Journal: Nature Communications

Article Title: MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57 Kip2 targeting

doi: 10.1038/s41467-023-44130-0

Figure Lengend Snippet: a Representative western blot on cells transfected with either Scrambled (siSCR) or SKP2 siRNA (siSKP2). Vinculin, GAPDH and β-Actin are the loading controls. b mRNA levels of MYOG , MYOD1 and CDKN1C on cells treated as in ( a ) were reported as fold increase over siSCR (1 arbitrary unit, not reported). Data presented as mean values ± SD, Student’s two-tailed t -test. c mRNA levels of MyH2 on cells treated as in ( a ) in growth medium (GM) for 4 days post-transfection were expressed as fold increase over siSCR (1 arbitrary unit). Data presented as mean values ± SD, Student’s two-tailed t -test. d Representative immunofluorescence on cells treated as in ( a ) for 6 days in GM showing expression of Myosin Heavy Chain (MyHC) (green). DAPI (blue) stained nuclei. Scale Bar = 100 μm. e Histogram depicts the quantification of MyHC-positive cells treated as in ( d ). Data presented as mean values ± SD, Student’s two-tailed t -test. f Representative western blot on cells infected with either shSCR, shSKP2.1 or shSKP2.2. Vinculin is the loading control. g Representative immunofluorescence on cells treated as in ( f ) for 6 days, showing expression of MyHC (green). DAPI (blue) stained nuclei. Scale Bar = 100 μm. h Histogram depicts the quantification of MyHC-positive cells treated as in ( g ). Data presented as mean values ± SD, two-way ANOVA. i Representative western blot on cells treated with SMIP004 20 μM for 168 h. Vinculin is the loading control. j mRNA levels of MYOG , MYOD1 and CDKN1C on cells treated as in ( i ) were reported as fold increase over DMSO (1 arbitrary unit, not reported). Data presented as mean values ± SD, Student’s two-tailed t -test. k mRNA levels of MyH2 on cells treated as in ( i ), cultured in GM, were expressed as fold increase over DMSO (1 arbitrary unit). Data presented as mean values ± SD, Student’s two-tailed t -test. l Representative immunofluorescence of cells treated as in ( i ) showing expression of MyHC (green). DAPI (blue) stained nuclei. Scale Bar = 100 μm. m Histogram depicts the quantification of MyHC-positive cells treated as in ( i ). Data presented as mean values ± SD, Student’s two-tailed t -test. All presented data derive from n = 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Sections were incubated overnight at +4 °C with mouse monoclonal α-SKP2 antibody (dilution 1:50; 32-3300, Thermofisher Scientific, Lafayette, CO, USA), mouse monoclonal α-p27 Kip1 antibody (dilution 1:50; SX53G8.5, sc-53871, Santa Cruz); mouse monoclonal α-p57 Kip2 antibody (dilution 1:100; KP39, sc-56341, Santa Cruz); mouse monoclonal α-MYOG antibody (dilution 1:100; F5D-c, DSHB); Rabbit polyclonal α-Cleaved Caspase 3 antibody (dilution 1:300; #9661, Cell Signaling Technology); Rabbit polyclonal α- p21 Cipl (1:50, 12D1, Cell Signaling Technology Cat# 2947); or mouse monoclonal α-MyHC antibody (dilution 1:50; MF20, DSHB) and α-Ki67 antibody (ready to use; IR626, Dako).

Techniques: Western Blot, Transfection, Two Tailed Test, Immunofluorescence, Expressing, Staining, Infection, Control, Cell Culture

Journal: Nature Communications

Article Title: MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57 Kip2 targeting

doi: 10.1038/s41467-023-44130-0

Figure Lengend Snippet: a Representative western blot ( n = 3 independent experiments) of the indicated proteins on RD and JR1 cells treated for 8 h with either vehicle (DMSO), 10.0 or 7.5 μM of MG132. Vinculin is the loading control. b Representative western blot ( n = 3 independent experiments) of co-Immunoprecipitation of endogenous SKP2 in RD and JR1 cells showing SKP2 and p57 Kip2 . c Representative confocal pictures ( n = 3 independent experiments) of Proximal Ligation Assay (PLA) for SKP2 and p57 Kip2 on RD and JR1 cells. SKP2/p57 Kip2 direct close proximity/interactions are visualized as red dots (upper). Nuclei were stained with DAPI (blue) and membrane glycoproteins with WGA (green) (see Methods). PLA negative controls were performed by using either p57 Kip2 (middle) or SKP2 (lower) antibodies alone. Scale Bar = 10 μm. d Representative western blot ( n = 3 independent experiments) of the indicated proteins on RD and JR1 cells transfected with either SCR, SKP2, p57 Kip2 or SKP2 + p57 Kip2 siRNA at 72 h post-transfection. Vinculin is the loading control. e Representative immunofluorescence of RD and JR1 cells treated as in ( d ) and cultured for 6 days in growth medium (GM, supplemented with 10% serum), showing expression of Myosin Heavy Chain (MyHC) (green). Nuclei were stained with DAPI (blue). Scale Bar = 100 μm. f Histogram depicts the quantification of MyHC-positive cells treated as in ( e ). n = 3 independent experiments, data presented as mean values ± SD, two-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: Sections were incubated overnight at +4 °C with mouse monoclonal α-SKP2 antibody (dilution 1:50; 32-3300, Thermofisher Scientific, Lafayette, CO, USA), mouse monoclonal α-p27 Kip1 antibody (dilution 1:50; SX53G8.5, sc-53871, Santa Cruz); mouse monoclonal α-p57 Kip2 antibody (dilution 1:100; KP39, sc-56341, Santa Cruz); mouse monoclonal α-MYOG antibody (dilution 1:100; F5D-c, DSHB); Rabbit polyclonal α-Cleaved Caspase 3 antibody (dilution 1:300; #9661, Cell Signaling Technology); Rabbit polyclonal α- p21 Cipl (1:50, 12D1, Cell Signaling Technology Cat# 2947); or mouse monoclonal α-MyHC antibody (dilution 1:50; MF20, DSHB) and α-Ki67 antibody (ready to use; IR626, Dako).

Techniques: Western Blot, Control, Immunoprecipitation, Ligation, Staining, Membrane, Transfection, Immunofluorescence, Cell Culture, Expressing

Journal: Nature Communications

Article Title: MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57 Kip2 targeting

doi: 10.1038/s41467-023-44130-0

Figure Lengend Snippet: a Representative western blot ( n = 3 independent experiments) of the indicated proteins on RD and JR1 cells infected with either Scrambled (shSCR), SKP2.1 (shSKP2.1) or SKP2.2 (shSKP2.2) lentiviral shRNA at 72 h post-selection. Vinculin is the loading control. b Representative light microscopy pictures of soft agar colony formation assay on RD and JR1 cells treated as in ( a ) and grown for 2 weeks. Scale bar = 50 μm. c Histogram depicts the quantification of soft agar colony numbers per field. n = 3 (RD) and n = 4 (JR1) independent experiments, data presented as mean values ± SD, one-way ANOVA. d Representative light microscopy pictures of single cell colony formation assay on RD and JR1 cells treated as in ( a ) and grown for 2 weeks. Scale bar = 50 μm. e Histogram depicts the quantification of colony numbers per field. n = 3 independent experiments, data presented as mean values ± SD, one-way ANOVA. f Representative light microscopy pictures of β-Galactosidase staining of RD and JR1 cells transfected with either SCR or SKP2 siRNA. Scale bar = 100 μm. g Histogram depicts the quantification of the percentage of senescent cells per field. n = 3 independent experiments, data presented as mean values ± SD, Student’s two-tailed t -test. h Images of JR1 shSCR, shSKP2.1, and shSKP2.2 tumors explanted from mice post euthanasia at 48 days post-inoculation. i Tumor volume of shSCR ( n = 11), shSKP2.1 ( n = 5) and shSKP2.2 ( n = 6) JR1 xenografts assessed by caliper measurement represented in mm 3 followed for 48 days post-inoculation. Data presented as mean values ± SD, two-way ANOVA. j Tumor weight of shSCR ( n = 11), shSKP2.1 ( n = 5) and shSKP2.2 ( n = 6) JR1 xenografts. Box plots show 25th to 75th quartiles, black bar shows the median, and whiskers go down to the smallest value and up to the largest. One-way ANOVA k Representative images ( n = 3 independent experiments) of H&E, p27 Kip1 , p57 Kip2 , MYOG, MyHC, and Ki67 immunohistochemistry of tumor sections from JR1 xenografts expressing either shSCR or shSKP2.2. Scale Bars = 100 μm. Source data are provided as a Source Data file.

Article Snippet: Sections were incubated overnight at +4 °C with mouse monoclonal α-SKP2 antibody (dilution 1:50; 32-3300, Thermofisher Scientific, Lafayette, CO, USA), mouse monoclonal α-p27 Kip1 antibody (dilution 1:50; SX53G8.5, sc-53871, Santa Cruz); mouse monoclonal α-p57 Kip2 antibody (dilution 1:100; KP39, sc-56341, Santa Cruz); mouse monoclonal α-MYOG antibody (dilution 1:100; F5D-c, DSHB); Rabbit polyclonal α-Cleaved Caspase 3 antibody (dilution 1:300; #9661, Cell Signaling Technology); Rabbit polyclonal α- p21 Cipl (1:50, 12D1, Cell Signaling Technology Cat# 2947); or mouse monoclonal α-MyHC antibody (dilution 1:50; MF20, DSHB) and α-Ki67 antibody (ready to use; IR626, Dako).

Techniques: Western Blot, Infection, shRNA, Selection, Control, Light Microscopy, Soft Agar Assay, Colony Assay, Staining, Transfection, Two Tailed Test, Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57 Kip2 targeting

doi: 10.1038/s41467-023-44130-0

Figure Lengend Snippet: a A model showing that in myoblasts and RMS cells that are both proliferating, H3K27ac (Ac) enrichment at SKP2 DNA regulatory regions allows MYOD, which is bound on these regions, to induce the expression of SKP2 thus supporting proliferation (left). Upon differentiation cues, reduction of H3K27ac enrichment results in a closed chromatin conformation, impairing MYOD ability to induce SKP2 transcription. These events promote differentiation, which is inhibited in RMS (right). b A model showing that in RMS cells, MYOD induces the expression of SKP2 , which in turn associates with the SCF/CRL1 complex to promote ubiquitylation and proteasomal degradation of p27 Kip1 and p57 Kip2 by direct interaction and p21 Cip1 in an indirect way, promoting oncogenic features (left). These SKP2 functions can be suppressed by SKP2 silencing (upper right) or by its functional inhibition by the SKP2 inhibitor SMIP004 or the NEDDylation inhibitor MLN4924 (lower right) to block RMS oncogenic properties. NAE NEDD8-activating enzyme, Ub Ubiquitin, N8 NEDD8. Created with BioRender.com.

Article Snippet: Sections were incubated overnight at +4 °C with mouse monoclonal α-SKP2 antibody (dilution 1:50; 32-3300, Thermofisher Scientific, Lafayette, CO, USA), mouse monoclonal α-p27 Kip1 antibody (dilution 1:50; SX53G8.5, sc-53871, Santa Cruz); mouse monoclonal α-p57 Kip2 antibody (dilution 1:100; KP39, sc-56341, Santa Cruz); mouse monoclonal α-MYOG antibody (dilution 1:100; F5D-c, DSHB); Rabbit polyclonal α-Cleaved Caspase 3 antibody (dilution 1:300; #9661, Cell Signaling Technology); Rabbit polyclonal α- p21 Cipl (1:50, 12D1, Cell Signaling Technology Cat# 2947); or mouse monoclonal α-MyHC antibody (dilution 1:50; MF20, DSHB) and α-Ki67 antibody (ready to use; IR626, Dako).

Techniques: Expressing, Functional Assay, Inhibition, Blocking Assay, Ubiquitin Proteomics