Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: a Heat map of differentially regulated transcripts obtained by bulk RNA sequencing of the hypothalamic from the DIO wild-type mice treated with vehicle or TubA for four consecutive days ( n = 3 Veh/TubA mice). b Connectivity Map (CMap) similarity scores of the perturbagens for the up- and down-regulated hypothalamic transcripts from TubA-treated mice used as query. Top 20 positively (left) and negatively (right) associated perturbagens are shown. c Heat maps of the differentially regulated genes from LepR+ cells obtained from lean wild-type mice after PBS or leptin treatment . d CMap scores of the perturbagens and the top associations for the leptin gene expression signature from ( c ). Individual ( e ) and averaged-combined ( f ) CMap similarity scores of TubA and leptin plotted. Top two positively associated and top two negatively associated perturbagens are marked.

Article Snippet: The wild-type Fak (#50515), autophosphorylation-site mutant FAK Y397F (#50516), FLAG-tagged Stat3 , and LacZ constructs were obtained from Addgene (Cambridge, MA).

Techniques: RNA Sequencing, Gene Expression

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: a , b DIO wild-type mice that have been on HFD diet were implanted a guide cannula into the lateral ventricle. Mice were treated with vehicle or PF centrally (icv), and vehicle or TubA (15 mg/kg) was administered peripherally once daily. The daily weight change curves ( a ) and cumulative weight change at the end of the treatment ( b ) ( n = 4 Veh/PF mice; n = 7 Veh/PF + TubA mice). c Growth curves and d average food intake of wild-type or Pyk2 KO mice on HFD. Body weight change of wild-type and Pyk2 KO DIO mice during ( e ) and at the end of ( f ) daily TubA treatments. Average food intake of the animals during vehicle acclimation ( g ) and TubA treatments ( h ). i Cumulative food intake of the wild-type and Pyk2 KO mice at the end of TubA treatments. j Blood glucose of the mice before and after TubA treatments. k Difference in the blood glucose of the mice following TubA treatment ( n = 6 WT mice; n = 5 Pyk2 KO mice). Data shown are representative of experiments conducted once ( a – d ) or twice ( e – k ) in separate cohorts of animals with similar results. Data are represented as mean ± SEM and analyzed by t -test ( d , f – i , k ), mixed effect analysis ( a ), one-way ANOVA ( b ), two-way ANOVA ( c , e , j ).

Article Snippet: The wild-type Fak (#50515), autophosphorylation-site mutant FAK Y397F (#50516), FLAG-tagged Stat3 , and LacZ constructs were obtained from Addgene (Cambridge, MA).

Techniques:

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: HEK-293T cells were transiently transfected with 0.25 µg Stat3 (lanes 2–10) and increasing amounts (0.25–1.75 μg) of wild-type or mutant Fak (FAK Y397F ) ( a ) or wild-type or kinase-dead (KD) Pyk2 ( b ). The FAK Y397F mutation prevents phosphorylation at Tyr 397 , abolishing kinase activity ( a , lanes 7–10). Total cell lysates (TCL) were immunoblotted with the indicated antibodies. The migration difference between wild-type and Pyk2 KD reflects the GFP tag in the KD construct. c STAT3, PYK2, or FAK were transiently overexpressed in HEK-293T cells as indicated. PYK2 or FAK was immunoprecipitated, and TCL or the immunoprecipitated fractions were analyzed by western blot using the indicated antibodies. d Leptin enhances the interaction of hypothalamic focal adhesion kinases with STAT3. Lean wild-type mice received vehicle or leptin (5 mg/kg). Hypothalamic were collected 30 min later, crosslinked (10% formalin), homogenized, and immunoprecipitated with STAT3 or IgG control antibodies. Immunoprecipitates were analyzed for the indicated proteins. Fak and Pyk2 mRNA ( e ) and protein ( f ) expression in N1 cells stably expressing shScr, shFak, or shPyk2 constructs. g LepRb-expressing N1 cells with shScr, shFak, or shPyk2 were stimulated with leptin (40 ng/mL), and pSTAT3 and total STAT3 were analyzed at the indicated times. h Quantification of pSTAT3/STAT3 signals; inset shows the area under the curve. i N1 cells transfected with LepRb and either shScr or shRNAs targeting both Fak and Pyk2 were stimulated with 40 ng/mL leptin. Total lysates were analyzed by immunoblot for pSTAT3 and total STAT3. j N1 cells stably expressing LepRb and a STAT3-driven luciferase reporter were treated with PF and leptin. Luminescence was measured 24 h later. k Relative leptin-induced luminescence at 0, 2, and 128 ng/mL leptin with PF concentrations of 0 (Veh), 330 nM, 1 µM, or 3.3 µM. Blots are visualized with a Bio-Rad ChemiDoc Touch Imaging System. Data shown are representative of an experiment conducted once ( a – f ) or twice ( g – k ) with similar results. Data are represented as mean ± SEM and analyzed by one-way ( h ) or two-way ANOVA ( e , k ).

Article Snippet: The wild-type Fak (#50515), autophosphorylation-site mutant FAK Y397F (#50516), FLAG-tagged Stat3 , and LacZ constructs were obtained from Addgene (Cambridge, MA).

Techniques: Transfection, Mutagenesis, Phospho-proteomics, Activity Assay, Migration, Construct, Immunoprecipitation, Western Blot, Control, Expressing, Stable Transfection, Luciferase, Imaging

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: a – k Hypothalamic sections from lean wild-type mice, ad libitum fed, overnight fasted, or overnight fasted and leptin (3 h) treated (ip) were stained with fluorescent RNAscope probes targeting LepR , Fak or Pyk2 transcripts. a The percent of LepR + cells with Fak and Pyk2 in the ARC, VMH, and DMH. b Representative images of hypothalamic sections covering ARC, VMH, and DMH for LepR , Fak , Pyk2 expressions. The scale bar represents 100 μm. Representative RNAscope images and region specific and LepR + specific transcript quantification in the VMH ( c – e ), DMH ( f – h ), and ARC ( i – k ) ( n = 5 mice per group). Red: LepR , yellow: Pyk2 , green: Fak , blue: DAPI. Arrowheads indicate triple colocalization of LepR , Pyk2 , and Fak . The scale bar represents 50 μm ( c ). The transcripts in the LepR + cells were expressed as the counted pixels within the LepR + cells, except for Pyk2 signal in the DMH, which is expressed as the total fluorescence due to high signal intensity. Data shown are representative of an experiment conducted once. Data are represented as mean ± SEM and are analyzed by one-way ANOVA ( d , e , g , h , j , k ).

Article Snippet: The wild-type Fak (#50515), autophosphorylation-site mutant FAK Y397F (#50516), FLAG-tagged Stat3 , and LacZ constructs were obtained from Addgene (Cambridge, MA).

Techniques: Staining, RNAscope, Fluorescence

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: a Fasted lean wild-type mice implanted with a cannula into the lateral ventricle were administered DMSO (Vehicle) or PF (2.5 µg) by intracerebroventricular (icv) route. One hour later, mice were administered either saline or leptin ip and food was provided. Food intake was measured at 3, 6, 16, and 2 h ( n = 20 DMSO + Vehicle mice; n = 21 DMSO + Leptin mice; n = 21 PF + Vehicle mice; n = 20 PF + Leptin mice). b Lean wild-type mice fasted overnight were administered vehicle or PF icv 15 min later, mice received an ip injection of either saline or leptin. 30 min later, hypothalamic punches were collected and snap frozen in liquid nitrogen for analysis by immunoblot. Blots are visualized with a Bio-Rad ChemiDoc Touch Imaging System. Quantification of the western blot is shown on the right ( n = 3 mice per group). c pSTAT3 Y705 IHC staining of the hypothalamic sections of lean wild-type mice. Animals were centrally infused with vehicle or PF, and treated with saline or leptin 15 min later. Brains were removed and prepared for IHC staining 30, 60 or 120 min after leptin injections. pSTAT3 positive cell number across the ARC, VMH, and DMH are quantified. The scale bar represents 100 μm. Data shown are representative of an experiment conducted once ( c ), twice ( b ) or three times ( a ) in separate cohorts of animals with similar results. Data are represented as mean ± SEM and analyzed by one-way ( b ) or two-way ANOVA ( a , c ).

Article Snippet: The wild-type Fak (#50515), autophosphorylation-site mutant FAK Y397F (#50516), FLAG-tagged Stat3 , and LacZ constructs were obtained from Addgene (Cambridge, MA).

Techniques: Saline, Injection, Western Blot, Imaging, Immunohistochemistry

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: a – c Fasted lean wild-type and Pyk2 KO mice were treated (ip) with vehicle or leptin, and food intake and body weights were followed for 24 h. a Time course of food intake, b food intake presented as normalized to the respective vehicle groups, and c weight change of the animals ( a – c , n = 6 mice per group). d , e Hypothalamic STAT3 phosphorylation was analyzed 30, 60, and 120 min post leptin injection in lean wild-type and Pyk2 KO mice ( n = 1 Vehicle mouse; n = 3 Leptin mice, per timepoint). Blots are visualized with a Bio-Rad ChemiDoc Touch Imaging System. Data shown are representative of experiments conducted twice in separate cohorts of animals with similar results. Data are represented as mean ± SEM and are analyzed by two-way ANOVA.

Article Snippet: The wild-type Fak (#50515), autophosphorylation-site mutant FAK Y397F (#50516), FLAG-tagged Stat3 , and LacZ constructs were obtained from Addgene (Cambridge, MA).

Techniques: Phospho-proteomics, Injection, Imaging

Journal: Nature Communications

Article Title: The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

doi: 10.1038/s41467-026-69008-9

Figure Lengend Snippet: Lean wild type mice were bilaterally injected with AAV particles encoding shScr or shPyk2/Fak in the mediobasal hypothalamus and DMH. Two weeks later mice were either maintained on regular chow or switched to HFD. a – c Growth curves of the cohorts on the indicated diets expressed either as absolute body weight or percent weight gain. Week 0 indicates 2 weeks post viral injection. d Food intake of the cohorts measured over 24 h around week 3. Body composition of the cohort measured around week 2 using NMR and plotted as absolute tissue mass ( e , g ) or normalized to body weight ( f , h ). i Blood glucose of the mice measured during the daytime after a 4 h fast ( a – i , n = 6 shScr Chow mice; n = 7 shScr HFD mice; n = 9 shFak/Pyk2 mice). Cohorts on chow diet (group caged) were treated with either vehicle or leptin (5 mg/kg, ip) prior to dark cycle and their food intake ( j ) ( n = 8 mice per group) and body weight ( k ) was followed for 24 h ( n = 25 shScr mice; n = 24 shFak/Pyk2 mice). l – o DIO cohorts were treated with vehicle or TubA (15 mg/kg, ip, daily). Absolute weight change ( l ), percent weight change ( m ), average food intake ( n ), and body weights ( o ) are measured daily ( l – o , n = 19 shScr mice; n = 16 shFak/Pyk2 mice). Data shown are representative of experiments conducted twice in separate cohorts of animals with similar results. Data are represented as mean ± SEM and are analyzed by one-way ( d , i ) or two-way ANOVA ( a – c , e – h , j – o ).

Article Snippet: The wild-type Fak (#50515), autophosphorylation-site mutant FAK Y397F (#50516), FLAG-tagged Stat3 , and LacZ constructs were obtained from Addgene (Cambridge, MA).

Techniques: Injection

Journal: Experimental and molecular pathology

Article Title: Focal adhesion kinase coordinates costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction.

doi: 10.1016/j.yexmp.2019.03.006

Figure Lengend Snippet: Fig. 9. Effects of FRNK-mediated inhibition of FAK on hypertrophy signaling. Box Whisker plots of the (specific) phosphorylation of Y397-FAK (A), S2448-mTOR (B), T183/Y185-JNK (C) and S63-cJUN (D) in not tenotomized soleus muscle 7 days after co-transfection of muscle pairs with pCMV & pCMV-FAK and pCMV-FRNK & pCMV-FAK. Circles indicate outliers. + and ++, p < .05 and p < .01 vs. pCMV & pCMV-FAK (repeated-measures ANOVA). Values represented normalized intensities respective to the pCMV & pCMV-FAK transfected muscles. E) Example western blot showing the detection of FAK and FRNK (top) in 10 μg protein homogenate from a soleus muscle pair being co-transfected with pCMV & pCMV-FAK and pCMV-FRNK & pCMV-FAK, respectively. Below, respective actin band.

Article Snippet: An antibody directed against the Nterminal region of FAK (AF4467, R&D systems) and an antibody that detects the phosphorylation at tyrosine 397 (P00151, Boster Biological Technology) were biotinylated (Biotin-NH2 labeling Kit, KT-221, Kamiya Biomedical Company) and used as capture antibodies.

Techniques: Inhibition, Whisker Assay, Phospho-proteomics, Cotransfection, Transfection, Muscles, Western Blot

Journal: Experimental and molecular pathology

Article Title: Focal adhesion kinase coordinates costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction.

doi: 10.1016/j.yexmp.2019.03.006

Figure Lengend Snippet: Fig. 10. Inter-relationships. Hierarchical networks of the correlations between measured molecular and cellular parameters in all assessed soleus mus- cles. Correlated parameters (nodes) are connected though lines (edges; solid dark-grey line: positive correlation, back- ward slashed grey line: negative correlation). Abbreviations: body_m_e, body mass at the end of the experiment; body_m_s, body mass at the start of the experiment; %con_tis, percentage area of connective tissue; %f-fast, percentage of fast (II) type muscle fibers; %f-hybrid, percentage of hybrid (I/II) type muscle fibers; %f-inuc, percentage of fibers with internal nu- clei; %f-slow, percentage of slow (I) type muscle fibers; FAK, levels of FAK; FRNK, levels of FRNK; g-VN, gamma-vinculin; GM_mass, mass of gastrocnemius muscle; MCSA, mean cross sectional area of muscle fibers; m-VN, meta-vinculin; s-pFAK, specific phosphorylation of Y397-FAK; s-pJNK, specific phosphorylation of T183/Y185-JNK; s-pmTOR, specific phosphorylation of S2448-mTOR; s-pP70S6K, specific phos- phorylation of T421/S424-P70S6K; SOL_mass, mass of soleus muscle.

Article Snippet: An antibody directed against the Nterminal region of FAK (AF4467, R&D systems) and an antibody that detects the phosphorylation at tyrosine 397 (P00151, Boster Biological Technology) were biotinylated (Biotin-NH2 labeling Kit, KT-221, Kamiya Biomedical Company) and used as capture antibodies.

Techniques: Phospho-proteomics