A and C . Root mean square deviation ( RMSD ) analysis of the movement of ( A ) αC - helix and ( C ) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation ( orange ) compared to the wild type ( WT ) kinase ( black ) . B and D . Time - lapse images show movement within the αC - helix ( B ) and DFG motif ( D ) of the inactive structure of the WT ( black outline ) and R351W ( orange outline ) constructs .

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A and C. Root mean square deviation (RMSD) analysis of the movement of (A) αC-helix and (C) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation (orange) compared to the wild type (WT) kinase (black). B and D. Time-lapse images show movement within the αC-helix (B) and DFG motif (D) of the inactive structure of the WT (black outline) and R351W (orange outline) constructs.


E . left , western blot analysis of transient overexpression of wild type ABL1 ( WT ) and ABL1 - R351W , compared to empty vector control ( EV ) in HEK293T cells . Right , quantification of p - CRKL signal based on densitometry analysis of blots from 6 independent experiments . Error bars represent ± S . D . , n = 6 , * P = 0 . 0004 by two - tailed unpaired Student ' s t - test .

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E. left, western blot analysis of transient overexpression of wild type ABL1 (WT) and ABL1-R351W, compared to empty vector control (EV) in HEK293T cells. Right, quantification of p-CRKL signal based on densitometry analysis of blots from 6 independent experiments. Error bars represent ±S.D., n=6, *P=0.0004 by two-tailed unpaired Student's t-test.


F . Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors ( Ding et al , 2008 ) in H157 cells . Values indicate quantified band intensity for p - CRKL , relative to α - tubulin and HA - tagged ABL1 .

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F. Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors (Ding et al, 2008) in H157 cells. Values indicate quantified band intensity for p-CRKL, relative to α-tubulin and HA-tagged ABL1.


A . Immunofluorescence imaging of endogenous ABL1 in the panel of NSCLC cell lines ; scale bars = 20 μm . B and C Quantification of nuclear versus cytoplasmic localization of ( B ) endogenous ABL1 in NSCLC cell lines and ( C ) tetracycline - inducible expression of ABL1 - WT , ABL1 - R351W and ABL1 - G340L in Beas - 2B cells . Error bars represent ± S . D . , n = 6 - 10 images , * P < 0 . 005 by two - tailed unpaired Student ' s t - test ( Beas - 2B vs H1915 P = 6 . 6 × 10 - 5 , vs H2110 P = 0 . 0001 ; WT vs R351W P = 0 . 003 , vs G340L P = 0 . 002 ) .

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A. Immunofluorescence imaging of endogenous ABL1 in the panel of NSCLC cell lines; scale bars=20 μm. B and C Quantification of nuclear versus cytoplasmic localization of (B) endogenous ABL1 in NSCLC cell lines and (C) tetracycline-inducible expression of ABL1-WT, ABL1-R351W and ABL1-G340L in Beas-2B cells. Error bars represent ±S.D., n=6-10 images, *P<0.005 by two-tailed unpaired Student's t-test (Beas-2B vs H1915 P=6.6×10-5, vs H2110 P=0.0001; WT vs R351W P=0.003, vs G340L P=0.002).


IL - 1β concentrations in supernatants of macrophages , unstimulated ( control ) ( B ) or stimulated with SP / N ( C ) or LPS / N ( D ) .

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IL-1β concentrations in supernatants of macrophages, unstimulated (control) (B) or stimulated with SP/N (C) or LPS/N (D).


E . SARS - CoV - 2 spike ELISA of the corresponding plasma : ( EC50 titer ) of samples collected at T0 ( n = 35 individuals ) , T1 ( n = 28 individuals ) or T2 ( n = 44 individuals ) .

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E. SARS-CoV-2 spike ELISA of the corresponding plasma: (EC50 titer) of samples collected at T0 (n = 35 individuals), T1 (n = 28 individuals) or T2 (n = 44 individuals).


F . Immunofluorescence microscopy images of ASC specks ( green ) in T2 macrophages . Nuclei were stained with DAPI ( blue ) . Scale bar indicates 20 µm . Arrow indicates ASC speck formation with magnified illustration in the right corner of the image .

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F. Immunofluorescence microscopy images of ASC specks (green) in T2 macrophages. Nuclei were stained with DAPI (blue). Scale bar indicates 20 µm. Arrow indicates ASC speck formation with magnified illustration in the right corner of the image.


G , H . ASC specks were quantified microscopically at T0 ( G ) ( n = 7 individuals ) and T2 ( H ) ( n = 8 individuals ) upon indicated stimuli .

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G, H. ASC specks were quantified microscopically at T0 (G) (n = 7 individuals) and T2 (H) (n = 8 individuals) upon indicated stimuli.


I . Cleaved gasdermin D ( lysate ) and cleaved IL - 1β ( supernatant ) detected by Western Blot in macrophages stimulated with LPS / N or SP / N at T0 and T2 .

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I. Cleaved gasdermin D (lysate) and cleaved IL-1β (supernatant) detected by Western Blot in macrophages stimulated with LPS/N or SP/N at T0 and T2.


J , K . Cell death quantification of macrophages by flow cytometry in unstimulated or SP / N stimulated cells at T0 ( n = 5 individuals ) ( J ) and T2 ( n = 5 individuals ) ( K ) using the viability dye Zombie UV . LPS / N was used as positive control .

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J, K. Cell death quantification of macrophages by flow cytometry in unstimulated or SP/N stimulated cells at T0 (n = 5 individuals) (J) and T2 (n = 5 individuals) (K) using the viability dye Zombie UV. LPS/N was used as positive control.


L . LDH release of macrophages ( n = 6 individuals ) stimulated with SP / N at T2 compared to unstimulated cells . M . IL - 1β concentrations of MCC950 treated T2 macrophage ( n = 44 individuals ) stimulation with SP / N .

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L. LDH release of macrophages (n = 6 individuals) stimulated with SP/N at T2 compared to unstimulated cells. M. IL-1β concentrations of MCC950 treated T2 macrophage (n = 44 individuals) stimulation with SP/N.


B . Volcano plot showing DEGs in the same samples . Negative log10 padj - values are plotted against the log2 fold - change . Selected genes are labeled .

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B. Volcano plot showing DEGs in the same samples. Negative log10 padj-values are plotted against the log2 fold-change. Selected genes are labeled.


C , D . CLEC4D ( C ) and CLEC4E ( D ) surface expression on patient - derived macrophages prior ( n = 5 individuals ) or post ( n = 5 individuals ) vaccination were quantified by flow cytometry . Graphs indicate the mean fluorescence intensity ( MFI ) .

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C, D. CLEC4D (C) and CLEC4E (D) surface expression on patient-derived macrophages prior (n = 5 individuals) or post (n = 5 individuals) vaccination were quantified by flow cytometry. Graphs indicate the mean fluorescence intensity (MFI).


E , F . SP or LPS as a control PAMP were incubated with affinity purified CLRs ( ratio : 10 vs . 1 for SP ) for 1h and then added to primary T2 macrophages ( n = 5 individuals ) . Subsequently , IL - 1β was quantified in the supernatants . ( E ) shows absolute values for SP ; ( F ) shows the fold change calculated with the corresponding unstimulated control for SP and LPS .

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E, F. SP or LPS as a control PAMP were incubated with affinity purified CLRs (ratio: 10 vs. 1 for SP) for 1h and then added to primary T2 macrophages (n = 5 individuals). Subsequently, IL-1β was quantified in the supernatants. (E) shows absolute values for SP; (F) shows the fold change calculated with the corresponding unstimulated control for SP and LPS.


G . Monocytes were isolated prior ( n = 5 individuals ) or after vaccination ( n = 5 individuals ) and differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin . H . Monocytes were isolated prior ( T0 ; n = 35 individuals ) or after vaccination [ 2 weeks after 1st ( T1 ; n = 28 individuals ) , 2 weeks ( T2 ; n = 38 individuals ) and 10 weeks ( T3 ; n = 33 individuals ) after 2nd vaccination ] and differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages , stimulated with the SP S2 subunit SP are shown .

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G. Monocytes were isolated prior (n = 5 individuals) or after vaccination (n = 5 individuals) and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin. H. Monocytes were isolated prior (T0; n = 35 individuals) or after vaccination [2 weeks after 1st (T1; n = 28 individuals), 2 weeks (T2; n = 38 individuals) and 10 weeks (T3; n =33 individuals) after 2nd vaccination] and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages, stimulated with the SP S2 subunit SP are shown.


B . Phospho - SYK Immunofluorescence - microscopy in macrophages before and after vaccination . Nuclei stained with DAPI . Scale bars indicate 20 µm .

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B. Phospho-SYK Immunofluorescence-microscopy in macrophages before and after vaccination. Nuclei stained with DAPI. Scale bars indicate 20 µm.


C . Quantification of pSYK positive macrophages by flow cytometry before ( n = 3 individuals ) and 14 d after vaccination ( n = 3 individuals ) . Total SYK was measured as control .

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C. Quantification of pSYK positive macrophages by flow cytometry before (n = 3 individuals) and 14 d after vaccination (n = 3 individuals). Total SYK was measured as control.


D . Western blot analysis of phospho - NF - κB and NLRP3 in cell lysates of macrophages ( T0 ; T2 ) stimulated with LPS / N or SP / N .

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D. Western blot analysis of phospho-NF-κB and NLRP3 in cell lysates of macrophages (T0; T2) stimulated with LPS/N or SP/N.


E . Macrophages were incubated with IKK - β - inhibitor KINK - 1 ( n = 10 individuals ) and SYK - inhibitors entospletinib ( n = 20 individuals ) , R406 ( n = 13 individuals ) prior to SP / nigericin stimulation . IL - 1β concentrations measured with ELISA . DMSO - treated , SP / N - stimulated and untreated unstimulated macrophages were used as control ( both n = 44 individuals ) .

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E. Macrophages were incubated with IKK-β-inhibitor KINK-1 (n = 10 individuals) and SYK-inhibitors entospletinib (n = 20 individuals), R406 (n = 13 individuals) prior to SP/nigericin stimulation. IL-1β concentrations measured with ELISA. DMSO-treated, SP/N-stimulated and untreated unstimulated macrophages were used as control (both n = 44 individuals).


F . IL - 1β quantification in THP - 1 wild - type cells incubated with SYK inhibitors entospletinib ( n = 4 independent experiments ) and R406 ( n = 2 independent experiments ) or in THP - 1 SYKKO cells stimulated with SP / N ( n = 5 independent experiments ) .

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F. IL-1β quantification in THP-1 wild-type cells incubated with SYK inhibitors entospletinib (n = 4 independent experiments) and R406 (n = 2 independent experiments) or in THP-1 SYKKO cells stimulated with SP/N (n = 5 independent experiments).


G . ASC formation quantified by immunofluorescence - microscopy in SP / N stimulated primary macrophages after vaccination ( n = 5 individuals ) .

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G. ASC formation quantified by immunofluorescence-microscopy in SP/N stimulated primary macrophages after vaccination (n = 5 individuals).


H . Quantification of cell death in T2 macrophages ( n = 4 individuals ) treated with R406 prior to SP / N - stimulation .

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H. Quantification of cell death in T2 macrophages (n = 4 individuals) treated with R406 prior to SP/N-stimulation.


I - K . mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP / N - stimulated macrophages without inhibitor ( I [ exemplary results ] , J [ T0 : n = 5 individuals ; T2 : n = 5 individuals ] )

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I-K. mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages without inhibitor (I [exemplary results], J [T0: n = 5 individuals; T2: n = 5 individuals])


mROS quantified MitoSOX red staining at T0 and T2 in SP / N - stimulated macrophages with R406 treatment ( n = 4 individuals ) ( K ) .

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mROS quantified MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages with R406 treatment (n = 4 individuals) (K).


L . Western blot analysis of SOD2 in macrophages at T0 and T2 treated with SP / N or LPS / N .

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L. Western blot analysis of SOD2 in macrophages at T0 and T2 treated with SP/N or LPS/N.


M . Mitochondrial membrane potential determined with flow cytometry of tetramethylrhodamine methyl ester ( TMRM ) stained macrophages at T0 ( n = 5 individuals ) and T2 ( n = 5 individuals ) stimulated with SP / N . N . T2 - macrophages ( n = 4 individuals ) were treated with the SYK - inhibitor R406 . Data information : For statistical analysis , one - or two - way ANOVA with tukey post hoc test was used . Box plots indicate the median and the upper and lower quartile . Outliers are plotted as individual dots ( outside the 10 - 90 percentile ) . Scatter dot plots show mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 . ( S - protein : SP , lipopolysaccharide : LPS , nigericin : N )

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M. Mitochondrial membrane potential determined with flow cytometry of tetramethylrhodamine methyl ester (TMRM) stained macrophages at T0 (n = 5 individuals) and T2 (n = 5 individuals) stimulated with SP/N. N. T2-macrophages (n = 4 individuals) were treated with the SYK-inhibitor R406. Data information: For statistical analysis, one- or two-way ANOVA with tukey post hoc test was used. Box plots indicate the median and the upper and lower quartile. Outliers are plotted as individual dots (outside the 10-90 percentile). Scatter dot plots show mean ± SD. *p<0.05; **p<0.01, ***p<0.001. (S-protein: SP, lipopolysaccharide: LPS, nigericin: N)


A - H . ( A ) T cells at T0 ( n = 6 individuals ) and T2 ( n = 10 individuals ) were incubated with SP / N - stimulated macrophages . Co - cultures of unstimulated macrophages were used as control ( con ) . CD4 + subpopulation analyses performed with flow cytometry ( Experimental setup : Fig EV3 ) . ( B ) Quantification of fold change of the CD4 + T cell subpopulation upon SP / N stimulation ( T0 ; T2 ) . Dotted line indicates the baseline ( fold change = 1 . 0 ) . ( C ) Fold change of CD4 + naïve T cell and CD4 + effector memory T cell populations ( D ) upon co - culture of macrophages stimulated with SP / N in presence of MCC950 or R406 . Identical analyses performed with CD8 + T cells ( E , F , G , H ) .

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A-H. (A) T cells at T0 (n = 6 individuals) and T2 (n = 10 individuals) were incubated with SP/N-stimulated macrophages. Co-cultures of unstimulated macrophages were used as control (con). CD4+ subpopulation analyses performed with flow cytometry (Experimental setup: Fig EV3). (B) Quantification of fold change of the CD4+ T cell subpopulation upon SP/N stimulation (T0; T2). Dotted line indicates the baseline (fold change = 1.0). (C) Fold change of CD4+ naïve T cell and CD4+ effector memory T cell populations (D) upon co-culture of macrophages stimulated with SP/N in presence of MCC950 or R406. Identical analyses performed with CD8+ T cells (E, F, G, H).


I , J . PD - 1 expression was analyzed in CD4 + ( I ) and CD8 + T cells ( J ) by flow cytometry upon co - culture with SP / N - stimulated T0 ( n = 6 individuals ) or T2 ( n = 11 individuals ) macrophages . Co - cultures of unstimulated macrophages were used as control .

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I, J. PD-1 expression was analyzed in CD4+ (I) and CD8+ T cells (J) by flow cytometry upon co-culture with SP/N-stimulated T0 (n = 6 individuals) or T2 (n = 11 individuals) macrophages. Co-cultures of unstimulated macrophages were used as control.


K , L . Interferon - γ quantified in supernatants of corresponding co - cultures , untreated ( T0 [ n = 6 individuals ] and T2 [ n = 11 individuals ] ) ( K ) and treated with MCC950 or R406 ( L ) at T2 ( n = 11 individuals ) .

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K, L. Interferon-γ quantified in supernatants of corresponding co-cultures, untreated (T0 [n = 6 individuals] and T2 [n = 11 individuals]) (K) and treated with MCC950 or R406 (L) at T2 (n = 11 individuals).


M . IL - 1β concentrations determined in SP / N - stimulated monocyte - derived macrophages at 34 weeks after 2nd vaccination ( first booster ) ( T4 = 7 individuals ) and 2 weeks after the 3rd vaccination ( second booster ) ( T5 = 11 individuals ) .

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M. IL-1β concentrations determined in SP/N-stimulated monocyte-derived macrophages at 34 weeks after 2nd vaccination (first booster) (T4 = 7 individuals) and 2 weeks after the 3rd vaccination (second booster) (T5 = 11 individuals).


A . The Armi complex immunoisolated from OSCs using anti - Armi antibodies contains Yb , SoYb , and Vret .

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A. The Armi complex immunoisolated from OSCs using anti-Armi antibodies contains Yb, SoYb, and Vret.


B . The Vret complex immunoisolated from OSCs using anti - Vret antibodies contains Yb , Armi , and SoYb .

B. The Vret complex immunoisolated from OSCs using anti-Vret antibodies contains Yb, Armi, and SoYb.


C . Silver staining of protein components in the Vret complex The lane α - Vret ( left ) contained only the anti - Vret antibodies used in these experiments .

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C. Silver staining of protein components in the Vret complex The lane α-Vret (left) contained only the anti-Vret antibodies used in these experiments.


D . Immunoprecipitation and western blotting were performed in OSCs upon RNAi treatment ( kd ) . Even without the association of Yb , Armi , SoYb , and Vret associated . Yb and Armi remained bound to each other upon SoYb or Vret depletion . The level of SoYb ( input ) decreased upon Vret depletion . Likewise , the level of Vret ( input ) decreased upon SoYb depletion .

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D. Immunoprecipitation and western blotting were performed in OSCs upon RNAi treatment (kd). Even without the association of Yb, Armi, SoYb, and Vret associated. Yb and Armi remained bound to each other upon SoYb or Vret depletion. The level of SoYb (input) decreased upon Vret depletion. Likewise, the level of Vret (input) decreased upon SoYb depletion.


E . Upon Armi depletion , SoYb and Vret were still bound to each other but this heterodimer no longer associated with Yb .

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E. Upon Armi depletion, SoYb and Vret were still bound to each other but this heterodimer no longer associated with Yb.


F . Immunofluorescence analyses in OSCs . Yb bodies disappeared upon Yb depletion , which led Armi , SoYb and Vret to be localized in the cytosol . Yb bodies were detected in OSCs upon Armi depletion , but SoYb and Vret localized in the cytosol . Armi was detected at Yb bodies upon SoYb or Vret depletion . Yb bodies are indicated by white arrows . Nuclei are shown in blue . Scale bar : 2 μm .

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F. Immunofluorescence analyses in OSCs. Yb bodies disappeared upon Yb depletion, which led Armi, SoYb and Vret to be localized in the cytosol. Yb bodies were detected in OSCs upon Armi depletion, but SoYb and Vret localized in the cytosol. Armi was detected at Yb bodies upon SoYb or Vret depletion. Yb bodies are indicated by white arrows. Nuclei are shown in blue. Scale bar: 2 μm.


B . In Yb - lacking OSCs ( Yb kd ) , WT Yb and ∆ eTud bound to Armi but ∆ eTud did not . Note that WT Yb and both mutants were engineered to be RNAi - resistant by mutations . β - gal was used as a negative control .

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B. In Yb-lacking OSCs (Yb kd), WT Yb and ∆eTud bound to Armi but ∆eTud did not. Note that WT Yb and both mutants were engineered to be RNAi-resistant by mutations. β-gal was used as a negative control.


C . In normal OSCs , WT Yb and ∆ eTud bound with Yb but ∆ Hel - C did not .

O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O

C. In normal OSCs, WT Yb and ∆eTud bound with Yb but ∆Hel-C did not.


D . Flag - Hel - C copurified with Myc - Hel - C but not with BSD ( negative control ) .

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D. Flag-Hel-C copurified with Myc-Hel-C but not with BSD (negative control).


E . Subcellular localization of WT Yb and deletion mutants ∆ Hel - C and ∆ eTud . Myc - Yb is shown in green . Endogenous Armi is shown in red . DAPI shows nuclei ( blue ) . Scale bar : 5 μm . Western blotting ( right ) showing WT Yb and deletion mutants ∆ Hel - C and ∆ eTud expressed in Yb - lacking OSCs .

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E. Subcellular localization of WT Yb and deletion mutants ∆Hel-C and ∆eTud. Myc-Yb is shown in green. Endogenous Armi is shown in red. DAPI shows nuclei (blue). Scale bar: 5 μm. Western blotting (right) showing WT Yb and deletion mutants ∆Hel-C and ∆eTud expressed in Yb-lacking OSCs.


F . CLIP shows that WT Yb and ∆ Hel - C bind RNA in vivo but ∆ eTud does not .

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F. CLIP shows that WT Yb and ∆Hel-C bind RNA in vivo but ∆eTud does not.


A . Rescue experiments showing that ∆ Hel - C but not ∆ eTud restored piRNA biogenesis attenuated by endogenous Yb depletion . Flag - Piwi was expressed upon endogenous Yb depletion .

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A. Rescue experiments showing that ∆Hel-C but not ∆eTud restored piRNA biogenesis attenuated by endogenous Yb depletion. Flag-Piwi was expressed upon endogenous Yb depletion.


B . The levels of transposon mdg1 Bars and error bars represent means ± SEM values of three independent experiments . p values were calculated by t - test . * : p < 0 . 05 .

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B. The levels of transposon mdg1 Bars and error bars represent means ± SEM values of three independent experiments. p values were calculated by t-test. *: p < 0.05.


C . Mapping of flam - piRNAs produced in the presence and absence of the Hel - C domain in OSCs . D . Mapping of tj - piRNAs produced in the presence and absence of the Hel - C domain in OSCs .

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C. Mapping of flam-piRNAs produced in the presence and absence of the Hel-C domain in OSCs. D. Mapping of tj-piRNAs produced in the presence and absence of the Hel-C domain in OSCs.


E . Scatterplot showing changes in the abundances of transposon - repressing piRNAs and non - transposon - repressing piRNAs in the presence and absence of the Hel - C domain . Orange and blue dots indicate the number of normalized piRNA reads mapped uniquely to transposons in the reverse orientation and coding genes in the sense orientation , respectively .

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E. Scatterplot showing changes in the abundances of transposon-repressing piRNAs and non-transposon-repressing piRNAs in the presence and absence of the Hel-C domain. Orange and blue dots indicate the number of normalized piRNA reads mapped uniquely to transposons in the reverse orientation and coding genes in the sense orientation, respectively.


F . The relative enrichments of RNAs coimmunopurified with WT Yb and ∆ Hel - C . Bars and error bars represent means ± SEM values of three independent experiments . In addition to tj , three genic piRNA sources that were characterized in previous studies ( dm / Myc [ 59 ] , c - Fos / kay [ 60 ] , and CG9257 [ 34 ] ) are also shown . rp49 was used as a negative control . p value was calculated by t - test . * * * : p < 0 . 001 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. The relative enrichments of RNAs coimmunopurified with WT Yb and ∆Hel-C. Bars and error bars represent means ± SEM values of three independent experiments. In addition to tj, three genic piRNA sources that were characterized in previous studies (dm/Myc [59], c-Fos/kay [60], and CG9257 [34]) are also shown. rp49 was used as a negative control. p value was calculated by t-test. ***: p < 0.001.


A . Yb bodies ( Yb in green ) disappeared after 1 , 6 - hexanediol treatment of OSCs , as did P bodies ( Dcp1 in red ) . Nuclei are shown in blue . Scale bar : 5 μm .

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A. Yb bodies (Yb in green) disappeared after 1,6-hexanediol treatment of OSCs, as did P bodies (Dcp1 in red). Nuclei are shown in blue. Scale bar: 5 μm.


B . Snapshots of live imaging of GFP - Yb expressed in OSCs Fusion - and fission - like dynamics of Yb bodies were observed . Saturated pixels are shown in red . Scale bar : 0 . 5 μm .

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B. Snapshots of live imaging of GFP-Yb expressed in OSCs Fusion- and fission-like dynamics of Yb bodies were observed. Saturated pixels are shown in red. Scale bar: 0.5 μm.


C . Yb Y23A and F129A mutants ( green ) impaired Yb body formation . Nuclei are shown in blue . Scale bar : 5 μm .

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C. Yb Y23A and F129A mutants (green) impaired Yb body formation. Nuclei are shown in blue. Scale bar: 5 μm.


D . Yb Y23A and F129A mutants failed to produce flam - piRNAs . miR - 310 was used as a loading control .

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D. Yb Y23A and F129A mutants failed to produce flam-piRNAs. miR-310 was used as a loading control.


E . Mapping of flam - and tj - piRNAs produced in the flamKG mutant and heterozygote ovaries .

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E. Mapping of flam- and tj-piRNAs produced in the flamKG mutant and heterozygote ovaries.


B , Total pup retrieval time in biological mothers ( n = 6 ) , foster mothers ( n = 7 ) , and virgin females ( n = 10 ) on 3 consecutive days .

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B, Total pup retrieval time in biological mothers (n=6), foster mothers (n=7), and virgin females (n=10) on 3 consecutive days.


C , D Fraction of parental ( C ) and ignoring ( D ) behaviors of virgin females , of the total session duration in each of the three pup exposure days ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are displayed as individual measurements with the line connecting the mean value ; * p < 0 . 05 .

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C,D Fraction of parental (C) and ignoring (D) behaviors of virgin females, of the total session duration in each of the three pup exposure days (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are displayed as individual measurements with the line connecting the mean value; *p < 0.05.


F , A representative image of a coronal brain section immunostained for c - Fos after pup retrieval on day 1 and day 3 . The mPFC region is indicated by dashed lines , which is also shown in the insets in higher magnification . Scale bar indicates 500 µm , 10 × magnification .

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F, A representative image of a coronal brain section immunostained for c-Fos after pup retrieval on day 1 and day 3. The mPFC region is indicated by dashed lines, which is also shown in the insets in higher magnification. Scale bar indicates 500 µm, 10× magnification.


G , Heat - map of the Z - score for the difference in the fraction of c - Fos expressing cells , between day 1 and day 3 of behavioral testing . Foster mothers ( n = 5 ) , virgins with pups ( n = 6 ) , virgins with object ( n = 7 ) .

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G, Heat-map of the Z-score for the difference in the fraction of c-Fos expressing cells, between day 1 and day 3 of behavioral testing. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7).


H , Representative coronal sections of the mPFC immunostained for c - Fos with the anatomical borders of the ACC indicated by dashed lines , in foster mothers ( FM ) and virgin females exposed to pups ( V pups ) or objects ( V object ) on day 1 and day 3 of behavioral testing . Scale bar 100 µm , 20 × magnification .

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H, Representative coronal sections of the mPFC immunostained for c-Fos with the anatomical borders of the ACC indicated by dashed lines, in foster mothers (FM) and virgin females exposed to pups (V pups) or objects (V object) on day 1 and day 3 of behavioral testing. Scale bar 100 µm, 20× magnification.


A , Reconstruction of the mouse brain following anterograde labeling with AAV - GFP in the ACC ( I ) ; representative coronal images showing site of injection and axonal distribution pattern in the dorsal striatum ( II ) and central lateral nucleus of the thalamus ( III , IV ) . Images in aI - aIV were taken from the Allen Mouse Brain Connectivity

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A, Reconstruction of the mouse brain following anterograde labeling with AAV-GFP in the ACC (I); representative coronal images showing site of injection and axonal distribution pattern in the dorsal striatum (II) and central lateral nucleus of the thalamus (III, IV). Images in aI-aIV were taken from the Allen Mouse Brain Connectivity Atlas


C , A representative coronal section showing labeling pattern at the site of injection . White arrows in the magnified image ( C , right ) point to double - labeled putative starter cells for transsynaptic retrograde labeling .

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C, A representative coronal section showing labeling pattern at the site of injection. White arrows in the magnified image (C, right) point to double-labeled putative starter cells for transsynaptic retrograde labeling.


D , Representative coronal sections showing bidirectional connectivity patterns with the thalamus ( I ) and monosynaptic input to the ACC from the claustrum ( II ) and ventral CA1 ( III ) . Scale bars 500 µm , 10 × magnification ( left images ) and 100 µm , 20 × magnification ( right images ) .

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D, Representative coronal sections showing bidirectional connectivity patterns with the thalamus (I) and monosynaptic input to the ACC from the claustrum (II) and ventral CA1 (III). Scale bars 500 µm, 10× magnification (left images) and 100 µm, 20× magnification (right images).


G , Representative coronal sections of the dorsal thalamus , following c - Fos immunolabeling on day 1 ( left ) and day 3 ( right ) , with the ILM subnuclei indicated ; CM central medial nucleus , CL central lateral nucleus , PCN paracentral nucleus , RH rhomboid nucleus . Scale bar 200 µm , 10 × magnification . Foster mothers ( n = 5 ) , virgins with pups ( n = 6 ) , virgins with object ( n = 7 ) .

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G, Representative coronal sections of the dorsal thalamus, following c-Fos immunolabeling on day 1 (left) and day 3 (right), with the ILM subnuclei indicated; CM central medial nucleus, CL central lateral nucleus, PCN paracentral nucleus, RH rhomboid nucleus. Scale bar 200 µm, 10× magnification. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7).


H , Fraction of c - Fos + cells in different ILM subnuclei , virgin exposed to pups ( left ) , in foster mothers ( middle ) , and virgin exposed to object ( right ) , on day 1 and day 3 of behavioral testing . Foster mothers ( n = 5 ) , virgins with pups ( n = 6 ) , virgins with object ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are expressed as mean ± s . e . m ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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H, Fraction of c-Fos+ cells in different ILM subnuclei, virgin exposed to pups (left), in foster mothers (middle), and virgin exposed to object (right), on day 1 and day 3 of behavioral testing. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are expressed as mean ± s.e.m; *p < 0.05, **p < 0.01, ***p < 0.001.


B , Representative coronal sections of the PFC , following the labeling schemes described in ( A ) , for mapping inputs onto ACC excitatory ( B , top ) and inhibitory ( B , bottom ) neurons . High magnification images ( B , right top and bottom ) show the double - labeled , putative starter cells for transsynaptic retrograde labeling . Scale bars represent 200 µm , 10 × magnification ( left top and bottom ) , and 50 µm ( right top and bottom ) , 20 × magnification .

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B, Representative coronal sections of the PFC, following the labeling schemes described in (A), for mapping inputs onto ACC excitatory (B, top) and inhibitory (B, bottom) neurons. High magnification images (B, right top and bottom) show the double-labeled, putative starter cells for transsynaptic retrograde labeling. Scale bars represent 200 µm, 10× magnification (left top and bottom), and 50 µm (right top and bottom), 20× magnification.


C , Representative coronal images of the thalamus , immunolabeled for c - Fos , following retrograde labeling from excitatory ( C , top ) or inhibitory ( C , bottom ) neurons in the ACC . White arrows in high magnification images indicate colocalization of c - Fos and td - Tomato ( C , right top and bottom ) . Scale bars represent 200 µm , 10 × magnification ( left top and bottom ) , and 25 µm ( right top and bottom ) , 20 × magnification .

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C, Representative coronal images of the thalamus, immunolabeled for c-Fos, following retrograde labeling from excitatory (C, top) or inhibitory (C, bottom) neurons in the ACC. White arrows in high magnification images indicate colocalization of c-Fos and td-Tomato (C, right top and bottom). Scale bars represent 200 µm, 10× magnification (left top and bottom), and 25 µm (right top and bottom), 20× magnification.


D , Quantification of the number of neurons in CL region projecting to excitatory or inhibitory ACC neurons , as a fraction of the total thalamic contribution ( top ) and fraction of neurons co - expressing tdTomato and c - Fos of the total tdTomato + population in CL ( bottom ) . Foster mothers ( n = 3 ) , virgins with pups ( n = 4 ) , virgins with object ( n = 4 ) . Data were analyzed using student ' s t - test and are displayed as mean ± s . e . m ;

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D, Quantification of the number of neurons in CL region projecting to excitatory or inhibitory ACC neurons, as a fraction of the total thalamic contribution (top) and fraction of neurons co-expressing tdTomato and c-Fos of the total tdTomato+ population in CL (bottom). Foster mothers (n=3), virgins with pups (n=4), virgins with object (n=4). Data were analyzed using student's t-test and are displayed as mean ± s.e.m;


, Representative in - situ hybridization images of Gal in the ILM taken from the Allen Mouse Brain Atlas representative coronal images showing the expression of Gal neurons in Gal - Cre : : FLEX - YFP female mice ( bottom right and left ) .

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, Representative in-situ hybridization images of Gal in the ILM taken from the Allen Mouse Brain Atlas representative coronal images showing the expression of Gal neurons in Gal-Cre::FLEX-YFP female mice (bottom right and left).


, Representative image of the ILM immunolabeled for GFP and c - Fos , following pup retrieval test . Yellow and white arrows indicate c - Fos expressing Gal - positive and Gal - negative neurons , respectively . Scale bars represent 100 µm , 20 × magnification .

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, Representative image of the ILM immunolabeled for GFP and c-Fos, following pup retrieval test. Yellow and white arrows indicate c-Fos expressing Gal-positive and Gal-negative neurons, respectively. Scale bars represent 100 µm, 20× magnification.


G , Graph plots showing the fraction of c - Fos expressing Gal - positive ( Galc - Fos + ) and Gal - negative neurons ( DAPIc - Fos + ) in the CL , PCN , and CM after pup retrieval test ( G , top ) or object exposure ( G , bottom ) . Data were analyzed using 2 - way ANOVA and expressed as mean ± s . e . m . ; n = 3 ; * p < 0 . 05 .

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G, Graph plots showing the fraction of c-Fos expressing Gal-positive (Galc-Fos+) and Gal-negative neurons (DAPIc-Fos+) in the CL, PCN, and CM after pup retrieval test (G, top) or object exposure (G, bottom). Data were analyzed using 2-way ANOVA and expressed as mean ± s.e.m.; n=3; *p < 0.05.


B , C , Quantification of pup retrieval latencies following ACC chemogenetic activation with hM3D ( n = 6 ) ( B ) or chemogenetic inhibition using hM4D ( n = 7 ) ( C ) compared to control groups in virgin females ( n = 8 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are displayed as mean ± s . e . m * p < 0 . 05 , * * p < 0 . 01 . D , Same as ( C ) but for mothers ( n = 7 )

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B,C, Quantification of pup retrieval latencies following ACC chemogenetic activation with hM3D (n=6) (B) or chemogenetic inhibition using hM4D (n=7) (C) compared to control groups in virgin females (n=8). Data were analyzed using 2-way ANOVA with repeated measures and are displayed as mean ± s.e.m* p< 0.05, ** p< 0.01. D, Same as (C) but for mothers (n=7)


E , Representative images showing AAV - DIO - CAG - hM3D - 2A - tdTomato and c - Fos expression in the PFC of virgin females , 2 hours following CNO / vehicle administration . Masks demonstrating distribution of DAPI + c - Fos ( green ) , DAPI + dTomato ( magenta ) and their colocalization ( white ) . Scale bars 100 µm , 20 × magnification .

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E, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the PFC of virgin females, 2 hours following CNO/vehicle administration. Masks demonstrating distribution of DAPI + c-Fos (green), DAPI + dTomato (magenta) and their colocalization (white). Scale bars 100 µm, 20× magnification.


F , Fraction c - Fos + cells of the total DAPI ( top ) or hM3D - 2A - dTomato ( bottom ) population in the ACC , following CNO / vehicle administration . ( n = 3 ) . Data were analyzed using student ' s t - test and are displayed as mean ± s . e . m ; * p < 0 . 05 , * * p < 0 . 01 .

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F, Fraction c-Fos+ cells of the total DAPI (top) or hM3D-2A-dTomato (bottom) population in the ACC, following CNO/vehicle administration. (n=3). Data were analyzed using student's t-test and are displayed as mean ± s.e.m; * p< 0.05, ** p< 0.01.


G , Representative images of the CL , immunolabeled for c - Fos , following hM3D - mediated neuronal activation of the ACC . Scale bars 200 µm , 20 × magnification .

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G, Representative images of the CL, immunolabeled for c-Fos, following hM3D-mediated neuronal activation of the ACC. Scale bars 200 µm, 20× magnification.


H , Spearman correlation between the hM3D - mediated activation of the ACC ( as measured in ( F , top ) ) and the CL region .

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H, Spearman correlation between the hM3D-mediated activation of the ACC (as measured in (F, top)) and the CL region.


B , Representative images showing AAV - DIO - CAG - hM3D - 2A - tdTomato and c - Fos expression in the CL , 2 h after CNO administration . Scale bars 200 µm , 10 × magnification ( left ) and 50 µm , 20 × magnification ( right ) .

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B, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the CL, 2 h after CNO administration. Scale bars 200 µm, 10× magnification (left) and 50 µm, 20× magnification (right).


C , Quantification of pup retrieval latencies in virgin females following CLGal + chemogenetic activation with hM3D compared to GFP - expressing control group ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are presented as mean ± s . e . m ; * p < 0 . 05 .

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C, Quantification of pup retrieval latencies in virgin females following CLGal+ chemogenetic activation with hM3D compared to GFP-expressing control group (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are presented as mean ± s.e.m; * p < 0.05.


E . Immunoblot ( IB ) analysis of stable Ub replacement cell lines treated or not with DOX for 72 h . Data information : Data are representative of three ( E independent experiments with similar outcome .

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E. Immunoblot (IB) analysis of stable Ub replacement cell lines treated or not with DOX for 72 h. Data information: Data are representative of three (E independent experiments with similar outcome.


F , G . HA - Ub conjugates isolated as in ( D ) were immunoblotted with indicated antibodies . Data information : Data are representative of three independent experiments with similar outcome .

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F,G. HA-Ub conjugates isolated as in (D) were immunoblotted with indicated antibodies. Data information: Data are representative of three independent experiments with similar outcome.


B . Representative images ( upper ) and QIBC analysis ( lower ) ( solid lines , median ; dashed lines , quartiles ; > 10000 cells analyzed per condition ) of Ub ( G76V ) - GFP expression in stable U2OS / Ub ( G76V ) - GFP cells treated or not with p97i for 4 h . Data information : Data are representative of three independent experiments with similar outcome .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

B. Representative images (upper) and QIBC analysis (lower) (solid lines, median; dashed lines, quartiles; >10000 cells analyzed per condition) of Ub(G76V)-GFP expression in stable U2OS/Ub(G76V)-GFP cells treated or not with p97i for 4 h. Data information: Data are representative of three independent experiments with similar outcome.


D . DOX - treated U2OS / shUb / HA - Ub ( WT ) and U2OS / shUb / HA - Ub ( K27R ) cell lines were transfected with shUb - resistant Ub ( G76V ) - GFP construct and treated or not with p97i for 4 h . Cell extracts were subjected to GFP IP followed by immunoblotting with indicated antibodies . Data information : Data are representative of three independent experiments with similar outcome .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines were transfected with shUb-resistant Ub(G76V)-GFP construct and treated or not with p97i for 4 h. Cell extracts were subjected to GFP IP followed by immunoblotting with indicated antibodies. Data information: Data are representative of three independent experiments with similar outcome.


E . Immunoblot analysis of DOX - treated U2OS / shUb / HA - Ub cell lines treated or not with MG132 for 4 h . Data information : Data are representative of three independent experiments with similar outcome .

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E. Immunoblot analysis of DOX-treated U2OS/shUb/HA-Ub cell lines treated or not with MG132 for 4 h. Data information: Data are representative of three independent experiments with similar outcome.


G . DOX - treated U2OS / shUb / HA - Ub ( WT ) and U2OS / shUb / HA - Ub ( K27R ) cell lines transfected with indicated Ub ( G76V ) - GFP expression constructs were subjected to GFP IP under denaturing conditions followed by immunoblotting with indicated antibodies . Data information : Data are representative of four ( G ) independent experiments with similar outcome .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines transfected with indicated Ub(G76V)-GFP expression constructs were subjected to GFP IP under denaturing conditions followed by immunoblotting with indicated antibodies. Data information: Data are representative of four (G) independent experiments with similar outcome.


A . FLAG IPs from U2OS cells transfected with indicated FLAG - tagged expression constructs were incubated with purified K27 di - Ub and analyzed by immunoblotting . Data information : Data are representative of three ( A independent experiments with similar outcome .

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A. FLAG IPs from U2OS cells transfected with indicated FLAG- tagged expression constructs were incubated with purified K27 di-Ub and analyzed by immunoblotting. Data information: Data are representative of three (A independent experiments with similar outcome.


C . Cells transfected as in ( B ) and treated or not with p97i for 4 h were subjected to GFP IP followed by immunoblotting . Data information : Data are representative of three independent experiments with similar outcome .

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C. Cells transfected as in (B) and treated or not with p97i for 4 h were subjected to GFP IP followed by immunoblotting. Data information: Data are representative of three independent experiments with similar outcome.


I . U2OS / Ub ( G76V ) - GFP and U2OS / Ub ( G76V ) - 20AA - GFP cell lines were transfected or not with FLAG - UCHL3 C95S expression construct , treated with MG132 for 2 h and subjected to GFP IP followed by immunoblotting . Data information : Data are representative of three independent experiments with similar outcome .

O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. U2OS/Ub(G76V)-GFP and U2OS/Ub(G76V)-20AA-GFP cell lines were transfected or not with FLAG-UCHL3 C95S expression construct, treated with MG132 for 2 h and subjected to GFP IP followed by immunoblotting. Data information: Data are representative of three independent experiments with similar outcome.


A . SHOXprotein representation and luciferase assay . The variants found in SHOX are indicated in red . Luciferase assays to test the impact of SHOX mutations on its transcriptional activity were performed on FGFR3 promoter in U2OS cells ( n = 4 ) . Experiments were performed in triplicates . pcDNA4 - TO empty vector was used as control . R153L represents a mutation known to affect SHOXprotein activity and was used as positive control ( Schneider et al . , 2005 ) . Homeodomain , DNA binding domain ; Src Homology 3 domain ; OAR , OtpAristalessRax domain . RLU , Relative Light Units .

O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. SHOXprotein representation and luciferase assay. The variants found in SHOX are indicated in red. Luciferase assays to test the impact of SHOX mutations on its transcriptional activity were performed on FGFR3 promoter in U2OS cells (n = 4). Experiments were performed in triplicates. pcDNA4-TO empty vector was used as control. R153L represents a mutation known to affect SHOXprotein activity and was used as positive control (Schneider et al., 2005). Homeodomain, DNA binding domain; Src Homology 3 domain; OAR, OtpAristalessRax domain. RLU, Relative Light Units.


B . CYP26C1protein representation and luciferase assay . Variants found in CYP26C1 are indicated in green . Cignal - RARE system luciferase assays to test the impact of CYP26C1 variants on its RA degradation activity were performed in U2OS cells treated with 250 nM all - trans retinoic acid ( ATRA ) for 24 hours ( n = 4 ) . Experiments were performed in triplicates . pIRES2 - EGFP empty vector was used as control . The residue C459 represents the Iron binding residue ( Q6VOL0 , UniProtKB ) and was mutated to Ala and used as a positive control . TM , Transmembrane helix ; P450 , cytochrome p450 domain . RLU , Relative Light Units .

O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. CYP26C1protein representation and luciferase assay. Variants found in CYP26C1 are indicated in green. Cignal-RARE system luciferase assays to test the impact of CYP26C1 variants on its RA degradation activity were performed in U2OS cells treated with 250 nM all-trans retinoic acid (ATRA) for 24 hours (n = 4). Experiments were performed in triplicates.pIRES2-EGFP empty vector was used as control. The residue C459 represents the Iron binding residue (Q6VOL0, UniProtKB) and was mutated to Ala and used as a positive control. TM, Transmembrane helix; P450, cytochrome p450 domain. RLU, Relative Light Units.


A . Right panel , western blot showing the expression of CYP26C1 in human primary chondrocytes on protein level . Overexpression of CYP26C1 in U2OS cells was carried out to compare protein sizes . + , cDNA ; - , water ; M , marker ; HPC , human primary chondrocytes .

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A. Right panel, western blot showing the expression of CYP26C1 in human primary chondrocytes on protein level. Overexpression of CYP26C1 in U2OS cells was carried out to compare protein sizes. +, cDNA; -, water; M, marker; HPC, human primary chondrocytes.


A . Left panel , RT - PCR showing the expression of CYP26C1 mRNA in human primary chondrocytes . SOX9 was used as chondrocyte marker .

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A. Left panel, RT-PCR showing the expression of CYP26C1 mRNA in human primary chondrocytes. SOX9 was used as chondrocyte marker.


B . Relative expression of SHOX mRNA normalized to the reference genes SDHA and HPRT in human primary chondrocytes treated with ATRA 100 nM for 6h ( n = 5 ) . One outlier with high relative expression ( 2 . 3 fold ) was excluded from ATRA 100 nM treatment ( two - sided Grubb ' s test , Z value 2 . 34 , p - value < 0 . 05 ) .

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B. Relative expression of SHOX mRNA normalized to the reference genes SDHA and HPRT in human primary chondrocytes treated with ATRA 100 nM for 6h (n = 5). One outlier with high relative expression (2.3 fold) was excluded from ATRA 100 nM treatment (two-sided Grubb's test, Z value 2.34, p-value < 0.05).


C . SHOX promoter was cloned in pGL3basic for luciferase reporter experiments as previously described ( Verdin et al , 2015 ) . In silico analysis of SHOX promoter identified three putative RXRa binding sites which were mutated to test their direct effect on SHOX expression upon treatment with ATRA 250 nM in U2OS cells ( n = 4 ) . Experiments were performed in triplicates . RLU , Relative Light Units .

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C. SHOX promoter was cloned in pGL3basic for luciferase reporter experiments as previously described (Verdin et al, 2015). In silico analysis of SHOX promoter identified three putative RXRa binding sites which were mutated to test their direct effect on SHOX expression upon treatment with ATRA 250 nM in U2OS cells (n = 4). Experiments were performed in triplicates. RLU, Relative Light Units.


A - C . Wild type embryos injected with control MO or with shox MO . ( A ) Lateral views of the embryos at 55 hours post fertilization ( hpf ) . ( B ) Dorsal view and magnification on the lateral view of the embryos . Dotted line , pectoral fins . shox morphants show smaller fins compared to controls ( n = 30 embryos ) .

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A-C. Wild type embryos injected with control MO or with shox MO. (A) Lateral views of the embryos at 55 hours post fertilization (hpf). (B) Dorsal view and magnification on the lateral view of the embryos. Dotted line, pectoral fins. shox morphants show smaller fins compared to controls (n = 30 embryos).


A - C . Wild type embryos injected with control MO or with shox MO . C . Expression of col2a1 at 55 hpf was examined by in situ hybridization in wild type embryos injected with control MO and with shox MO . Arrow and dotted line indicate the pectoral fin .

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A-C. Wild type embryos injected with control MO or with shox MO. C. Expression of col2a1 at 55 hpf was examined by in situ hybridization in wild type embryos injected with control MO and with shox MO. Arrow and dotted line indicate the pectoral fin.


A - C . Wild type embryos injected with control MO or with shox MO . D . Pectoral fin area was measured by imageJ ( n = 30 embryos ) .

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A-C. Wild type embryos injected with control MO or with shox MO. D. Pectoral fin area was measured by imageJ (n = 30 embryos).


A - D . Wild type embryos injected with control MO or with cyp26c1 MO . ( A ) Lateral views of the embryos at 55 hours post fertilization ( hpf ) . ( B ) Dorsal view and magnification on the lateral view of the embryos . Dotted line , pectoral fins ; * , otic vesicle . cyp26c1 morphants show smaller fins compared to controls ( n = 30 embryos ) .

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A-D. Wild type embryos injected with control MO or with cyp26c1 MO. (A) Lateral views of the embryos at 55 hours post fertilization (hpf). (B) Dorsal view and magnification on the lateral view of the embryos. Dotted line, pectoral fins; *, otic vesicle. cyp26c1 morphants show smaller fins compared to controls (n = 30 embryos).


A - D . Wild type embryos injected with control MO or with cyp26c1 MO . ( C , D ) Expression of col2a1 ( C ) and shox ( D ) at 55 hpf was examined by in situ hybridization in embryos injected with control MO or with cyp26c1 MO . ( C ) Dorsal view and magnification on the pectoral fins of col2a1expression . Arrow and dotted line indicate the pectoral fin . ( D ) Dorsal view and magnification on the pectoral fins of shox expression . Arrow and dotted line indicate the pectoral fin .

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A-D. Wild type embryos injected with control MO or with cyp26c1 MO. (C, D) Expression of col2a1(C) and shox (D) at 55 hpf was examined by in situ hybridization in embryos injected with control MO or with cyp26c1 MO. (C) Dorsal view and magnification on the pectoral fins of col2a1expression. Arrow and dotted line indicate the pectoral fin. (D) Dorsal view and magnification on the pectoral fins of shox expression. Arrow and dotted line indicate the pectoral fin.


E . Pectoral fin area was measured by imageJ ( n = 30 embryos ) .

E. Pectoral fin area was measured by imageJ (n = 30 embryos).


F . Cignal - RARE system luciferase assay testing cyp26c1 MO on RA acid levels in zebrafishembryos ( n = 5 replicates ) . Embryos were separated in groups of 20 - 30 and luciferase assayed . RLU , Relative Light Units .

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F. Cignal-RARE system luciferase assay testing cyp26c1 MO on RA acid levels in zebrafishembryos (n = 5 replicates). Embryos were separated in groups of 20-30 and luciferase assayed. RLU, Relative Light Units.


G . Relative shox mRNA expression normalized to reference genes eef1a and b - actin in zebrafishembryos injected with control MO or cyp26c1 MO ( n = 4 ) . RNA was extracted from 10 - 15 injected embryos at 55 hours post fertilization .

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G. Relative shox mRNA expression normalized to reference genes eef1a and b-actin in zebrafishembryos injected with control MO or cyp26c1 MO (n = 4). RNA was extracted from 10-15 injected embryos at 55 hours post fertilization.


H . Relative shox mRNA expression normalized to reference genes eef1a and b - actin in zebrafishembryos treated with 100 nM ATRA ( n = 4 ) . Embryos were collected at 24 hpf and treated with mock control or ATRA . RNA was extracted from 10 - 15 embryos after 6 hours treatment .

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H. Relative shox mRNA expression normalized to reference genes eef1a and b-actin in zebrafishembryos treated with 100 nM ATRA (n = 4). Embryos were collected at 24 hpf and treated with mock control or ATRA. RNA was extracted from 10-15 embryos after 6 hours treatment.


A - C . Wild type embryos injected with control MO , subphenotypic doses of shox MO , cyp26c1 MO or a combination of shox + cyp26c1 MOs . ( A ) Dorsal views of the embryos at 55 hours post fertilization ( hpf ) . Dotted line , pectoral fins . shox + cyp26c1 double morphants show smaller fins compared to control and single MOs ( n = 30 embryos ) .

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A-C. Wild type embryos injected with control MO, subphenotypic doses of shox MO, cyp26c1 MO or a combination of shox + cyp26c1 MOs. (A) Dorsal views of the embryos at 55 hours post fertilization (hpf). Dotted line, pectoral fins. shox + cyp26c1 double morphants show smaller fins compared to control and single MOs (n = 30 embryos).


A - C . Wild type embryos injected with control MO , subphenotypic doses of shox MO , cyp26c1 MO or a combination of shox + cyp26c1 MOs . ( B , C ) Dorsal view and magnification on the pectoral fins of col2a1expression at 55 hpf . Arrow and dotted line indicate the pectoral fin .

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A-C. Wild type embryos injected with control MO, subphenotypic doses of shox MO, cyp26c1 MO or a combination of shox + cyp26c1 MOs. (B, C) Dorsal view and magnification on the pectoral fins of col2a1expression at 55 hpf. Arrow and dotted line indicate the pectoral fin.


D . Pectoral fins area was measured by imageJ ( n = 30 embryos ) .

D. Pectoral fins area was measured by imageJ (n = 30 embryos).


E . Relative expression of shox mRNA normalized to reference genes eef1a and b - actin in zebrafishembryos injected with control MO , shox MO , cyp26c1 MO , or shox + cyp26c1 MOs ( n = 4 ) . RNA was extracted from 10 - 15 injected embryos at 55 hours post fertilization .

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E. Relative expression of shox mRNA normalized to reference genes eef1a and b-actin in zebrafishembryos injected with control MO, shox MO, cyp26c1 MO, or shox + cyp26c1 MOs (n = 4). RNA was extracted from 10-15 injected embryos at 55 hours post fertilization.


B Western blots of whole - cell extracts showing efficient degradation of PDS5A or PDS5B ( left , right ) with dTAG treatment ( at different time points ) .

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B Western blots of whole-cell extracts showing efficient degradation of PDS5A or PDS5B (left, right) with dTAG treatment (at different time points).


D ChIP - seq signal for FLAG - PDS5A and FLAG - PDSB ( ChIP assays were performed with antibodies against Flag and PDS5A / B ) centered at TSS of promoters ( ± 3 kb ) across all peaks called for each of the proteins in DMSO treated PDS5A - dTAG or PDS5B - dTAG cells , showing the drastic reduction in PDS5A / B enrichment on chromatin after degradation . Top : normalized signal intensities for each protein .

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D ChIP-seq signal for FLAG-PDS5A and FLAG-PDSB (ChIP assays were performed with antibodies against Flag and PDS5A/B) centered at TSS of promoters (± 3 kb) across all peaks called for each of the proteins in DMSO treated PDS5A-dTAG or PDS5B-dTAG cells, showing the drastic reduction in PDS5A/B enrichment on chromatin after degradation. Top: normalized signal intensities for each protein.


F ChIP - qPCR using Flag antibody demonstrating complete depletion of PDS5A or PDS5B ( left , right ) in H19 / IGF2 imprinting locus and the promoter region of PCDH10 , NC represents a negative binding site . Data are represented as mean ± SD from three biological replicates .

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F ChIP-qPCR using Flag antibody demonstrating complete depletion of PDS5A or PDS5B (left, right) in H19/IGF2 imprinting locus and the promoter region of PCDH10, NC represents a negative binding site. Data are represented as mean±SD from three biological replicates.


B Heatmaps of signal intensities for FLAG - PDS5A , FLAG - PDS5B , CTCF , and RAD21 ChIP - seq data across all defined FLAG - PDS5A peaks .

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B Heatmaps of signal intensities for FLAG-PDS5A, FLAG-PDS5B, CTCF, and RAD21 ChIP-seq data across all defined FLAG-PDS5A peaks.


C Averaged ChIP - seq signal densities of FLAG - PDS5A , FLAG - PDS5B , RAD21 , and CTCF on PDS5A & PDS5B common sites and PDS5A / PDS5B unique sites .

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C Averaged ChIP-seq signal densities of FLAG-PDS5A, FLAG-PDS5B, RAD21, and CTCF on PDS5A & PDS5B common sites and PDS5A/PDS5B unique sites.


F Reciprocal Re - ChIP for PDS5A and PDS5B . The co - binding of PDS5A and PDS5B at PDS5A & PDS5B common sites were assayed by Re - ChIP - qPCR . The second ChIP for Re - ChIP was performed using antibodies against PDS5A , FLAG ( PDS5B ) , RAD21 , control IgG . RAD21 served as a positive control . Data are shown as fold enrichment versus IgG ( mean ± SD from three biological replicates ) .

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F Reciprocal Re-ChIP for PDS5A and PDS5B. The co-binding of PDS5A and PDS5B at PDS5A&PDS5B common sites were assayed by Re-ChIP-qPCR. The second ChIP for Re-ChIP was performed using antibodies against PDS5A, FLAG(PDS5B), RAD21, control IgG. RAD21 served as a positive control. Data are shown as fold enrichment versus IgG (mean±SD from three biological replicates).


A Validation of PDS5A ; PDS5B - dTAG cells for simultaneous depletion of PDS5A and PDS5B .

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A Validation of PDS5A;PDS5B-dTAG cells for simultaneous depletion of PDS5A and PDS5B.


B Chromatin fractionation assay showing the change of chromatin - bound level of RAD21 or CTCF after co - depletion of PDS5A and PDS5B .

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B Chromatin fractionation assay showing the change of chromatin-bound level of RAD21 or CTCF after co-depletion of PDS5A and PDS5B.


C Heatmaps displaying the ChIP - seq binding intensities for significantly changed RAD21 peaks after joint PDS5 proteins depletion ( left ) . Veen diagram showing RAD21 peaks identified in cells before and after co - depletion of PDS5A and PDS5B .

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C Heatmaps displaying the ChIP-seq binding intensities for significantly changed RAD21 peaks after joint PDS5 proteins depletion (left). Veen diagram showing RAD21 peaks identified in cells before and after co-depletion of PDS5A and PDS5B.


D Changes of RAD21 signals at all loop anchors defined in PDS5A - dTAG cells or PDS5B - dTAG cells with DMSO treatment after PDS5A and PDS5B co - depletion , or either depletion . For PDS5A ; PDS5B - dTAG cells , we used the genomic positions of loop anchors identified in DMSO treated PDS5A - dTAG cells . E Differential RAD21 ChIP - seq signals of representative regions showing significantly down - regulated peaks after co - depletion of PDS5A and PDS5B . F Differential RAD21 ChIP - seq signals of representative regions showing significantly up - regulated peaks after co - depletion of PDS5A and PDS5B .

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D Changes of RAD21 signals at all loop anchors defined in PDS5A-dTAG cells or PDS5B-dTAG cells with DMSO treatment after PDS5A and PDS5B co-depletion, or either depletion. For PDS5A;PDS5B-dTAG cells, we used the genomic positions of loop anchors identified in DMSO treated PDS5A-dTAG cells. E Differential RAD21 ChIP-seq signals of representative regions showing significantly down-regulated peaks after co-depletion of PDS5A and PDS5B. F Differential RAD21 ChIP-seq signals of representative regions showing significantly up-regulated peaks after co-depletion of PDS5A and PDS5B.


A , B Volcano plots showing gene expression changes after PDS5A ( A ) and PDS5B ( B ) degradation . Genes with significant changes ( padj < 0 . 05 ) are colored blue ( upregulated ) or red ( downregulated ) .

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A, B Volcano plots showing gene expression changes after PDS5A (A) and PDS5B (B) degradation. Genes with significant changes (padj < 0.05) are colored blue (upregulated) or red (downregulated).


C The overlap and discrepancy of PDS5A and PDS5B misregulated genes ( left ) . Heatmap displaying Benjamini - Hochberg adjusted p - value of shared genes down - regulated and up - regulated after PDS5A or PDS5B depletion ( right ) .

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C The overlap and discrepancy of PDS5A and PDS5B misregulated genes (left). Heatmap displaying Benjamini-Hochberg adjusted p-value of shared genes down-regulated and up-regulated after PDS5A or PDS5B depletion (right).


A . Evaluation of siRNA - mediated depletion . U2OS cells were treated with control , Plk4 or hSAS - 6 siRNA and immunoblotting was performed with the indicated antibodies . Asterisk indicates non - specific bands . The positions of molecular weight markers ( kDa ) are shown on the right .

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A. Evaluation of siRNA-mediated depletion. U2OS cells were treated with control, Plk4 or hSAS-6 siRNA and immunoblotting was performed with the indicated antibodies. Asterisk indicates non-specific bands. The positions of molecular weight markers (kDa) are shown on the right.


B . U2OS cells were transfected with the indicated siRNAs . Cell peripheries are marked with dotted lines . Enlarged single cell images ( marked with arrowheads in the top row ) are shown at the bottom . Scale bars , 5 μm ( bottom ) , 10 μm ( top ) .

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B. U2OS cells were transfected with the indicated siRNAs. Cell peripheries are marked with dotted lines. Enlarged single cell images (marked with arrowheads in the top row) are shown at the bottom. Scale bars, 5 μm (bottom),10 μm (top).


C , D , E . Quantification of the dispersion of satellite components upon depletion of Plk4 but not hSAS - 6 knockdown . The results of PCM1 ( C ) , hMsd1 / SSX2IP ( D ) or BBS4 ( E ) are shown . Data represent the mean + SD ( > 200 cells , n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * p < 0 . 01 , n . s . ( not significant ) .

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C, D, E. Quantification of the dispersion of satellite components upon depletion of Plk4 but not hSAS-6 knockdown. The results of PCM1 (C), hMsd1/SSX2IP (D) or BBS4 (E) are shown. Data represent the mean + SD (>200 cells, n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. **p<0.01, n.s. (not significant).


F , G . U2OS cells were arrested in G1 and treated with control or Plk4 siRNA , followed by immunofluorescence microscopy by using indicated antibodies . Enlarged images ( marked by arrowheads ) are shown on the right . Quantification of PCM1 distribution is shown in G . > 200 cells were counted and classified into two categories : normal ( around and away from the centrosome ) or dispersed . Data represent the mean + SD ( n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * p < 0 . 01 . Scale bar , 1 μm ( F , right ) , 10 μm ( F ) .

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F, G. U2OS cells were arrested in G1 and treated with control or Plk4 siRNA, followed by immunofluorescence microscopy by using indicated antibodies. Enlarged images (marked by arrowheads) are shown on the right. Quantification of PCM1 distribution is shown in G. >200 cells were counted and classified into two categories: normal (around and away from the centrosome) or dispersed. Data represent the mean + SD (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. **p<0.01. Scale bar, 1 μm (F, right), 10 μm (F).


H , I . siRNA - treated U2OS cells were transfected with empty vectors ( EV ) or plasmids containing Plk4 or siRNA - resistant Plk4 - myc WT * or KD * and immunostained with the indicated antibodies . Cell peripheries are marked with solid lines , in which cells with yellow lines are successfully transfected ( positive in myc signals ) , while those with white lines represent non - transfected cells . Quantification of PCM1 distribution is shown in I . Data represent the mean + SD ( > 200 cells , n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * * * p < 0 . 0001 , * * p < 0 . 01 , n . s . ( not significant ) . Scale bar , 5 μm ( H ) .

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H, I. siRNA-treated U2OS cells were transfected with empty vectors (EV) or plasmids containing Plk4 or siRNA-resistant Plk4-myc WT* or KD* and immunostained with the indicated antibodies. Cell peripheries are marked with solid lines, in which cells with yellow lines are successfully transfected (positive in myc signals), while those with white lines represent non-transfected cells. Quantification of PCM1 distribution is shown in I. Data represent the mean + SD (>200 cells, n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. ****p<0.0001, **p<0.01, n.s. (not significant). Scale bar, 5 μm (H).


A , B . Either empty vector plasmids ( EV ) or those producing EGFP - PCM1 ( A ) or myc - Plk4 ( B ) were transfected into U2OS cells . Immunoprecipitation ( IP ) was performed with anti - GFP ( A ) or anti - myc antibodies ( B ) , followed by immunoblotting with the indicated antibodies . DIC , the dynein intermediate chain .

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A, B. Either empty vector plasmids (EV) or those producing EGFP-PCM1 (A) or myc-Plk4 (B) were transfected into U2OS cells. Immunoprecipitation (IP) was performed with anti-GFP (A) or anti-myc antibodies (B), followed by immunoblotting with the indicated antibodies. DIC, the dynein intermediate chain.


C . Bacterially expressed and purified His - PCM1 ( a . a . 1 - 1128 ) was incubated with MBP alone or MBP - Plk4 ( WT or KD ) in vitro , and pulled down by magnetic beads coupled with an anti - MBP antibody , followed by staining with Coomassie Blue ( left ) or immunoblotting with an anti - His antibody ( right ) . Asterisks show non - specific bands . The mobility difference between Plk4 - WT and Plk4 - KD was reported previously [ 43 , 48 ] . We reproducibly observed the upward mobility shift of His - PCM1 upon binding to MBP - Plk4 - WT , but not Plk4 - KD .

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C. Bacterially expressed and purified His-PCM1 (a.a. 1-1128) was incubated with MBP alone or MBP-Plk4 (WT or KD) in vitro, and pulled down by magnetic beads coupled with an anti-MBP antibody, followed by staining with Coomassie Blue (left) or immunoblotting with an anti-His antibody (right). Asterisks show non-specific bands. The mobility difference between Plk4-WT and Plk4-KD was reported previously [43, 48]. We reproducibly observed the upward mobility shift of His-PCM1 upon binding to MBP-Plk4-WT, but not Plk4-KD.


E . Total cell extracts were prepared from U2OS cells treated with PCM1 siRNA and transfected with RNAi - resistant EGFP - PCM1 - WT * . Cells were cultured asynchronously ( As ) or arrested in G1 , G2 or M phase , followed by immunoprecipitation with an anti - GFP antibodies . Pulled down samples were immunoblotted with the indicated antibodies .

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E. Total cell extracts were prepared from U2OS cells treated with PCM1 siRNA and transfected with RNAi-resistant EGFP-PCM1-WT*. Cells were cultured asynchronously (As) or arrested in G1, G2 or M phase, followed by immunoprecipitation with an anti-GFP antibodies. Pulled down samples were immunoblotted with the indicated antibodies.


A , B , C , D . siRNA - treated U2OS cells ( PCM1 for A and B ; Plk4 for C and D ) were transfected with plasmids producing various EGFP - PCM1 constructs ( WT * , S372A * , S372D * or S372E * ) , fixed and stained with the indicated antibodies ( arrowheads point to the centrosome ) ( A and C ) . Quantification data are shown in B and D . > 200 cells were counted and classified into three categories : normal ( around and away from the centrosome ) , aggregated or dispersed . Data represent the mean + SD ( n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , n . s . ( not significant ) . Note that the percentage of cells displaying " dispersed " is analysed . Scale bars , 5 μm ( A and C ) .

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A, B, C, D. siRNA-treated U2OS cells (PCM1 for A and B; Plk4 for C and D) were transfected with plasmids producing various EGFP-PCM1 constructs (WT*, S372A*, S372D* or S372E*), fixed and stained with the indicated antibodies (arrowheads point to the centrosome) (A and C). Quantification data are shown in B and D. >200 cells were counted and classified into three categories: normal (around and away from the centrosome), aggregated or dispersed. Data represent the mean + SD (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. ***p<0.001, **p<0.01, *p<0.05, n.s. (not significant). Note that the percentage of cells displaying "dispersed" is analysed. Scale bars, 5 μm (A and C).


E . PCM1 siRNA - treated U2OS cells were transfected with two types of plasmids ( EGFP - and mCherry - tagged ) that produce PCM1 - WT * and PCM1 - S372A * . Immunoprecipitation and subsequent immunoblotting were performed with the indicated antibodies . Quantification data is shown on the right . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , n . s . not significant .

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E. PCM1 siRNA-treated U2OS cells were transfected with two types of plasmids (EGFP- and mCherry-tagged) that produce PCM1-WT* and PCM1-S372A*. Immunoprecipitation and subsequent immunoblotting were performed with the indicated antibodies. Quantification data is shown on the right. Statistical analysis was performed using two-tailed unpaired student's t-tests. *p<0.05, **p<0.01, n.s. not significant.


F . U2OS cells were treated with PCM1 siRNA and transfected with empty vectors ( EV ) or EGFP plasmids that produce PCM1 - WT * or PCM1 - S372A * . Immunoprecipitation was performed with an anti - GFP antibody , followed by immunoblotting with the indicated antibodies .

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F. U2OS cells were treated with PCM1 siRNA and transfected with empty vectors (EV) or EGFP plasmids that produce PCM1-WT* or PCM1-S372A*. Immunoprecipitation was performed with an anti-GFP antibody, followed by immunoblotting with the indicated antibodies.


A . Immunofluorescence microscopy was performed using the indicated antibodies in hTERT - RPE - 1 cells treated with control , PCM1 or Plk4 siRNA . Regions around the basal body ( insets ) are enlarged on the right hand side . Scale bars , 5 μm ( left ) and 1 μm ( right ) . B . Quantification . At least 150 cells were counted ( n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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A. Immunofluorescence microscopy was performed using the indicated antibodies in hTERT-RPE-1 cells treated with control, PCM1 or Plk4 siRNA. Regions around the basal body (insets) are enlarged on the right hand side. Scale bars, 5 μm (left) and 1 μm (right).B. Quantification. At least 150 cells were counted (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. ***p<0.001, ****p<0.0001.


C . hTERT - RPE - 1 cells treated with PCM1 siRNA were transfected by an empty vector ( EV ) or plasmids producing PCM1 - WT * , - S327A * or - S372E * , and immunofluorescence microscopy performed . Enlarged images of the regions around the basal body are shown in insets . Scale bars , 5 μm and 1 μm ( inset ) .

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C. hTERT-RPE-1 cells treated with PCM1 siRNA were transfected by an empty vector (EV) or plasmids producing PCM1-WT*, -S327A* or -S372E*, and immunofluorescence microscopy performed. Enlarged images of the regions around the basal body are shown in insets. Scale bars, 5 μm and 1 μm (inset).


D . Quantification . At least 200 cells were counted ( n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . not significant .

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D. Quantification. At least 200 cells were counted (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. not significant.


Graphs show changes in cytotoxicity between WT and GR T24 and UMUC3 cells exposed to increasing concentrations of GEM , and WST assays were performed 48 hr after treatment ( n = 3 , biological replicates ) .

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Graphs show changes in cytotoxicity between WT and GR T24 and UMUC3 cells exposed to increasing concentrations of GEM, and WST assays were performed 48 hr after treatment (n=3, biological replicates).


Major metabolites altered in the glycolytic pathway in CR cells and GR cells compared to WT cells . The data are shown as the means ± SDs ( n = 4 ) and were analyzed by Student ' s t test .

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Major metabolites altered in the glycolytic pathway in CR cells and GR cells compared to WT cells. The data are shown as the means ± SDs (n=4) and were analyzed by Student's t test.


Western blot analysis of GLUT1 , PKM2 , LDHA , and β - actin compared among WT , CR , and GR cells .

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Western blot analysis of GLUT1, PKM2, LDHA, and β-actin compared among WT, CR, and GR cells.


Levels of endpoint metabolites of the purine synthesis pathway in GR cells relative to those in WT cells as determined by CE - MS metabolomics . The data are shown as the means ± SDs ( n = 3 , biological replicates ) and were analyzed by Student ' s t test . n . s . = non - significant Levels of endpoint metabolites of the pyrimidine synthesis pathway in GR cells relative to those in WT cells as determined by CE - MS metabolomics . The data are shown as the means ± SDs ( n = 3 , biological replicates ) and were analyzed by Student ' s t test . * p < 0 . 05 , * * p < 0 . 01 .

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Levels of endpoint metabolites of the purine synthesis pathway in GR cells relative to those in WT cells as determined by CE-MS metabolomics. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. n.s.= non-significant   Levels of endpoint metabolites of the pyrimidine synthesis pathway in GR cells relative to those in WT cells as determined by CE-MS metabolomics. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. *p<0.05, **p<0.01.


Western blot analysis of Hif - 1α , G6PD , TIGAR , TKT , CTPS1 , and β - actin in WT and GR cells under normoxia and hypoxia ( left : T24 cells , right : UMUC3 cells ) .

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Western blot analysis of Hif-1α, G6PD, TIGAR, TKT, CTPS1, and β-actin in WT and GR cells under normoxia and hypoxia (left: T24 cells, right: UMUC3 cells).


CE - MS - based metabolite detection for diphosphate nucleosides in WT and GR cells . The data are shown as the means ± SDs ( n = 3 , biological replicates ) . The levels in GR cells are presented relative to those in WT control cells and analyzed by Student ' s t test . * * * p < 0 . 001 . N . D . = non - detectable .

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CE-MS-based metabolite detection for diphosphate nucleosides in WT and GR cells. The data are shown as the means ± SDs (n=3, biological replicates). The levels in GR cells are presented relative to those in WT control cells and analyzed by Student's t test. ***p<0.001. N.D.= non-detectable.


Levels of intermediate metabolites of the tricarboxylic acid cycle ( TCA ) cycle in GR cells relative to those in WT cells based on targeted CE - MS metabolomics . The data are shown as the means ± SDs ( n = 3 , biological replicates ) and were analyzed by Student ' s t test . * p < 0 . 05 , * * p < 0 . 01 .

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Levels of intermediate metabolites of the tricarboxylic acid cycle (TCA) cycle in GR cells relative to those in WT cells based on targeted CE-MS metabolomics. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. *p<0.05, **p<0.01.


Relative 2 - hydroxyglutarate ( 2 - HG ) production level based on targeted CE - MS metabolomics comparing among WT and GR cells ( left panel : T24 , right panel : UMUC3 ) . The data are shown as the means ± SDs ( n = 3 , biological replicates ) and were analyzed by Student ' s t test . * p < 0 . 05 , * * p < 0 . 01 . Relative mRNA expression levels of IDH2 and EGLN1 genes compared between WT and GR cells ( left panel : T24 , right panel : UMUC3 ) . The expression levels were analyzed by Student ' s t test and plotted relative to expression levels in WT cells . The data are shown as the means ± SEs ( n = 3 , biological replicates . * p < 0 . 05 , * * * p < 0 . 001 .

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Relative 2-hydroxyglutarate (2-HG) production level based on targeted CE-MS metabolomics comparing among WT and GR cells (left panel: T24, right panel: UMUC3). The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. *p<0.05, **p<0.01. Relative mRNA expression levels of IDH2 and EGLN1 genes compared between WT and GR cells (left panel: T24, right panel: UMUC3). The expression levels were analyzed by Student's t test and plotted relative to expression levels in WT cells. The data are shown as the means ± SEs (n=3, biological replicates. *p<0.05, ***p<0.001.