Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A ) Sliver staining of the mass spectrometry sample. * represent the Gli3 full-length, ** represent the Gli3 repressor. ( B and C ) The mass spectrometry results of the methylation modification on K436 ( B ) and K595 ( C ) in the Gli3 full-length sample. DOI: http://dx.doi.org/10.7554/eLife.15690.004

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Staining, Mass Spectrometry, Methylation, Modification

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A ) Sequence alignment of the reported Set7 substrates with Gli3K436 and Gli3K595 (upper). Schematic representation of Gli3 protein and the truncated peptide used in in vitro methylation assay ( B ) and GST pull-down assay ( C ) (lower). ( B ) In vitro methylation assay with 3 H-S-adenosine-methionine ( 3 H-SAM), bacteria purified Set7 and MBP fusion protein. * and ** represent the MBP-K436 and MBP-K595 respectively. ( C ) GST pull-down assay using GST-Set7 and MBP tagged Gli3 truncated fragments described in ( A ). * and ** represent the MBP-K436 and MBP-K595 respectively. ( D–F ) Western blot of immunoprecipitates (top three panels) and lysates (bottom) from HEK293T cells expressing indicated siRNAs or proteins. * and ** represent the full-length and repressor forms of Gli3 respectively. ( G–I ) Western blot of immunoprecipitates (top three panels) and lysates (bottom) from NIH-3T3 cells stably expressing indicated shRNAs or proteins. ( I’ ) Schematic representation of 6 PKA targeted serines which were mutated to nonphosphorylatable alanines in Gli3 PA . ( J ) GST pull-down assay using GST-Set7 and flag-Gli3 in NIH-3T3 cells in the presence of Shh. Ctrl, Control. Me-K436, antibody anti methylated Gli3-K436; Me-K595, antibody anti methylated Gli3-K595. WCL, whole cell lysis. The protein level of Gli3 in ( D–I ) are normalized to the same. DOI: http://dx.doi.org/10.7554/eLife.15690.003

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Sequencing, In Vitro, Methylation, Pull Down Assay, Bacteria, Purification, Western Blot, Expressing, Stable Transfection, Control, Lysis

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A ) and K595 ( B ) in Gli3 from different species suggested that these two sites are evolution conserved. DOI: http://dx.doi.org/10.7554/eLife.15690.005

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques:

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A ) The sequences of peptides used for preparing antibodies. ( B ) Dot blots of peptides described in ( A ). Me-K436, antibody against methylated Gli3-K436; Me-K595, antibody against methylated Gli3-K595. DOI: http://dx.doi.org/10.7554/eLife.15690.006

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Methylation

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: Methylation signals on Gli3 full length were detected by western blots. DOI: http://dx.doi.org/10.7554/eLife.15690.007

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Methylation, Western Blot

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A–C ) Shh luciferase reporter assays in NIH-3T3 cells expressing indicated siRNAs or proteins. ( A’ ) Set7 level changes in NIH-3T3 cells expressing indicated Set7 siRNAs. n.s. p>0.05, ***p<0.005 versus siCtrl respectively in ( A ). ( D and E ) qPCR to detect Shh target gene, Gli1 and Ptch1 , in Shh treated NIH-3T3 cells after Set7 knockdown. ***p<0.005 versus siCtrl. ( F ) Western blot to detect protein level changes of Set7 and Gli1 in ( D and E ). ( G ) Shh luciferase reporter assay in Shh treated NIH-3T3 cells transfected with indicated siRNAs. ***p<0.001, n.s. p>0.05 versus siCtrl siCtrl respectively. ( H–J ) mRNA level changes of Gli1 and Ptch1 ( H and I ) and Shh luciferase activity changes ( J ) in Shh treated NIH-3T3 cells transfected with unmethylatable mutants, Gli3 K436R and Gli3 K595R . *p<0.05, **p<0.01, ***p<0.001 versus Gli3WT ( K and L ) Shh luciferase assay in Shh treated NIH-3T3 cells expressing indicated siRNAs or/and proteins., **p<0.01, ***p<0.001 versus siCtrl Gli3WT or PCDNA Gli3WT respectively.. Results in ( A–E , G – L ) are shown as mean ± SEM (n=3). All the qPCR results are normalized to GAPDH. Ctrl, Control. DOI: http://dx.doi.org/10.7554/eLife.15690.008

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Luciferase, Expressing, Knockdown, Western Blot, Reporter Assay, Transfection, Activity Assay, Control

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: The protein density of Gli3 full length and methylation signals is analyzed using ImageJ. DOI: http://dx.doi.org/10.7554/eLife.15690.010

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Methylation

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A and B ) Immunofluorescent staining of Ac-tubulin (blue) and Gli3 (red) in the absence ( A-A’’ ) or presence ( B-B’’ ) of Shh. ( C and D ) Statistic results of Gli3 accumulation at cilia tips in NIH-3T3 cells with Set7 knockdown ( C ) or Set7/Set7 H297A over-expression ( D ). In all experiments of ( C and D ), cells were treated with Shh. Data are represented as mean ± SEM (n=3). Ctrl, Control. DOI: http://dx.doi.org/10.7554/eLife.15690.014

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Staining, Knockdown, Over Expression, Control

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A and B ) Western blot ( A ) and qPCR ( B ) of Gli3 in the condition of Set7 knockdown or overexpression in NIH-3T3 cells. n.s. p>0.05 versus siCtrl (left) or PCDNA (right) respectively. ( C and D ) Western blot of Gli3 WT , Gli3 K436R or Glli3 K595R in the condition of Set7 knockdown or overexpression in NIH-3T3 cells. ( E and F ) Western blot ( E ) and statistic ( F ) of Gli3 WT , Gli3 K436R or Gli3 K595R in NIH-3T3 cells treated with cycloheximide (CHX) for the indicated times. ( G ) Schematic representation of three Gli3 binding sites on the promoter regions of Gli1 . ( H ) qPCR of Set7 in Gli3 WT -flag stable cell lines after Set7 knockdown in ( I ). **p<0.01, ***p<0.00 versus siCtrl. ( I ) ChIP-qPCR analysis using Ctrl IgG (grey) or anti-flag antibody (black) in NIH-3T3 cells transfected with Ctrl or Set7 siRNAs. ChIP signal levels are represented as fold change of input chromatin. ( J ) Western blot of flag tagged Gli3 WT and mutants in the stable cell lines used in ( K ). ( K ) ChIP-qPCR analysis using anti-flag antibody in NIH-3T3 cells expressing Gli3 WT -flag, Gli3 K436R -flag or Gli3 K595R -flag. ChIP signal levels are represented as fold change of input chromatin. *p<0.05, **p<0.01, ***p<0.001, n.s. p>0.05 versus anti flag siCtrl ( I ) or Gli3 WT -flag ( K ). Data are represented as mean ± SEM (n=3). All the qPCR results are normalized to GAPDH. In all the experiments, cells are treated with Shh. Ctrl, Control. DOI: http://dx.doi.org/10.7554/eLife.15690.013

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Western Blot, Knockdown, Over Expression, Binding Assay, Stable Transfection, ChIP-qPCR, Transfection, Expressing, Control

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A ) ChIP-qPCR analysis at Gli1 loci using control IgG or anti-Gli3 antibody in NIH-3T3 cells transfected with control or Set7 siRNAs. ChIP signal levels are represented as fold change of input chromatin. **p<0.01, ***p<0.001, versus anti Gli3 siCtrl. ( B ) qPCR of Set7 in NIH-3T3 cells with Set7 knockdown. RNAi efficiency was shown. Data are represented as mean ± SEM (n=3). **p<0.01 versus siCtrl. Ctrl, Control. All the qPCR results are normalized to GAPDH. DOI: http://dx.doi.org/10.7554/eLife.15690.015

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: ChIP-qPCR, Control, Transfection, Knockdown

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A ) and Ptch1 ( B ) loci using control IgG or anti-flag antibody in NIH-3T3 Gli3-flag stable cells transfected with control or Set7 siRNA. ChIP signal levels are represented as fold change of input chromatin. *p<0.05, **p<0.01, ***p<0.001, versus anti flag siCtrl. DOI: http://dx.doi.org/10.7554/eLife.15690.016

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Control, Transfection

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A and B ) Shh luciferase reporter assay ( A ) and qPCR ( B ) of Gli1, Ptch1 and Set7 in A549 cells in the condition of Set7 knockdown. ( C ) Western of flagged Gli3 WT , Gli3 K436R , Gli3 K595R or Gli3 KRKR in A549 stable cells used in ( D - M ). ( D and E ) qPCR ( D ) of Gli1 and Ptch1 and Shh luciferase reporter assay ( E ) in A549 cells expressing Gli3 WT , Gli3 K436R , Gli3 K595R or Gli3 KRKR . ( F ) MTT assays in A549 cells expressing indicated proteins. ( G and G’ ) Photography ( G ) and statistic results ( G’ ) of anchorage-independent growth assay in A549 cells expressing indicated proteins. ( H and I ) Statistic ( H ) and photography ( I ) results of tumor size in A549 cells expressing indicated proteins. ( J ) Tumor growth curves of A549 cells express indicated protein in a xenograft mouse model. ( K–M ) Migration ability evaluated by wound healing assay ( K ) and transwell migration assay ( L ). Invasiveness evaluated by matrigel invasion assay ( M ) in A549 cells expressing indicated proteins. All the qPCR results are normalized to GAPDH. Data in ( D–H , J ) were represented as mean ± SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Gli3 WT . Ctrl, Control. DOI: http://dx.doi.org/10.7554/eLife.15690.017

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Luciferase, Reporter Assay, Knockdown, Western Blot, Expressing, Growth Assay, Migration, Wound Healing Assay, Transwell Migration Assay, Invasion Assay, Control

Journal: eLife

Article Title: Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

doi: 10.7554/eLife.15690

Figure Lengend Snippet: ( A ) Western blot of flagged Gli3 PAWT , Gli3 PAK436R , Gli3 PAK595R or Gli3 PAKRKR in A549 stable cells used in ( B-H ). ( B and C ) Hh luciferase reporter assay ( B ) and qPCR ( C ) of Gli1 and Ptch1 in A549 cells expressing flagged Gli3 PAWT , Gli3 PAK436R , Gli3 PAK595R or Gli3 PAKRKR . ( D ) MTT assays in A549 cells expressing indicated proteins. ( E and E’ ) Photography ( E ) and statistic results ( E’ ) of anchorage-independent growth assay in A549 cells expressing indicated proteins. ( F-H ) Migration ability evaluated by wound healing assay ( F ) and transwell migration assay ( G ). Invasiveness evaluated by matrigel invasion assay ( H ) in A549 cells expressing indicated proteins. ( G’ and H’ ) Statistic results of migration ( G’ ) and invasion ( H’ ) ability of A549 cells expressing indicated proteins. Data in ( B-D, G’, H’ ) were represented as mean ± SEM (n=3). Ctrl, Control. All the qPCR results are normalized to GAPDH. DOI: http://dx.doi.org/10.7554/eLife.15690.019

Article Snippet: The Gli3 PA plasmid was obtained from Addgene (Plasmid# 226672).

Techniques: Western Blot, Luciferase, Reporter Assay, Expressing, Growth Assay, Migration, Wound Healing Assay, Transwell Migration Assay, Invasion Assay, Control