Journal: Cancer & Metabolism

Article Title: Treatment of ErbB2 breast cancer by mitochondrial targeting

doi: 10.1186/s40170-020-00223-8

Figure Lengend Snippet: Suppression mTORC1 activity by MEDICA. a BT474 cells infected with empty or NDI1 virus were treated for 24 h with 150uM MEDICA as indicated. Representative blots. b , c BT474 cells infected with empty, shSestrin2, or shAMPK were treated for 28 h with MEDICA as indicated. Representative blots. d , e BT474 cells infected with control or shREDD1 were treated for 28 h with MEDICA as indicated. Representative blots. f Suppression of mitochondrial complex I activity by MEDICA results in suppression of mTORC1 activity and lipid raft disruption with loss of ErbB members. The double hit of MEDICA results in abrogating HER2 survival

Article Snippet: ShREDD1 (NM-019058) was from Sigma Mission.

Techniques: Activity Assay, Infection

Journal: EMBO Molecular Medicine

Article Title: REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids

doi: 10.15252/emmm.201404601

Figure Lengend Snippet: A–G B6D2 mice were treated topically with acetone (vehicle control) or glucocorticoid FA (2 μg/animal), every 72 h for 2 weeks. Human volunteers were treated with 0.05% CPB cream applied to the right arm skin once or daily for 2 weeks. Untreated skin from the left arm was used as a control. H&E staining of mouse skin (A) and human skin (E). Scale bars are 20 μm (A) and 40 μm (E). Morphometric analysis of epidermal thickness and number of basal keratinocytes in mouse skin treated with FA 1, 2, and 4 times (B), and human skin treated daily for 2 weeks (F). REDD1 mRNA expression in mouse epidermis 8 h after 1 st , 2 nd , and 4 th applications of FA (C, Q-PCR), in mouse epidermis and s.c. adipose, 24 h after FA (D, Q-PCR), and in human skin 24 h after single (G, volunteers V4 and V5) and 2-week (G, volunteers V1, V2, V3, RT–PCR) treatment with CBP. RPL27 was used as a cDNA normalization control. In human skin, the means ± SD were calculated in each individual sample compared to the untreated skin from the same individual (30 measurements/condition). In mouse skin, the means ± SD were calculated for three individual skin samples/condition in one representative experiment (30 measurements/condition) out of three experiments. Q-PCR results are the means ± SD calculated for three individual RNA samples/condition. Statistical analysis for differences between treatment and control was done by the unpaired two-tailed t -test.

Article Snippet: To knockdown REDD1 expression in keratinocytes, we used lentiviral construct expressing shREDD1 targeting REDD1 3′ UTR sequences homologous in human and mouse (clone V2LHS_176476, Thermo Scientific GIPZ Lentiviral shRNA Library).

Techniques: Control, Cream, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids

doi: 10.15252/emmm.201404601

Figure Lengend Snippet: B6D2 mice were treated topically as in Fig . Skin was harvested 4–24 h after single FA application or during chronic treatment (8 h after 1 st , 2 nd , and 4 th applications). A Immunohistochemical staining of mouse skin treated with acetone or FA for 8 h for REDD1 (upper panels) and phosphorylated mTOR-Ser2448 (lower panels). Scale bars are 20 μm. B Western blot analysis of REDD1 protein and phosphorylation of down-stream mTOR target proteins 4E-BP1 and rpS6 in murine epidermis. GAPDH is used as a normalization control. C, D Glucocorticoids induce autophagy in epidermis. Western blot analysis of Beclin-1 and conversion of light chain 3 (LC3) from LC3-I to LC3-II (C). Quantification of LC3-I to LC3-II conversion and Beclin-1 expression (D). The means ± SD were calculated using Western blots from two independent experiments (each lane is whole-cell protein from three pulled individual samples of epidermis).

Article Snippet: To knockdown REDD1 expression in keratinocytes, we used lentiviral construct expressing shREDD1 targeting REDD1 3′ UTR sequences homologous in human and mouse (clone V2LHS_176476, Thermo Scientific GIPZ Lentiviral shRNA Library).

Techniques: Immunohistochemical staining, Staining, Western Blot, Phospho-proteomics, Control, Expressing

Journal: EMBO Molecular Medicine

Article Title: REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids

doi: 10.15252/emmm.201404601

Figure Lengend Snippet: REDD1 KO and isogenic wild-type B6x129 mice were treated with acetone (vehicle control) or FA (2 μg/animal) every 72 h for 2 weeks. A, B H&E staining (A) and Masson's trichrome staining: Dermis/collagen fibers are blue, muscle is red, nuclei are dark red, and cytoplasm is red/pink (B). Arrows point to epidermis and brackets indicate subcutaneous adipose (A) and dermis (D). Scale bars are 20 μm. C, D Morphometric analysis of epidermal thickness and dermal cellularity as described in Materials and Methods. Changes in epidermal thickness (C) are presented as % to wild-type control epidermis. Changes in dermal cellularity (D) are presented as % to corresponding control skin. The means ± SD were calculated for three individual skin samples in one representative experiment (30 measurements/condition) out of two experiments. Statistical analysis for differences between treatment and control and between control wild-type and REDD1 KO epidermal thickness was done by the unpaired two-tailed t -test.

Article Snippet: To knockdown REDD1 expression in keratinocytes, we used lentiviral construct expressing shREDD1 targeting REDD1 3′ UTR sequences homologous in human and mouse (clone V2LHS_176476, Thermo Scientific GIPZ Lentiviral shRNA Library).

Techniques: Control, Staining, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids

doi: 10.15252/emmm.201404601

Figure Lengend Snippet: REDD1 KO and wild-type animals were treated as in Fig . A–C Expression of p63 (A) and CD34 (C). Scale bars are 10 μm. Analysis of p63 staining (B). The number of p63 + basal keratinocytes/total number of basal keratinocytes is presented as % to the corresponding control epidermis. D Similar GR expression in epidermis of wild-type and REDD1 KO mice determined by Q-PCR and Western blotting. Rpl27 and GAPDH used as a normalization controls, respectively. Data information: The means ± SD were calculated for three individual skin samples in one representative experiment (30 measurements/condition) out of two experiments. Q-PCR results are presented as the means ± SD for three individual RNA samples/condition. Statistical analysis for differences between treatment and control was done by the unpaired two-tailed t -test.

Article Snippet: To knockdown REDD1 expression in keratinocytes, we used lentiviral construct expressing shREDD1 targeting REDD1 3′ UTR sequences homologous in human and mouse (clone V2LHS_176476, Thermo Scientific GIPZ Lentiviral shRNA Library).

Techniques: Expressing, Staining, Control, Western Blot, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids

doi: 10.15252/emmm.201404601

Figure Lengend Snippet: Ear edema was induced by croton oil (CO) as in Materials and Methods. FA was applied 1 h before CO, and four-millimeter ear punch was weighed 9 h after CO application to assess swelling. In the additional experiment, ears were harvested 9 h after CO application and used for histological analysis. A H&E staining. Scale bars are 20 μm. B Ear punch weight. Results are presented as % to corresponding (wild-type or REDD1 KO) control ear weight. The means ± SD were calculated for six individual ear punches/condition in one representative experiment (out of three experiments). Statistical analysis for differences between treatment and corresponding control was done by the unpaired two-tailed t -test.

Article Snippet: To knockdown REDD1 expression in keratinocytes, we used lentiviral construct expressing shREDD1 targeting REDD1 3′ UTR sequences homologous in human and mouse (clone V2LHS_176476, Thermo Scientific GIPZ Lentiviral shRNA Library).

Techniques: Staining, Control, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids

doi: 10.15252/emmm.201404601

Figure Lengend Snippet: Organotypic raft cultures (ORC) made from NHEK infected with REDD1 shRNA and control pGIPZ lentiviruses were treated with glucocorticoid CBP (5 μM) or vehicle control (0.05% DMSO) for 7 days. A, B H&E and BrdU staining of raft cultures (BrdU + cells are indicated by arrowheads). Scale bars are 10 μm. C Western blot analysis of REDD1, GR, and mTOR substrate 4E-BP1 phosphorylation. GAPDH was used as a loading control. D Q-PCR analysis of REDD1 expression in rafts. E Analysis of CBP effect on basal keratinocyte number (left) and keratinocyte proliferation (number of BrdU + basal keratinocytes/total number of basal keratinocytes, right) is presented as % to corresponding control rafts. Data information: The means ± SD for BrdU + cells and basal keratinocytes in (E) were calculated for two individual rafts in one representative experiment (20 measurements/condition) out of two experiments. Q-PCR results in (D) are the means ± SD calculated for two individual RNA samples/condition. Statistical analysis for differences between treatment and control was done by the unpaired two-tailed t -test.

Article Snippet: To knockdown REDD1 expression in keratinocytes, we used lentiviral construct expressing shREDD1 targeting REDD1 3′ UTR sequences homologous in human and mouse (clone V2LHS_176476, Thermo Scientific GIPZ Lentiviral shRNA Library).

Techniques: Infection, shRNA, Control, BrdU Staining, Western Blot, Phospho-proteomics, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids

doi: 10.15252/emmm.201404601

Figure Lengend Snippet: REDD1 KO and isogenic B6x129 mice were treated topically with FA (2 µg) or vehicle (acetone) for 24 h, and RNA was extracted from the epidermis and used for microarray analysis. A A heatmap of gene expression as analyzed by the Mouse Whole-Genome Gene Expression BeadChips (Illumina). Columns show normalized gene expression for individual animals (red: increased, green: decreased expression). B Venn diagrams show overlap of differentially expressed genes between REDD1 KO and wild-type mice (adjusted P < 0.05). Arrows connect sections of the heatmap with corresponding Venn diagrams. C Most enriched gene ontology (GO) categories for biological processes and molecular functions (fold-change enrichment ≥ 3, P < 0.01), associated with transrepression and transactivation of the glucocorticoid-responsive genes in REDD1 KO and wild-type mice. D Array validation by Q-PCR for genes from the different GO categories. Note: lack of overlap in GO categories between KO and wild-type mice associated with up-regulated genes and significant overlap in the function of down-regulated genes. Data information: We used for array analysis two individual RNA samples/condition. Statistical analysis of DNA arrays is described in Materials and Methods. Statistical analysis of array validation is shown in .

Article Snippet: To knockdown REDD1 expression in keratinocytes, we used lentiviral construct expressing shREDD1 targeting REDD1 3′ UTR sequences homologous in human and mouse (clone V2LHS_176476, Thermo Scientific GIPZ Lentiviral shRNA Library).

Techniques: Microarray, Gene Expression, Expressing, Biomarker Discovery

Journal: EMBO Molecular Medicine

Article Title: REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids

doi: 10.15252/emmm.201404601

Figure Lengend Snippet: HaCaT human keratinocytes were infected with shREDD1 or pGIPZ (control) lentiviruses and treated with glucocorticoid FA (10 −6 M) for the indicated time. Western blot analysis of REDD1, GR, and phosphorylated GR-Ser211. GAPDH used as a loading control. Reduced induction of Luciferase reporter in shREDD1-HaCaT cells. shREDD1- and pGIPZ-HaCaT cells were infected with GRE.Luc lentivirus and treated with FA (10 −6 M) for 24 h. The Luciferase induction is presented as a fold change to corresponding vehicle-treated control. The means ± SD were calculated for three individual wells/group in one representative experiment (out of three experiments). Statistical analysis for differences between groups was done by ANOVA. Immunofluorescence analysis of GR nuclear translocation and retention in shREDD1- and pGIPZ-HaCaT cells treated with FA (10 −6 M). Scale bars are 10 μm.

Article Snippet: To knockdown REDD1 expression in keratinocytes, we used lentiviral construct expressing shREDD1 targeting REDD1 3′ UTR sequences homologous in human and mouse (clone V2LHS_176476, Thermo Scientific GIPZ Lentiviral shRNA Library).

Techniques: Infection, Control, Western Blot, Luciferase, Immunofluorescence, Translocation Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Loss of REDD1 prevents chemotherapy‐induced muscle atrophy and weakness in mice

doi: 10.1002/jcsm.12795

Figure Lengend Snippet: Carboplatin caused C2C12 myotube atrophy and increased REDD1 expression. C2C12 myotubes were treated with carboplatin for 24 or 48 h. ( A ) C2C12 myotube diameter was decreased after carboplatin treatment compared with vehicle in C2C12 cells differentiated for either 4 or 6 days. Scale bar = 50 μm. ( B ) REDD1 mRNA expression was increased in C2C12 myotubes differentiation for 4 days and treated with carboplatin for either 24 or 48 h. ( C ) REDD1 protein expression was increased in C2C12 myotubes differentiation for 4 days and treated with carboplatin for either 24 or 48 h. ( A ) One‐way ANOVA with Tukey's post‐hoc test for multiple comparisons, or Student's t ‐test. ( B–C ) Two‐way ANOVA with Tukey's post‐hoc test for multiple comparisons. * P < .05 ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( A ) Approximately 100 myotubes assessed, n = 3 biological replicates for all groups. ( B–C ) n = 3 biological replicates for all groups.

Article Snippet: pLKO.1 (Addgene plasmid #10878 ) (shControl), pcDNA3‐EGFP (Addgene plasmid #13031), and REDD1 shRNA (shREDD1) construct TRCN0000176020 (Sigma, Darmstadt, Germany) were used for in vivo electroporation.

Techniques: Expressing

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Loss of REDD1 prevents chemotherapy‐induced muscle atrophy and weakness in mice

doi: 10.1002/jcsm.12795

Figure Lengend Snippet: Loss of REDD1 in vivo attenuates carboplatin‐induced cachexia. ( A ) REDD1 mRNA expression in the TA muscle of wild‐type or REDD1 knockout (REDD1 KO) mice treated with carboplatin. ( B ) Whole animal body weight of mice treated with vehicle or carboplatin. ( C ) Weight of the TA, gastrocnemius, EDL and soleus muscles of mice treated with vehicle or carboplatin. ( A ) Student's t ‐test * P < 0.05. ( B–C ) Two‐way ANOVA with Tukey's post‐hoc test for multiple comparisons. ( B ) * P < 0.05, ** P < 0.01 wild‐type + vehicle compared with wild‐type + carboplatin; ‡ P < 0.05 wild‐type + carboplatin compared with REDD1 KO + carboplatin. ( C )* P < 0.05, ** P < 0.01, **** P < 0.0001. n = 8 biological replicates for each group ( A–C ).

Article Snippet: pLKO.1 (Addgene plasmid #10878 ) (shControl), pcDNA3‐EGFP (Addgene plasmid #13031), and REDD1 shRNA (shREDD1) construct TRCN0000176020 (Sigma, Darmstadt, Germany) were used for in vivo electroporation.

Techniques: In Vivo, Expressing, Knock-Out

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Loss of REDD1 prevents chemotherapy‐induced muscle atrophy and weakness in mice

doi: 10.1002/jcsm.12795

Figure Lengend Snippet: Loss of REDD1 prevents carboplatin‐induced myofibre atrophy. ( A ) Representative cross sections and CSA quantification from the TA muscle of mice treated with vehicle or carboplatin. Scale bar = 50 μm. ( B ) Myofibre distribution frequency from the TA muscle of mice treated with vehicle or carboplatin. ( C ) Representative cross sections and quantification of MyHC fibre type from the TA muscle of mice treated with vehicle or carboplatin. ( A , C ) Two‐way ANOVA with Tukey's post‐hoc test for multiple comparisons. ** P < 0.01. n = 3 biological replicates for ( A–C ). Average CSA from ~200 fibres per replicate for ( A–B ) and fibre‐type quantitation ( C ).

Article Snippet: pLKO.1 (Addgene plasmid #10878 ) (shControl), pcDNA3‐EGFP (Addgene plasmid #13031), and REDD1 shRNA (shREDD1) construct TRCN0000176020 (Sigma, Darmstadt, Germany) were used for in vivo electroporation.

Techniques: Quantitation Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Loss of REDD1 prevents chemotherapy‐induced muscle atrophy and weakness in mice

doi: 10.1002/jcsm.12795

Figure Lengend Snippet: Loss of REDD1 attenuates carboplatin‐induced protein synthesis. Puromycin incorporation in vivo from the TA muscle of mice treated with vehicle or carboplatin. Two‐way ANOVA with Tukey's post‐hoc test for multiple comparisons * P < 0.05. n = 3 biological replicates for each group.

Article Snippet: pLKO.1 (Addgene plasmid #10878 ) (shControl), pcDNA3‐EGFP (Addgene plasmid #13031), and REDD1 shRNA (shREDD1) construct TRCN0000176020 (Sigma, Darmstadt, Germany) were used for in vivo electroporation.

Techniques: In Vivo

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Loss of REDD1 prevents chemotherapy‐induced muscle atrophy and weakness in mice

doi: 10.1002/jcsm.12795

Figure Lengend Snippet: Knockdown of REDD1 expression both in vitro and in vivo attenuates carboplatin‐induced myofibre atrophy. ( A ) Representative images of carboplatin‐treated C2C12 myotubes electroporated with REDD1 shRNA or control shRNA and quantification of GFP positive myotube diameter. ( B ) REDD1 mRNA expression from C2C12 treated with carboplatin. ( C ) Representative cross sections and CSA quantification of GFP positive fibres. ( D ) REDD1 mRNA expression from the TA of carboplatin‐treated mice electroporated with shControl or shREDD1. ( A – D ) Two‐way ANOVA with Tukey's post‐hoc test for multiple comparisons. ** P < 0.01, *** P < 0.001. n = 3 biological replicates for ( A – D ). Average myotube diameter from ~75 GFP positive myotubes per replicate ( A ). Average CSA from ~100 GFP positive fibres per replicate for ( C ). ( A , C ) Scale bar = 50 μm. n = 3 biological replicates for ( A – D ).

Article Snippet: pLKO.1 (Addgene plasmid #10878 ) (shControl), pcDNA3‐EGFP (Addgene plasmid #13031), and REDD1 shRNA (shREDD1) construct TRCN0000176020 (Sigma, Darmstadt, Germany) were used for in vivo electroporation.

Techniques: Expressing, In Vitro, In Vivo, shRNA

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Loss of REDD1 prevents chemotherapy‐induced muscle atrophy and weakness in mice

doi: 10.1002/jcsm.12795

Figure Lengend Snippet: Loss of REDD1 prevents carboplatin‐induced muscle weakness. ( A ) Deletion of REDD1 prevents carboplatin‐induced forelimb grip strength reduction in vivo . ( B ) Absolute force generated from the EDL muscle of mice treated with vehicle or carboplatin. ( C ) Specific force of the EDL muscle of mice treated with vehicle or carboplatin. ( A – C ) Two‐way ANOVA with Tukey's post‐hoc test for multiple comparisons. (A) *** P < 0.001, compared with wild‐type + vehicle. (B) * P < 0.05, ** P < 0.01 all groups compared with wild‐type + carboplatin. ( C ) * P < 0.05, ** P < 0.01 wild‐type + vehicle compared with wild‐type + carboplatin n = 8 biological replicates for all groups.

Article Snippet: pLKO.1 (Addgene plasmid #10878 ) (shControl), pcDNA3‐EGFP (Addgene plasmid #13031), and REDD1 shRNA (shREDD1) construct TRCN0000176020 (Sigma, Darmstadt, Germany) were used for in vivo electroporation.

Techniques: In Vivo, Generated