Journal: Experimental & Molecular Medicine

Article Title: REDD1 is a determinant of low-dose metronomic doxorubicin-elicited endothelial cell dysfunction through downregulation of VEGFR-2/3 expression

doi: 10.1038/s12276-021-00690-z

Figure Lengend Snippet: a , b REDD1 mRNA and protein levels in the HUVECs and HLECs treated with doxorubicin (DOX; 3 nM) for 24 h. c Microarray analysis of total and polysomal mRNAs purified from the HUVECs infected with control adenovirus (Ad-C) or Ad- Redd1 (Ad-R). Heat map of receptor genes involved in angiogenesis expressed as the fold change in total and polysomal mRNA levels in the Ad- Redd1 -transfected HUVECs compared with the Ad-C-transfected cells ( n = 3). d VEGFR-1, VEGFR-2, EGFR, and IGF-1Rβ expression levels in the HUVECs treated with DOX (3 nM), paclitaxel (PTX; 25 nM), or cisplatin (CisPt; 2 μM) for 24 h. e VEGFR-2 protein and mRNA levels in the HUVECs treated with chemotherapeutic drugs. f , g VEGFR-3, EGFR, and IGF-1Rβ expression levels ( f ) and VEGFR-3 expression at the protein and mRNA levels ( g ) in the HLECs treated with chemotherapeutic drugs for 24 h. Data are presented as the mean ± SD ( n = 4). *** P < 0.001; NS, not significant.

Article Snippet: Some cells were infected with Redd1 (Ad- Redd1 ) or empty adenovirus (Ad-Control; Genenmed, Inc., Seoul, South Korea) at a multiplicity of infection (MOI) of 50 for 4 h or transfected with 80 nM small interfering RNA (siRNA), either the control (#SC-37007) or Redd1 (#SC-45806; Santa Cruz Biotechnology, Dallas, TX), using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, followed by continuous culturing in fresh medium for 24 h. Tumor cells (HeLa, B16F1, and B16F10 cells) were cultured in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Microarray, Purification, Infection, Control, Transfection, Expressing

Journal: Experimental & Molecular Medicine

Article Title: REDD1 is a determinant of low-dose metronomic doxorubicin-elicited endothelial cell dysfunction through downregulation of VEGFR-2/3 expression

doi: 10.1038/s12276-021-00690-z

Figure Lengend Snippet: VEGFR-1/2 expression and VEGF-A-induced angiogenesis were examined in HUVECs treated with DOX following transfection with control (siC) or Redd1 siRNA (siR). a , b Western blots of REDD1 and VEGFR-1/2 ( a ) as well as phosphorylated mTOR, 4E-BP1, and S6K ( b ). c Polysome profiling using sucrose density gradient ultracentrifugation. d qRT-PCR of Vegfr-1/2 mRNAs associated with high-molecular-weight polysomes ( n = 4). e Western blots of phosphorylated VEGFR-2, ERK, and Akt in the HUVECs stimulated with VEGF-A. f Representative images of the migration (upper) and tube formation (lower) of the HUVECs stimulated with VEGF-A were obtained using Boyden chamber and Matrigel-based morphogenesis assays, respectively. g , h Quantitation of migration ( g ) and tube formation ( h ) was performed using ImageJ software ( n = 4). Data are presented as the mean ± SD. ** P < 0.01, *** P < 0.001; NS, not significant.

Article Snippet: Some cells were infected with Redd1 (Ad- Redd1 ) or empty adenovirus (Ad-Control; Genenmed, Inc., Seoul, South Korea) at a multiplicity of infection (MOI) of 50 for 4 h or transfected with 80 nM small interfering RNA (siRNA), either the control (#SC-37007) or Redd1 (#SC-45806; Santa Cruz Biotechnology, Dallas, TX), using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, followed by continuous culturing in fresh medium for 24 h. Tumor cells (HeLa, B16F1, and B16F10 cells) were cultured in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR, High Molecular Weight, Migration, Quantitation Assay, Software

Journal: Experimental & Molecular Medicine

Article Title: REDD1 is a determinant of low-dose metronomic doxorubicin-elicited endothelial cell dysfunction through downregulation of VEGFR-2/3 expression

doi: 10.1038/s12276-021-00690-z

Figure Lengend Snippet: a – f VEGFR-1/2 expression and VEGF-A-induced angiogenesis were examined in the HUVECs infected with control adenovirus (Ad-C) or Ad- Redd1 (Ad-R) at an MOI of 50. a , b Western blots of VEGFR-1/2, EGFR, and IGF-1Rβ ( a ) as well as phosphorylated mTOR, 4E-BP1, and S6K ( b ). c Polysome profiling using sucrose density gradient ultracentrifugation. d qRT-PCR of Vegfr-1/2 mRNAs associated with high-molecular-weight polysomes ( n = 4). e Western blots of phosphorylated VEGFR-2, ERK, and Akt in the HUVECs stimulated with VEGF-A. f Quantitative migration and tube formation of HUVECs in response to VEGF-A were assessed using Boyden chamber and Matrigel-based morphogenesis assays, respectively. ( n = 4). g Schematic representation of the 5′-UTRs of Egfr , Vegfr-1 , Vegfr-2 , and Vegfr-3 ; clover-like structures in the 5′-UTRs of Egfr (-54 to -25) and Vegfr-1 (−87 to −1) indicate authentic IRES and putative IRES sequences, respectively; +1 indicates the AUG start codon, and dotted lines indicate deleted regions of 5′-UTRs. NF, not found. h HEK-293 cells were cotransfected with the pGL4.13/5′-UTR- Firefly luciferase construct and Renilla luciferase pGL4.74 vector, followed by treatment with or without DOX or infection with Ad-C or Ad- Redd1 . Reporter activities were measured in cell lysates using a Dual Luciferase Assay Kit ( n = 5). Data are presented as the mean ± SD. * P < 0.05, *** P < 0.001; NS, not significant.

Article Snippet: Some cells were infected with Redd1 (Ad- Redd1 ) or empty adenovirus (Ad-Control; Genenmed, Inc., Seoul, South Korea) at a multiplicity of infection (MOI) of 50 for 4 h or transfected with 80 nM small interfering RNA (siRNA), either the control (#SC-37007) or Redd1 (#SC-45806; Santa Cruz Biotechnology, Dallas, TX), using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, followed by continuous culturing in fresh medium for 24 h. Tumor cells (HeLa, B16F1, and B16F10 cells) were cultured in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Expressing, Infection, Control, Western Blot, Quantitative RT-PCR, High Molecular Weight, Migration, Luciferase, Construct, Plasmid Preparation

Journal: Experimental & Molecular Medicine

Article Title: REDD1 is a determinant of low-dose metronomic doxorubicin-elicited endothelial cell dysfunction through downregulation of VEGFR-2/3 expression

doi: 10.1038/s12276-021-00690-z

Figure Lengend Snippet: a Representative images of Matrigel plugs containing saline or VEGF-A removed from the WT and Redd1 −/− mice metronomically treated with or without DOX. b Quantitated levels of hemoglobin (Hb) extracted from the plugs ( n = 6). c Representative immunostaining of CD31 + vessels in a Matrigel plug section. Scale bar, 100 μm. d Quantitated levels of CD31 + vessels in the plugs ( n = 4). e Representative images of Matrigel plugs containing saline or VEGF-C removed from the WT and Redd1 −/− mice metronomically treated with or without DOX. f Quantitated levels of Hb extracted from the plugs ( n = 6). g Representative immunostaining of CD31 + and LYVE-1 + vessels in a Matrigel plug section. Scale bar, 100 μm. h Quantitated levels of LYVE-1 + lymphatic vessels ( n = 4). Data are presented as the mean ± SD. * P < 0.05, *** P < 0.001; NS, not significant.

Article Snippet: Some cells were infected with Redd1 (Ad- Redd1 ) or empty adenovirus (Ad-Control; Genenmed, Inc., Seoul, South Korea) at a multiplicity of infection (MOI) of 50 for 4 h or transfected with 80 nM small interfering RNA (siRNA), either the control (#SC-37007) or Redd1 (#SC-45806; Santa Cruz Biotechnology, Dallas, TX), using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, followed by continuous culturing in fresh medium for 24 h. Tumor cells (HeLa, B16F1, and B16F10 cells) were cultured in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Saline, Immunostaining

Journal: Experimental & Molecular Medicine

Article Title: REDD1 is a determinant of low-dose metronomic doxorubicin-elicited endothelial cell dysfunction through downregulation of VEGFR-2/3 expression

doi: 10.1038/s12276-021-00690-z

Figure Lengend Snippet: a Comparison of tumor growth in the WT and Redd1 −/− mice metronomically treated with saline or DOX ( n = 10 per group). The arrow indicates the start of DPX treatment. b Representative images of tumor sections showing CD31 + blood vessels and DAPI-stained nuclei. Scale bar, 100 μm. c Quantification of CD31 + blood vessel density per high-power field (HPF) ( n = 10). d Representative images of tumor sections showing CD31 + blood vessels and REDD1 expression. Scale bar, 50 μm. e Quantification of their colocalization ( n = 10). f Representative images of tumor sections showing CD31 + vessels and VEGFR-2 expression. Scale bar, 100 μm. g Quantification of their colocalization ( n = 10). h Representative images of tumor sections showing LYVE-1 + lymphatic vessels and VEGFR-3 expression. Scale bar, 100 μm. i Quantification of colocalization of VEGFR-3 and LYVE-1 ( n = 10). j Quantification of LYVE-1 + lymphatic vessel density ( n = 10). Data are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant. r , Pearson’s correlation coefficient.

Article Snippet: Some cells were infected with Redd1 (Ad- Redd1 ) or empty adenovirus (Ad-Control; Genenmed, Inc., Seoul, South Korea) at a multiplicity of infection (MOI) of 50 for 4 h or transfected with 80 nM small interfering RNA (siRNA), either the control (#SC-37007) or Redd1 (#SC-45806; Santa Cruz Biotechnology, Dallas, TX), using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, followed by continuous culturing in fresh medium for 24 h. Tumor cells (HeLa, B16F1, and B16F10 cells) were cultured in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Comparison, Saline, Staining, Expressing

Journal: Experimental & Molecular Medicine

Article Title: REDD1 is a determinant of low-dose metronomic doxorubicin-elicited endothelial cell dysfunction through downregulation of VEGFR-2/3 expression

doi: 10.1038/s12276-021-00690-z

Figure Lengend Snippet: a Comparison of tumor growth in the B16F10 tumor-bearing WT and Redd1 −/− mice metronomically treated with saline or DOX ( n = 12 per group). b Kaplan–Meier survival curves of the WT and Redd1 −/− mice treated with saline or metronomic DOX ( n = 20 per group). c Representative images of lung-metastatic tyrosinase + colonies and CD31 + blood vessels. Scale bar, 50 μm. d Quantification of tyrosinase + colonies per high-power field (HPF) ( n = 10). e Representative images of spleen-metastatic tyrosinase + colonies and CD31 + blood vessels. Scale bar, 50 μm. f Quantification of tyrosinase + colonies ( n = 10). g Representative images of lymph node-metastatic tyrosinase + colonies and LYVE-1 + lymphatic vessels. Scale bar, 50 μm. h Quantification of tyrosinase + colonies ( n = 10). i Diagram depicting inhibition of tumor angiogenesis, lymphangiogenesis, vessel permeability, growth, and metastasis by LDMC-induced REDD1 expression and subsequent translational repression of Vegfr-2/3 mRNAs in tumor vascular and lymphatic endothelial cells (TVECs and TLECs). Data, except for those for the mouse survival curve ( b ), are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Some cells were infected with Redd1 (Ad- Redd1 ) or empty adenovirus (Ad-Control; Genenmed, Inc., Seoul, South Korea) at a multiplicity of infection (MOI) of 50 for 4 h or transfected with 80 nM small interfering RNA (siRNA), either the control (#SC-37007) or Redd1 (#SC-45806; Santa Cruz Biotechnology, Dallas, TX), using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, followed by continuous culturing in fresh medium for 24 h. Tumor cells (HeLa, B16F1, and B16F10 cells) were cultured in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Comparison, Saline, Inhibition, Permeability, Expressing

Journal: FEBS letters

Article Title: SP600125 negatively regulates the mammalian target of rapamycin via ATF4-induced Redd1 expression.

doi: 10.1016/j.febslet.2008.11.035

Figure Lengend Snippet: Fig. 2. SP600125-induced Redd1 expression inhibits mTOR activity. (A) H1299 cells were treated with the indicated concentrations of SP600125, PD98059, or SB203580 for 6 h. (B) H1299 cells were transfected with Redd1 or silencer negative control (CTL) siRNAs for 24 h, followed by 30 lM SP600125 for 6 h. Cell lysates were prepared, and the indicated protein levels were measured by Western blot analysis. Total RNA was isolated and RT-PCR analyses were carried out using primer pairs specific for Redd1.

Article Snippet: The antibody against Redd1 was obtained from the ProteinTech Group (Chicago, IL), while those against p-S6K (Thr389), S6K, p-S6 (Ser240/244), S6, and 4E-BP1 were from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Activity Assay, Transfection, Negative Control, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: FEBS letters

Article Title: SP600125 negatively regulates the mammalian target of rapamycin via ATF4-induced Redd1 expression.

doi: 10.1016/j.febslet.2008.11.035

Figure Lengend Snippet: Fig. 3. ATF4 is required for Redd1-induced inhibition of mTOR in response to SP600125. (A and B) H1299 cells were treated with the indicated concentrations of SP600125 for 6 h (A) and treated with 30 lM SP600125 for the indicated times (B). (C) H1299 cells were transiently transfected with control empty vector (pEF), myc-tagged mATF4WT (ATF4 WT) or myc-tagged mATF4DN (ATF4 DN) for 24 h. (D) H1299 and HeLa cells were transfected with ATF4 or silencer negative control (CTL) siRNAs for 24 h, followed by 30 lM SP600125 for 6 h. The indicated protein and mRNA levels were measured by Western blot analysis and RT-PCR.

Article Snippet: The antibody against Redd1 was obtained from the ProteinTech Group (Chicago, IL), while those against p-S6K (Thr389), S6K, p-S6 (Ser240/244), S6, and 4E-BP1 were from Cell Signaling Technology (Beverly, MA).

Techniques: Inhibition, Transfection, Control, Plasmid Preparation, Negative Control, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: REDD1 promotes obesity-induced metabolic dysfunction via atypical NF-κB activation

doi: 10.1038/s41467-022-34110-1

Figure Lengend Snippet: a Weight gain in Redd1 −/− mice and their WT littermates fed NC or HFD for 16 weeks ( n = 6 per group). b Mass of eWAT and iWAT in NC- or HFD-fed Redd1 −/− mice and their WT littermates ( n = 8 per group). c Representative images of perilipin (green) and F4/80 (purple) staining in the eWAT of NC- or HFD-fed Redd1 −/− mice and WT littermates ( n = 8 per group). Scale bar, 100 μm. d Average adipocyte size in the eWAT of NC- or HFD-fed Redd1 −/− mice and WT littermates ( n = 8 per group). e Relative area of F4/80-positive cells in the eWAT of NC- or HFD-fed Redd1 −/− mice and WT littermates ( n = 8 per group). f Relative number of crown-like structures (CLSs) in the eWAT of NC- or HFD-fed Redd1 −/− mice and WT littermates ( n = 8 per group). g NF-κB activity in the eWAT from NC- or HFD-fed Redd1 −/− mice and WT littermates ( n = 6 per group). h Plasma levels of inflammatory cytokines in NC- or HFD-fed Redd1 −/− mice and WT littermates ( n = 6 per group). Bar graphs represent mean ± s.e.m. Statistical significance was calculated using two-way ANOVA followed by the Holm–Sidak post hoc test. Source data are provided as a Source Data file.

Article Snippet: In addition, mouse adipose SVF cells and 3T3-L1 preadipocytes (5.0 × 10 5 cells/well, American Type Culture Collection) were plated and transfected with sh Redd1 (#sc-45807-SH, Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Staining, Activity Assay, Clinical Proteomics

Journal: Nature Communications

Article Title: REDD1 promotes obesity-induced metabolic dysfunction via atypical NF-κB activation

doi: 10.1038/s41467-022-34110-1

Figure Lengend Snippet: a Fasting plasma levels of glucose and insulin in NC- or HFD-fed Redd1 −/− mice and their WT littermates ( n = 6 per group). b Representative images of insulin (green)-stained pancreatic islets from NC- or HFD-fed Redd1 −/− mice and WT littermates ( n = 6 per group). Scale bar, 100 μm. c Quantification of average islet size ( n = 6 per group). d Calculation of the HOMA-IR scores ( n = 6 per group). e Assessment of GTT and ITT in mice fasting for 12 and 6 h, respectively, in NC- or HFD-fed Redd1 −/− mice and WT littermates ( n = 8 per group). f Representative western blots of Akt phosphorylation and plasma membrane-associated GLUT4 (PM-GLUT4) in eWAT and skeletal muscle from mice injected i.p. with saline or insulin ( n = 3). g Representative western blots of phosphorylated Akt and FOXO1 in the liver of mice injected with saline or insulin ( n = 6). h Quantification of the phosphorylated FOXO1 to total FOXO1 ratio ( n = 6 per group). i Quantification of G6pc , Pck1 , and Fbp1 mRNA levels in the liver ( n = 6 per group). Bar graphs represent mean ± s.e.m. Statistical significance was calculated using two-way ANOVA followed by the Holm–Sidak post hoc test. Source data are provided as a Source Data file.

Article Snippet: In addition, mouse adipose SVF cells and 3T3-L1 preadipocytes (5.0 × 10 5 cells/well, American Type Culture Collection) were plated and transfected with sh Redd1 (#sc-45807-SH, Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Clinical Proteomics, Staining, Western Blot, Phospho-proteomics, Membrane, Injection, Saline

Journal: Nature Communications

Article Title: REDD1 promotes obesity-induced metabolic dysfunction via atypical NF-κB activation

doi: 10.1038/s41467-022-34110-1

Figure Lengend Snippet: a Weight gain over time in Redd1 fl/fl ( R fl/fl ) and Redd1 Δ Adipoq ( R Δ Adipoq ) mice fed HFD for 16 weeks ( n = 6 per group). b Mass measurements for the eWAT and iWAT in HFD-fed Redd1 fl/fl and Redd1 Δ Adipoq mice ( n = 6 per group). c Representative images showing perilipin (green) and F4/80 (purple) staining in the eWAT of Redd1 fl/fl and Redd1 Δ Adipoq mice fed HFD ( n = 6 per group). Scale bar, 100 μm. d NF-κB activity in the eWAT from HFD-fed Redd1 fl/fl and Redd1 Δ Adipoq mice ( n = 6 per group). e , f Plasma levels of inflammatory cytokines in HFD-fed Redd1 fl/fl and Redd1 Δ Adipoq mice ( n = 6 per group). g Fasting plasma levels of glucose and insulin in HFD-fed Redd1 fl/fl and Redd1 Δ Adipoq mice ( n = 6 per group). h Assessment of GTT and ITT in HFD-fed Redd1 fl/fl and Redd1 Δ Adipoq mice after fasting for 12 and 6 h, respectively ( n = 5 per group). i , j Representative western blots of phosphorylated IRS-1 and Akt in the eWAT and skeletal muscle ( i ) and phosphorylated Akt and FOXO1 in the liver ( j ) of NC- or HFD-fed Redd1 fl/fl and Redd1 Δ Adipoq mice after i.p. injection of saline or insulin ( n = 3). k Relative expression levels of G6pc , Pck1 , and Fbp1 in the liver of HFD-fed Redd1 fl/fl and Redd1 Δ Adipoq mice compared with NC-fed mouse groups ( n = 6 per group). Bar graphs represent mean ± s.e.m. Statistical significance was calculated using an unpaired two-tailed t-test. Source data are provided as a Source Data file.

Article Snippet: In addition, mouse adipose SVF cells and 3T3-L1 preadipocytes (5.0 × 10 5 cells/well, American Type Culture Collection) were plated and transfected with sh Redd1 (#sc-45807-SH, Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Staining, Activity Assay, Clinical Proteomics, Western Blot, Injection, Saline, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: REDD1 promotes obesity-induced metabolic dysfunction via atypical NF-κB activation

doi: 10.1038/s41467-022-34110-1

Figure Lengend Snippet: a Weight gain in Redd1 fl/fl ( R fl/fl ) and Redd1 Δ LysM ( R Δ LysM ) mice fed HFD for 16 weeks ( n = 5 per group). b Measurement of fat (eWAT + iWAT) mass in HFD-fed Redd1 fl/fl and Redd1 Δ LysM mice ( n = 5 per group). c Representative images showing perilipin (green) and F4/80 (purple) staining in the eWAT of HFD-fed Redd1 fl/fl and Redd1 Δ LysM mice ( n = 5 per group). Scale bar, 100 μm. d NF-κB activity in the eWAT from HFD-fed Redd1 fl/fl and Redd1 Δ LysM mice ( n = 5 per group). e Plasma levels of inflammatory cytokines in HFD-fed Redd1 fl/fl and Redd1 Δ LysM mice ( n = 5 per group). f Fasting plasma levels of glucose and insulin in HFD-fed Redd1 fl/fl and Redd1 Δ LysM mice ( n = 5 per group). g Calculation of HOMA-IR scores in HFD-fed Redd1 fl/fl and Redd1 Δ LysM mice ( n = 5 per group). h Assessment of GTT and ITT in HFD-fed Redd1 fl/fl and Redd1 Δ LysM mice fasting for 12 and 6 h, respectively ( n = 5 per group). i , j Representative western blots of the insulin-responsive phosphorylation of IRS-1 and Akt in the eWAT and skeletal muscle ( i ) and phosphorylation of Akt and FOXO1 in the liver ( j ) of NC- or HFD-fed Redd1 fl/fl and Redd1 Δ LysM mice ( n = 3). k Relative expression levels of G6pc , Pck1 , and Fbp1 in the liver of HFD-fed Redd1 fl/fl and Redd1 Δ LysM mice compared with NC-fed mouse groups ( n = 5 per group). Bar graphs represent mean ± s.e.m. Statistical significance was calculated using an unpaired two-tailed t -test. Source data are provided as a Source Data file.

Article Snippet: In addition, mouse adipose SVF cells and 3T3-L1 preadipocytes (5.0 × 10 5 cells/well, American Type Culture Collection) were plated and transfected with sh Redd1 (#sc-45807-SH, Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Staining, Activity Assay, Clinical Proteomics, Western Blot, Phospho-proteomics, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: REDD1 promotes obesity-induced metabolic dysfunction via atypical NF-κB activation

doi: 10.1038/s41467-022-34110-1

Figure Lengend Snippet: a – c Representative oil red-O (ORO)-stained images of WT and Redd1 −/− SVF cells ( a ), shControl (shC)- or sh-Redd1-transfected 3T3-L1 cells ( b ), and WT ( Redd1 fl/f l , R fl/fl ) and Redd1 Δ Adipoq ( R Δ Adipoq ) SVF cells ( c ) when cultured in differentiation medium (MDI) and quantification of relative ORO intensity ( n = 4). d – f , Expression levels of adipogenic genes ( d ), REDD1 ( e ), and lipogenic genes ( f ) in R fl/fl and R Δ Adipoq SVF cells cultured in MDI medium and quantification of relative ORO intensity ( n = 4). g Assessment of NF-κB–Luc activity in 3T3-L1 cells transfected either with siRNA for control, Ikka , Ikkb , or NF-κB p65 ( p65 ) or with pcDNA3.1/His- Ikba ( n = 5). h , i Representative images and realative quantification of ORO-stained images ( h ) and expression levels of Pparg and Cebpa ( i ) in 3T3-L1 cells infected with control adenovirus (Ad-C) or adenoviral Redd1 (Ad- R ) after transfection with vector alone or pcDNA3.1/His- Ikba ( n = 4). j NF-κB–Luc activity in mouse peritoneal macrophages infected with Ad-C or Ad- Redd1 ( n = 4). k Cytokine production in mouse peritoneal macrophages infected with Ad-C or Ad- Redd1 ( n = 5). Bar graphs represent mean ± s.e.m. Statistical significance was calculated using one-way ANOVA ( g , h ) and two-way ANOVA ( a , i ) followed by the Holm–Sidak post hoc test and an unpaired two-tailed t -test ( b – f , j , k ). Source data are provided as a Source Data file.

Article Snippet: In addition, mouse adipose SVF cells and 3T3-L1 preadipocytes (5.0 × 10 5 cells/well, American Type Culture Collection) were plated and transfected with sh Redd1 (#sc-45807-SH, Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Staining, Transfection, Cell Culture, Expressing, Activity Assay, Control, Infection, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: REDD1 promotes obesity-induced metabolic dysfunction via atypical NF-κB activation

doi: 10.1038/s41467-022-34110-1

Figure Lengend Snippet: a Predictive binding conformation between REDD1 and IκBα using computational protein-protein molecular docking methods. b Co-immunoprecipitation analysis of the interaction between REDD1 and IκBα in HEK293 cells transfected with pcDNA3.1/His- Ikba (His- Ikba ) and either pFlag-CMV-1- Redd1 ( Redd1 ) or Redd1 mutants ( R KKAA and R KKRAAA ) ( n = 3). c Representative confocal images of NF-κB p65 nuclear translocalization in HEK293 cells infected with Ad-C, Ad- Redd1 , or its mutants ( n = 4). Scale bar, 50 μm. d Assessment of NF-κB–Luc activity in 3T3-L1 cells infected with Ad-control, Ad- Redd1 , or its mutants ( n = 4). e Representative ORO-stained images of 3T3-L1 cells infected with Ad-control, Ad- Redd1 , or Ad- Redd1 KKAA and quantification of relative ORO intensity ( n = 4). f Expression levels of Pparg and Cebpa in 3T3-L1 cells infected with Ad-control, Ad- Redd1 , or Ad- Redd1 KKAA ( n = 4). g Production of MCP-1 and TNF-α in macrophages infected with Ad-control, Ad- Redd1 , or Ad- Redd1 KKAA ( n = 4). Bar graphs represent mean ± s.e.m. Statistical significance was calculated using one-way ANOVA ( d , e ) and two-way ANOVA ( f , g ) followed by the Holm–Sidak post hoc test. Source data are provided as a Source Data file.

Article Snippet: In addition, mouse adipose SVF cells and 3T3-L1 preadipocytes (5.0 × 10 5 cells/well, American Type Culture Collection) were plated and transfected with sh Redd1 (#sc-45807-SH, Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Infection, Activity Assay, Control, Staining, Expressing

Journal: Nature Communications

Article Title: REDD1 promotes obesity-induced metabolic dysfunction via atypical NF-κB activation

doi: 10.1038/s41467-022-34110-1

Figure Lengend Snippet: a Weight gain in WT and Redd1 KKAA mice after being fed HFD for 16 weeks ( n = 10 per group). b eWAT and iWAT mass measurements in HFD-fed Redd1 KKAA mice and their WT littermates ( n = 10 per group). c Expression levels of Pparg and Cebpa in the eWAT of Redd1 KKAA mice and WT littermates fed HFD for 10 weeks ( n = 8 per group). d Representative images showing perilipin (green) and F4/80 (purple) staining in the eWAT of HFD-fed Redd1 KKAA mice and WT littermates ( n = 5 per group). Scale bar, 100 μm. e NF-κB activity in the eWAT from HFD-fed Redd1 KKAA mice and WT littermates ( n = 6 per group). f , g Plasma levels of inflammatory cytokines in HFD-fed Redd1 KKAA mice and WT littermates ( n = 8 per group). h Fasting plasma levels of glucose and insulin in HFD-fed Redd1 KKAA mice and WT littermates ( n = 8 per group). i Assessment of GTT and ITT in HFD-fed Redd1 KKAA mice and WT littermates after fasting for 12 and 6 h, respectively ( n = 6 per group). j , k Representative western blots of insulin-responsive Akt phosphorylation and plasma membrane-associated GLUT4 (PM-GLUT4) levels in the eWAT and skeletal muscle ( j ) and Akt and FOXO1 phosphorylation in the liver ( k ) of HFD-fed Redd1 KKAA mice and WT littermates ( n = 3). l Relative expression levels of G6pc , Pck1 , and Fbp1 in the liver of HFD-fed Redd1 KKAA mice and WT littermates compared with NC-fed mice ( n = 6 per group). Bar graphs represent mean ± s.e.m. Statistical significance was calculated using an unpaired two-tailed t -test. Source data are provided as a Source Data file.

Article Snippet: In addition, mouse adipose SVF cells and 3T3-L1 preadipocytes (5.0 × 10 5 cells/well, American Type Culture Collection) were plated and transfected with sh Redd1 (#sc-45807-SH, Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Expressing, Staining, Activity Assay, Clinical Proteomics, Western Blot, Phospho-proteomics, Membrane, Two Tailed Test

Journal: Nature Communications

Article Title: REDD1 promotes obesity-induced metabolic dysfunction via atypical NF-κB activation

doi: 10.1038/s41467-022-34110-1

Figure Lengend Snippet: a Representative images of H&E-stained liver tissues from HFD-fed Redd1 −/− , Redd1 ΔAdipoq , Redd1 Δ LysM , Redd1 KKAA , and control mice, and quantification of hepatic steatosis from H&E-stained liver tissues ( n = 6 per group). Scale bars, 100 μm. b Expression levels of Acc , Fasn , and Scd-1 in the liver of HFD-fed Redd1 −/− , Redd1 ΔAdipoq , Redd1 Δ LysM , Redd1 KKAA , and their control mice ( n = 6 per group). Bar graphs represent mean ± s.e.m. Statistical significance was calculated using an unpaired two-tailed t -test. Source data are provided as a Source Data file.

Article Snippet: In addition, mouse adipose SVF cells and 3T3-L1 preadipocytes (5.0 × 10 5 cells/well, American Type Culture Collection) were plated and transfected with sh Redd1 (#sc-45807-SH, Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Staining, Control, Expressing, Two Tailed Test