-2B cells were transfected with TIGAR, Sestrin2, DAPK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression LC3BI/II, Beclin1, p62 and VEGF was examined at 12 h after PM2.5 exposure. ( D , E ) Beas-2B cells were transfected with TIGAR siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). Then autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent ( D ) or by quantitative flow cytrometric assay ( E ) at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected as described in ( D ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( G ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). Then, the induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( H ) Beas-2B cells were transfected and treated as described in ( D ). Then the activation of Src/STAT3 pathway was examined at 12 h after PM2.5 exposure. ( I ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Article Snippet: The following primary antibodies were used in this study, including Beclin1 (CST, 3495), LC3BI/II (CST, 3868), p62 (CST, 8025), p53-p-Ser15(CST, 9284), p53 (CST, 2524), Src-p-Tyr416 (CST, 6943), Src (CST, 2109), STAT3-p-Tyr705 (CST, 9145), STAT3 (CST, 9139), LKB1-p-Ser28 (CST, 3482), LKB1 (CST, 3047), ATR (CST, 2790), CHK1 (CST, 2360), VEGF(sc-65617), TIGAR (sc-166291), Sestrin2 (CST, 8487), DAPK1 (CST, 3008), VCAM-1(SC-13160), P-Selectin (SC-8419), E-Selectin (SC-71017), and β-actin (sc-69879) antibodies.

Techniques: Transfection, Control, Expressing, Confocal Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Stable Transfection, Luciferase, Activity Assay, Activation Assay

LKB1 contributed to autophagy-dependent VEGF induction in Beas-2B cells upon PM2.5 exposure. ( A ) Beas-2B cells were transfected with LKB1 siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). The expression levels of the autophagic hallmarks and VEGF were examined at 12 h after PM2.5 exposure. ( B , C ) Beas-2B cells were transfected and treated with PM2.5 (100 μg/mL) as described in ( A ). Then, the cells were stained with Cyto-ID Green Autophagy Detection Reagent, and the autophagy was examined by subjecting the cells to confocal microscopy assay or a flow cytometric analysis to quantitatively measure the autophagic fluorescence intensity inside the cells ( ** P < 0.01). ( D ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with LKB1 siRNA or control siRNA and then treated with PM2.5 (100 μg/mL). The induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( E ) Beas-2B cells were transfected and treated with PM2.5 (100 μg/mL) as described in ( A ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( F ) Beas-2B cells were transfected and treated with PM2.5 (100 μg/mL) as described in ( A ). Then the activation of Src/STAT3 pathway was examined at 12 h after PM2.5 exposure. ( F ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with LKB1 siRNA or control siRNA and then treated with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Journal: Scientific Reports

Article Title: LKB1/p53/TIGAR/autophagy-dependent VEGF expression contributes to PM2.5-induced pulmonary inflammatory responses

doi: 10.1038/s41598-019-53247-6

Figure Lengend Snippet: LKB1 contributed to autophagy-dependent VEGF induction in Beas-2B cells upon PM2.5 exposure. ( A ) Beas-2B cells were transfected with LKB1 siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). The expression levels of the autophagic hallmarks and VEGF were examined at 12 h after PM2.5 exposure. ( B , C ) Beas-2B cells were transfected and treated with PM2.5 (100 μg/mL) as described in ( A ). Then, the cells were stained with Cyto-ID Green Autophagy Detection Reagent, and the autophagy was examined by subjecting the cells to confocal microscopy assay or a flow cytometric analysis to quantitatively measure the autophagic fluorescence intensity inside the cells ( ** P < 0.01). ( D ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with LKB1 siRNA or control siRNA and then treated with PM2.5 (100 μg/mL). The induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( E ) Beas-2B cells were transfected and treated with PM2.5 (100 μg/mL) as described in ( A ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( F ) Beas-2B cells were transfected and treated with PM2.5 (100 μg/mL) as described in ( A ). Then the activation of Src/STAT3 pathway was examined at 12 h after PM2.5 exposure. ( F ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with LKB1 siRNA or control siRNA and then treated with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Article Snippet: The following primary antibodies were used in this study, including Beclin1 (CST, 3495), LC3BI/II (CST, 3868), p62 (CST, 8025), p53-p-Ser15(CST, 9284), p53 (CST, 2524), Src-p-Tyr416 (CST, 6943), Src (CST, 2109), STAT3-p-Tyr705 (CST, 9145), STAT3 (CST, 9139), LKB1-p-Ser28 (CST, 3482), LKB1 (CST, 3047), ATR (CST, 2790), CHK1 (CST, 2360), VEGF(sc-65617), TIGAR (sc-166291), Sestrin2 (CST, 8487), DAPK1 (CST, 3008), VCAM-1(SC-13160), P-Selectin (SC-8419), E-Selectin (SC-71017), and β-actin (sc-69879) antibodies.

Techniques: Transfection, Control, Expressing, Staining, Confocal Microscopy, Fluorescence, Stable Transfection, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Activation Assay

Induction of autophagy-related transcriptional targets of p53 upon PM2.5 exposure. ( A ) Beas-2B cells were treated with different doses of PM2.5 for 12 h and then the expression levels of TIGAR, Sestrin2 and DAPK1 were detected. ( B ) Beas-2B cells were treated with PM2.5 (100 μg/mL) for the indicated time periods and then the expression levels of TIGAR, Sestrin2 and DAPK1 were detected. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 and then cell viability was determined by PI/Annexin V staining assay ( C ) and by using Cell Counting Kit (CCK-8) ( D ) at 24 h after PM2.5 exposure. ( E ) Beas-2B cells were transfected with p53 siRNA or the control siRNA and then treated with PM2.5 (100 μg/mL). The expression levels of p53, TIGAR, Sestrin2 and DAPK1 were detected at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected and treated as described in ( E ). Then the transcription of TIGAR, Sestrin2 and DAPK1 were detected.

Journal: Scientific Reports

Article Title: LKB1/p53/TIGAR/autophagy-dependent VEGF expression contributes to PM2.5-induced pulmonary inflammatory responses

doi: 10.1038/s41598-019-53247-6

Figure Lengend Snippet: Induction of autophagy-related transcriptional targets of p53 upon PM2.5 exposure. ( A ) Beas-2B cells were treated with different doses of PM2.5 for 12 h and then the expression levels of TIGAR, Sestrin2 and DAPK1 were detected. ( B ) Beas-2B cells were treated with PM2.5 (100 μg/mL) for the indicated time periods and then the expression levels of TIGAR, Sestrin2 and DAPK1 were detected. ( C , D ) Beas-2B cells were treated with different doses of PM2.5 and then cell viability was determined by PI/Annexin V staining assay ( C ) and by using Cell Counting Kit (CCK-8) ( D ) at 24 h after PM2.5 exposure. ( E ) Beas-2B cells were transfected with p53 siRNA or the control siRNA and then treated with PM2.5 (100 μg/mL). The expression levels of p53, TIGAR, Sestrin2 and DAPK1 were detected at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected and treated as described in ( E ). Then the transcription of TIGAR, Sestrin2 and DAPK1 were detected.

Article Snippet: The following primary antibodies were used in this study, including Beclin1 (CST, 3495), LC3BI/II (CST, 3868), p62 (CST, 8025), p53-p-Ser15(CST, 9284), p53 (CST, 2524), Src-p-Tyr416 (CST, 6943), Src (CST, 2109), STAT3-p-Tyr705 (CST, 9145), STAT3 (CST, 9139), LKB1-p-Ser28 (CST, 3482), LKB1 (CST, 3047), ATR (CST, 2790), CHK1 (CST, 2360), VEGF(sc-65617), TIGAR (sc-166291), Sestrin2 (CST, 8487), DAPK1 (CST, 3008), VCAM-1(SC-13160), P-Selectin (SC-8419), E-Selectin (SC-71017), and β-actin (sc-69879) antibodies.

Techniques: Expressing, Annexin V Staining Assay, Cell Counting, CCK-8 Assay, Transfection, Control

TIGAR was involved in autophagy-dependent VEGF induction upon PM2.5 exposure. ( A – C ) Beas-2B cells were transfected with TIGAR, Sestrin2, DAPK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression LC3BI/II, Beclin1, p62 and VEGF was examined at 12 h after PM2.5 exposure. ( D , E ) Beas-2B cells were transfected with TIGAR siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). Then autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent ( D ) or by quantitative flow cytrometric assay ( E ) at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected as described in ( D ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( G ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). Then, the induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( H ) Beas-2B cells were transfected and treated as described in ( D ). Then the activation of Src/STAT3 pathway was examined at 12 h after PM2.5 exposure. ( I ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Journal: Scientific Reports

Article Title: LKB1/p53/TIGAR/autophagy-dependent VEGF expression contributes to PM2.5-induced pulmonary inflammatory responses

doi: 10.1038/s41598-019-53247-6

Figure Lengend Snippet: TIGAR was involved in autophagy-dependent VEGF induction upon PM2.5 exposure. ( A – C ) Beas-2B cells were transfected with TIGAR, Sestrin2, DAPK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression LC3BI/II, Beclin1, p62 and VEGF was examined at 12 h after PM2.5 exposure. ( D , E ) Beas-2B cells were transfected with TIGAR siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). Then autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent ( D ) or by quantitative flow cytrometric assay ( E ) at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected as described in ( D ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( G ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). Then, the induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( H ) Beas-2B cells were transfected and treated as described in ( D ). Then the activation of Src/STAT3 pathway was examined at 12 h after PM2.5 exposure. ( I ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Article Snippet: The following primary antibodies were used in this study, including Beclin1 (CST, 3495), LC3BI/II (CST, 3868), p62 (CST, 8025), p53-p-Ser15(CST, 9284), p53 (CST, 2524), Src-p-Tyr416 (CST, 6943), Src (CST, 2109), STAT3-p-Tyr705 (CST, 9145), STAT3 (CST, 9139), LKB1-p-Ser28 (CST, 3482), LKB1 (CST, 3047), ATR (CST, 2790), CHK1 (CST, 2360), VEGF(sc-65617), TIGAR (sc-166291), Sestrin2 (CST, 8487), DAPK1 (CST, 3008), VCAM-1(SC-13160), P-Selectin (SC-8419), E-Selectin (SC-71017), and β-actin (sc-69879) antibodies.

Techniques: Transfection, Control, Expressing, Confocal Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Stable Transfection, Luciferase, Activity Assay, Activation Assay

Increased mTORC1 activity in germ cells from Lkb1 newborn cKO testis. ( a ) Western blot of testis proteins at different postnatal ages using specific antibodies against p-S6K1 (T389), p-rpS6 (S235/6), rpS6 and β -tubulin. rpS6, and β -tubulin were used as internal controls. ( b ) Expression of PI3K-, mTOR- and SPC-related markers by western blot in P8 testes from control and Lkb1 cKO mice. ( c ) Immunohistochemistry for VASA, GATA4, p-rpS6 in P8 control testis. ( d ) Immunohistochemistry for Lin28a and p-rpS6 in P8 control testis. Serial sections were used in order to compare cellular localizations of these markers in the same tubule. Asterisks, different distribution of Lin28a and p-rpS6 in the same tubule resulting in strong positive for Lin28a and weak staining for p-rpS6. Arrows, cells expressing both Lin28a and p-rpS6. ( e ) Immunohistochemistry for Plzf and p-rpS6 in control and cKO mice. ( f ) Mean number of Plzf- or p-rpS6-positive cells in each tubule (positive cells/total tubules). The data were shown as mean±S.E.M. of at least three replicates. * P <0.05. Scale bars=50 μ m

Journal: Cell Death & Disease

Article Title: Differential regulation of spermatogenic process by Lkb1 isoforms in mouse testis

doi: 10.1038/cddis.2017.527

Figure Lengend Snippet: Increased mTORC1 activity in germ cells from Lkb1 newborn cKO testis. ( a ) Western blot of testis proteins at different postnatal ages using specific antibodies against p-S6K1 (T389), p-rpS6 (S235/6), rpS6 and β -tubulin. rpS6, and β -tubulin were used as internal controls. ( b ) Expression of PI3K-, mTOR- and SPC-related markers by western blot in P8 testes from control and Lkb1 cKO mice. ( c ) Immunohistochemistry for VASA, GATA4, p-rpS6 in P8 control testis. ( d ) Immunohistochemistry for Lin28a and p-rpS6 in P8 control testis. Serial sections were used in order to compare cellular localizations of these markers in the same tubule. Asterisks, different distribution of Lin28a and p-rpS6 in the same tubule resulting in strong positive for Lin28a and weak staining for p-rpS6. Arrows, cells expressing both Lin28a and p-rpS6. ( e ) Immunohistochemistry for Plzf and p-rpS6 in control and cKO mice. ( f ) Mean number of Plzf- or p-rpS6-positive cells in each tubule (positive cells/total tubules). The data were shown as mean±S.E.M. of at least three replicates. * P <0.05. Scale bars=50 μ m

Article Snippet: The following antibodies are used: LKB1 (CST; catalog no. 3047; 1 : 1000), p-S6K1 (phosphorylated at Thr389; CST; catalog no. 9234; 1 : 1000), rpS6 (CST; catalog no. 2217; 1 : 1000), p-rpS6 (CST), β -tubulin (CST; catalog no. 2128; 1 : 2000), Lin28a (Abcam; catalog no. 46026; 1 : 2000), Plzf (R&D; catalog no. AF2944; 1 : 1000), Akt (CST; catalog no. 2920; 1 : 2000) and p-Akt (CST; catalog no. 4060; 1 : 2000).

Techniques: Activity Assay, Western Blot, Expressing, Immunohistochemistry, Staining

Rapamycin treatment failed to rescue the depletion of SPCs in the tubules of Lkb1 cKO mice. Rapamycin was given to P10 control and cKO mice ( n =5 in each group) for 1 week, and testes were evaluated at 5 weeks of age. con+Ve, control mice injected with vehicle control saline; con+Rapa, control mice injected with rapamycin; cKO+Ve, cKO mice injected with vehicle control; cKO+Rapa, cKO mice injected with rapamycin. ( a ) Testis weight per body weight just after injection. ( b ) Testis weight per body weight at 5 weeks of age. ( c ) Western blot analysis of p-rpS6 level just after injection. ( d ) Western blot analysis of p-rpS6 and p-Akt level at 5 weeks. (e ) Seminiferous tubules immunostained for Plzf. Scale bar=50 μ m. Images on the right column are higher magnifications of the relative tubules in the left. Arrows, representative Plzf-positive SPCs in the tubules. ( f ) Average number of Plzf-positive cells per tubule in each of the four groups. Data presented as mean±S.E.M. of at least three replicates. * P <0.05, ** P <0.01

Journal: Cell Death & Disease

Article Title: Differential regulation of spermatogenic process by Lkb1 isoforms in mouse testis

doi: 10.1038/cddis.2017.527

Figure Lengend Snippet: Rapamycin treatment failed to rescue the depletion of SPCs in the tubules of Lkb1 cKO mice. Rapamycin was given to P10 control and cKO mice ( n =5 in each group) for 1 week, and testes were evaluated at 5 weeks of age. con+Ve, control mice injected with vehicle control saline; con+Rapa, control mice injected with rapamycin; cKO+Ve, cKO mice injected with vehicle control; cKO+Rapa, cKO mice injected with rapamycin. ( a ) Testis weight per body weight just after injection. ( b ) Testis weight per body weight at 5 weeks of age. ( c ) Western blot analysis of p-rpS6 level just after injection. ( d ) Western blot analysis of p-rpS6 and p-Akt level at 5 weeks. (e ) Seminiferous tubules immunostained for Plzf. Scale bar=50 μ m. Images on the right column are higher magnifications of the relative tubules in the left. Arrows, representative Plzf-positive SPCs in the tubules. ( f ) Average number of Plzf-positive cells per tubule in each of the four groups. Data presented as mean±S.E.M. of at least three replicates. * P <0.05, ** P <0.01

Article Snippet: The following antibodies are used: LKB1 (CST; catalog no. 3047; 1 : 1000), p-S6K1 (phosphorylated at Thr389; CST; catalog no. 9234; 1 : 1000), rpS6 (CST; catalog no. 2217; 1 : 1000), p-rpS6 (CST), β -tubulin (CST; catalog no. 2128; 1 : 2000), Lin28a (Abcam; catalog no. 46026; 1 : 2000), Plzf (R&D; catalog no. AF2944; 1 : 1000), Akt (CST; catalog no. 2920; 1 : 2000) and p-Akt (CST; catalog no. 4060; 1 : 2000).

Techniques: Injection, Western Blot

TIGAR was involved in autophagy-dependent VEGF induction upon PM2.5 exposure. ( A – C ) Beas-2B cells were transfected with TIGAR, Sestrin2, DAPK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression LC3BI/II, Beclin1, p62 and VEGF was examined at 12 h after PM2.5 exposure. ( D , E ) Beas-2B cells were transfected with TIGAR siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). Then autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent ( D ) or by quantitative flow cytrometric assay ( E ) at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected as described in ( D ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( G ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). Then, the induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( H ) Beas-2B cells were transfected and treated as described in ( D ). Then the activation of Src/STAT3 pathway was examined at 12 h after PM2.5 exposure. ( I ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Journal: Scientific Reports

Article Title: LKB1/p53/TIGAR/autophagy-dependent VEGF expression contributes to PM2.5-induced pulmonary inflammatory responses

doi: 10.1038/s41598-019-53247-6

Figure Lengend Snippet: TIGAR was involved in autophagy-dependent VEGF induction upon PM2.5 exposure. ( A – C ) Beas-2B cells were transfected with TIGAR, Sestrin2, DAPK1 siRNA or their control siRNAs, respectively. Then the cells were exposed to PM2.5 (100 μg/mL) and the expression LC3BI/II, Beclin1, p62 and VEGF was examined at 12 h after PM2.5 exposure. ( D , E ) Beas-2B cells were transfected with TIGAR siRNA or control siRNA and then exposed to PM2.5 (100 μg/mL). Then autophagy was examined under confocal microscopy after the cells were stained with Cyto-ID Green Autophagy Detection Reagent ( D ) or by quantitative flow cytrometric assay ( E ) at 12 h after PM2.5 exposure. ( F ) Beas-2B cells were transfected as described in ( D ). Cell supernatants were collected and the expression of VEGF was examined by ELISA at 24 h after PM2.5 exposure (** P < 0.01). ( G ) Beas-2B cells stably transfected with VEGF promoter-driven luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). Then, the induction of VEGF promoter-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01). ( H ) Beas-2B cells were transfected and treated as described in ( D ). Then the activation of Src/STAT3 pathway was examined at 12 h after PM2.5 exposure. ( I ) Beas-2B cells stably transfected with the STAT3-dependent luciferase reporter were transfected with TIGAR siRNA or control siRNA followed by treating with PM2.5 (100 μg/mL). The induction of the STAT3-dependent luciferase activity was determined at 12 h after PM2.5 exposure (** P < 0.01).

Article Snippet: The following primary antibodies were used in this study, including Beclin1 (CST, 3495), LC3BI/II (CST, 3868), p62 (CST, 8025), p53-p-Ser15(CST, 9284), p53 (CST, 2524), Src-p-Tyr416 (CST, 6943), Src (CST, 2109), STAT3-p-Tyr705 (CST, 9145), STAT3 (CST, 9139), LKB1-p-Ser28 (CST, 3482), LKB1 (CST, 3047), ATR (CST, 2790), CHK1 (CST, 2360), VEGF(sc-65617), TIGAR (sc-166291), Sestrin2 (CST, 8487), DAPK1 (CST, 3008), VCAM-1(SC-13160), P-Selectin (SC-8419), E-Selectin (SC-71017), and β-actin (sc-69879) antibodies.

Techniques: Transfection, Control, Expressing, Confocal Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Stable Transfection, Luciferase, Activity Assay, Activation Assay