Journal: Pediatric diabetes

Article Title: Relationship of Adiponectin and Leptin with Autoimmunity in Children with New Onset Type 1 Diabetes (T1D): A Pilot Study

doi: 10.1111/pedi.12267

Figure Lengend Snippet: Longitudinal Adiponectin Levels

Article Snippet: Adiponectin and Leptin assays Serum total adiponectin levels were determined by radioimmunoassay (Human adiponectin RIA kit, Linco Research, St Charles, Missouri, USA).

Techniques:

Journal: Pediatric diabetes

Article Title: Relationship of Adiponectin and Leptin with Autoimmunity in Children with New Onset Type 1 Diabetes (T1D): A Pilot Study

doi: 10.1111/pedi.12267

Figure Lengend Snippet: Racial differences in longitudinal adipocytokine values

Article Snippet: Adiponectin and Leptin assays Serum total adiponectin levels were determined by radioimmunoassay (Human adiponectin RIA kit, Linco Research, St Charles, Missouri, USA).

Techniques:

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet: The BMP scavenger ERFE is upregulated in cachexia through STAT3 activation (A) Schematic representation of the C26 cancer cachexia model where mice injected with C26 cells develop severe cachexia within 11 days. (B) Immunoblot of phosphorylated Smad1/5/8 (pSmad1/5/8) and vinculin in the quadriceps of control and C26 tumor-bearing mice (n = 5/6). (C) Densiometric quantification of the immunoblot of pSmad1/5/8 and Smad1 in the quadriceps of control and C26 tumor-bearing mice (n = 5/6). (D) mRNA levels of ERFE relative to GAPDH in the gastrocnemii of C26 tumor-bearing mice and relative control mice 11 days post cancer cells inoculation (n = 4/5). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) ERFE protein concentration detected by ELISA in the serum of C26 and control mice (n = 4). (F) Expression levels of ERFE relative to beta-actin in human skeletal muscle biopsies from pre-cachectic and cachectic colorectal and pancreatic cancer patients compared to control individuals (control, n = 41; pre-cachectic, n = 77; cachectic, n = 63). Values are reported as fold change over control group (control vs. pre-cachexia q value = 0.0021; control vs. cachexia q value = 0.0007). (G) Expression levels of Erfe in C2C12 myotubes treated for 48h with 20% C26 CM (n = 6) and representative immunofluorescence of atrophic C2C12 myotubes treated with C26 CM and stained for myoglobin. (H) Experimental design of the ChIP-qPCR assay performed on C2C12 myotubes treated with C26 CM or atrophic skeletal muscles of C26 tumor-bearing mice at day 11. (I) STAT3 predicted binding sites on Erfe promoter. (J) ChIP-qPCR of pSTAT3 binding Erfe promoter on TSS (+629/+639 bp) in C2C12 myotubes treated with C26 CM compared with control cells (n = 2). (K) In vivo ChIP-qPCR of pSTAT3 binding Erfe promoter on TSS (+629/+639 bp). ChIP-qPCR assay was performed by pooling 4 quadriceps per condition for control or C26 tumor-bearing mice (n = 2). (L) Expression levels of Erfe on C2C12 myotubes treated with Stattic 5 μM and/or 50% C26 CM for 6 h (n = 3). (M) Luminescence (firefly/renilla) of NIH/3T3 transfected with Erfe promoter-Luc reporter vector and knocked down for STAT3 (siSTAT3). Cells were treated overnight with 50% C26 CM prior to performing the assay (n = 2). See also Figures S1 and . Data information: Statistical significance was tested with unpaired two-tailed Student’s t -test in (C)–(E) and (G); with non-parametric Krustal-Wallis test followed by Benjamini, Krieger, and Yekutieli multiple comparison tests in (F) and with two-way ANOVA followed by Sidak’s multiple comparison test in (J) and (K) and with one-way ANOVA followed by Sidak’s multiple comparison test in (L) and (M).

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: Activation Assay, Injection, Western Blot, Control, Protein Concentration, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining, ChIP-qPCR, Muscles, Binding Assay, In Vivo, Transfection, Plasmid Preparation, Two Tailed Test, Comparison

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet: Muscle-specific knockdown of BMP inhibitors curbs muscle wasting in C26 tumor-bearing mice (A) Schematic representation of the pathway. The BMP scavenger ERFE is upregulated in cachexia and with the intracellular inhibitor FKBP12 contributes to the downregulation of pSmad1/5/8 in atrophic muscles. (B) Quantification of myotubes diameters after 24 h treatment with recombinant Fc-ERFE (1 μg/mL), inactive globular Fc-gERFE (1 μg/mL), and the relative Fc-CTR (1 μg/mL) (n = 3/4). (C) Myotubes diameters transfected with siRNA for Fkbp12 or scramble sequence (siCtr) for 48 h and treated for 24 h with 10% C26 CM (n = 3/4). (D) Expression levels in C2C12 myotubes knocked-down for FKBP12 with siRNA (n = 4/6). (E) Experimental design of muscle-specific AAV9 delivery in the skeletal muscle of C26 tumor-bearing mice. AAV9-shRNA (10 11 vp) viral particles were injected in the skeletal muscles of BALB/c mice, using the contralateral limb as control (AAV9-shScr). One month after AAV injection, BALB/c mice were subcutaneously inoculated with 750,000 C26 cancer cells. The mice were euthanized 11 days after C26 injection. (F) mRNA levels of ERFE in the quadriceps of AAV9-shERFE-injected mice vs. the contralateral AAV9-shScr limb (n = 7). (G) Quadriceps weight normalized for tibial length compared with the contralateral limb (n = 7). (H) Atrogin1 expression in the quadriceps analyzed in Figures 2 F and 2G (n = 7). (I) CSA distribution of AAV9-shERFE-injected mice (n = 3). (J) mRNA levels of FKBP12 in the quadriceps of AAV9-shFKBP12-injected mice vs. the contralateral AAV9-shScramble limb (n = 7). (K) Quadriceps weight normalized for tibial length compared with the contralateral limb (n = 7). (L) Atrogin1 expression in the quadriceps analyzed in Figures 2 J and 2K. (M) CSA distribution of AAV9-shFKBP12-injected mice (n = 3). See also Figure S3 . Data information: Statistical significance was tested with unpaired two-tailed Student’s t -test in (D); with one-way ANOVA followed by Sidak’s multiple comparison test in (B) and (C). One-sample t -test was performed in (F)–(H) and (J)–(L), while two-way ANOVA followed by Sidak’s multiple comparison test in (I) and (M).

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: Knockdown, Muscles, Recombinant, Transfection, Sequencing, Expressing, shRNA, Injection, Control, Two Tailed Test, Comparison

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet: FK506 bypasses BMP pathway inhibition by activating pSmad1/5/8 through FKBP12 binding (A) Representation of the proposed approach to bypass the "BMP resistance." FK506 binds FKBP12 displacing it from the BMPR-I and restoring pSmad1/5/8 signaling. (B and C) Diameters quantification of C2C12 myotubes treated for 24 h with FK506 (1 μg/mL) and/or 10% C26 CM (B) (n = 5) or treated with recombinant Fc-ERFE (1 μg/mL) and FK506 in Fc-CTR (1 μg/mL) (C) (n = 4). (D) Immunoblot of C2C12 myotubes serum starved for 2 h and treated with FK506 (1 μg/mL) for 1 h (n = 4). (E) Dose-response and immunoblot of pSmad1/5/8 in C2C12 myotubes serum starved for 2 h and treated with increasing doses of murine BMP6 (from 0.01 to 3 ng/mL) with or without FK506 (1 μg/mL) for 1 h (n = 4). (F) Luminescence signal detected in C2C12 transfected with pGL3-BMP responsive element (BRE)-Luciferase and TK-Renilla for 24 h, switched to low-serum DMEM 2% fetal bovine serum for 4 h and treated with FK506 (10 μg/mL) and BMP6 (30 ng/mL) for 5 h (n = 6). (G) Immunoblot of pSmad1/5 and Smad1 of C2C12 myotubes knocked-down for ALK2 or ALK3 (siAlk2 or siAlk3) for 48 h and treated with FK506 (1 μg/mL) for 1 h upon 2 h of serum starvation (n = 2). (H) mRNA levels of ALK2 and ALK3 in C2C12 myotubes knocked-down for ALK2 or ALK3 with siRNA (n = 2/3). (I) Diameters of C2C12 myotubes treated with DMH1 (500 nM) in combination with FK506 (1 μg/mL) and/or 10% C26 CM for 24 h in differentiation medium (n = 3/4). (J and K) In vitro SuNSET assay and relative quantifications performed with C2C12 myotubes treated with FK506 (1 μg/mL), 10% C26 CM (J) (n = 4) or Fc-CTR (1 μg/mL), Fc-ERFE (1 μg/mL), and inactive globular gERFE (1 μg/mL). (K) (n = 3) For 24 h in differentiation medium. For the last 4 h of treatment, puromycin (1 μM) was added to the cells. (L) Myotubes diameters imaged at 24 h post FK506 (1 μg/mL) and/or Torin1 (100 μM) treatment (n = 3). (M and N) mRNA levels of Atrogin1 (M) (n = 3/5) and MuRF1 (N) (n = 3) in C2C12 myotubes treated for 48 h with FK506 (1 μg/mL) and/or 20% C26 CM. See also Figure S4 . Data information: All graphs’ statistical significance was tested with one-way ANOVA followed by Sidak’s multiple comparison test.

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: Inhibition, Binding Assay, Recombinant, Western Blot, Transfection, Luciferase, In Vitro, Comparison

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet: FK506 prevents the degeneration of NMJs morphology and preserves muscle strength (A) Final grip strength as percentage of initial grip strength at day 0 in FK506 or vehicle-treated C26 tumor-bearing and control mice at day 11 post-C26 injection (n = 5/7). (B and C) Volume of the NMJs quantified in the EDL muscle and stained with α-bungarotoxin-Alexa 488 (B) and relative representative picture of the experimental groups (C) (n = 3, 28–30 NMJs quantified per mouse). (D) Expression levels of Chrne gene in the gastrocnemii of FK506 or vehicle treated C26 and control mice at day 11 post-C26 injection (n = 3/4). (E) Graphical representation of the molecular mechanism. In cancer cachexia, systemic inflammation induces catabolism by hyperactivating TGFβ-Smad2/3 axis and by increasing the expression of BMP signaling inhibitors. Activated pSTAT3 induces the upregulation of the BMP scavenger ERFE, which in combination with the intracellular inhibitor FKBP12, contributes to the downregulation of the BMP-pSmad1/5/8 axis and therefore reducing protein synthesis in atrophic muscles. Our therapeutic approach proposes the use of low-dose FK506, that by binding the immunophilin FKBP12 induces its displacement from the BMPR-I, rescuing pSmad1/5/8 signaling and protecting from muscle atrophy in cancer cachexia. See also Figure S6 . Data information: Statistical significance was tested with one-way ANOVA followed by Sidak’s multiple comparison test.

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: Control, Injection, Staining, Expressing, Muscles, Binding Assay, Comparison

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet:

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: FLAG-tag, Virus, Recombinant, Reporter Assay, BIA-KA, Enzyme-linked Immunosorbent Assay, Purification, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Software