( C and D ) Immobilized GST - TAB2 was pre - incubated with K63 - linked polyUb chains and then eluted from beads , followed by incubation with eluted GST - TAK1 and GST - p38IP as shown in ( B ) . Incubation mixture were immunoprecipitated with anti - TAB2 or anti - TAK1 , and then immunoblotted with anti - ub , anti - TAB2 , anti - TAK1 and anti - p38IP . ( E ) Statistical analysis of polyUb binding to TAB2 or TAK1 in ( C and D ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two - tailed unpaired t - test , mean and s . e . m . ) .

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 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 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 O O O O O O O O O O O O O O O O

(C and D) Immobilized GST-TAB2 was pre-incubated with K63-linked polyUb chains and then eluted from beads, followed by incubation with eluted GST-TAK1 and GST-p38IP as shown in (B). Incubation mixture were immunoprecipitated with anti-TAB2 or anti-TAK1, and then immunoblotted with anti-ub, anti-TAB2, anti-TAK1 and anti-p38IP. (E) Statistical analysis of polyUb binding to TAB2 or TAK1 in (C and D). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).


( F ) Immobilized GST - TAB2 and GST - TAK1 were respectively pre - incubated with K63 - linked polyUb chains or not as indicated , then eluted from beads , followed by coincubation as indicated . Incubation mixture were immunoprecipitated with anti - TAB2 , and then immunoblotted with anti - TAB2 and anti - TAK1 . The relative intensity of immunoprecipitated TAK1 was calculated and shown in the right . * * * P < 0 . 001 ; n . s . , not significant ( two - tailed unpaired t - test , mean and s . e . m . ) .

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 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

(F) Immobilized GST-TAB2 and GST-TAK1 were respectively pre-incubated with K63-linked polyUb chains or not as indicated, then eluted from beads, followed by coincubation as indicated. Incubation mixture were immunoprecipitated with anti-TAB2, and then immunoblotted with anti-TAB2 and anti-TAK1. The relative intensity of immunoprecipitated TAK1 was calculated and shown in the right. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).


( G ) Immobilized GST - TAK1 was pre - incubated with K63 - linked polyUb chains or not , then eluted from beads , followed by incubation with eluted GST - p38IP . Incubation mixture were immunoprecipitated with anti - TAK1 , and then immunoblotted with anti - TAK1 and anti - p38IP . The relative intensity of immunoprecipitated p38IP was calculated and shown in the right . * * * P < 0 . 001 ( two - tailed unpaired t - test , mean and s . e . m . ) .

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

(G) Immobilized GST-TAK1 was pre-incubated with K63-linked polyUb chains or not, then eluted from beads, followed by incubation with eluted GST-p38IP. Incubation mixture were immunoprecipitated with anti-TAK1, and then immunoblotted with anti-TAK1 and anti-p38IP. The relative intensity of immunoprecipitated p38IP was calculated and shown in the right. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).


( B ) HEK293T cells transfected with N - terminal ( left panels ) or C - terminal ( right panels ) deletion mutants of p38IP as indicated in ( A ) were lysed after 24 hours transfection and immunoprecipitated with IgG or anti - TAK1 , followed by incubation with protein G beads , and then immunoblotted with anti - YFP and anti - TAK1 .

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) HEK293T cells transfected with N-terminal (left panels) or C-terminal (right panels) deletion mutants of p38IP as indicated in (A) were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.


( D ) GST pull down assay . 3μg recombinant GST - T1B protein which immobilized on glutathione Sepharose beads was incubated with the cell lysates that obtained by adding 1ml lysis buffer to the HEK293T cells in 10 cm cell culture dish with a final protein concentration around 1μg / μl , after 4 hrs incubation , washed beads with cell lysis buffer five times and subjected to SDS - PAGE for immunoblotting with anti - TAK1 .

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

(D) GST pull down assay. 3μg recombinant GST-T1B protein which immobilized on glutathione Sepharose beads was incubated with the cell lysates that obtained by adding 1ml lysis buffer to the HEK293T cells in 10 cm cell culture dish with a final protein concentration around 1μg/μl, after 4 hrs incubation, washed beads with cell lysis buffer five times and subjected to SDS-PAGE for immunoblotting with anti-TAK1.


( E ) HEK293T cells transfected with YFP - p38IP T1B were lysed after 24 hours transfection and immunoprecipitated with IgG or anti - TAK1 , followed by incubation with protein G beads , and then immunoblotted with anti - YFP and anti - TAK1 .

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

(E) HEK293T cells transfected with YFP-p38IP T1B were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.


( F ) Jurkat TAg cells transfected with YFP vector or YFP - T1B were stimulated with 10μg / ml anti - CD3 for indicated times , then lysed and immunoprecipitated with anti - TAK1 , followed by immunoblotting with the indicated antibodies .

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

(F) Jurkat TAg cells transfected with YFP vector or YFP-T1B were stimulated with 10μg/ml anti-CD3 for indicated times, then lysed and immunoprecipitated with anti-TAK1, followed by immunoblotting with the indicated antibodies.


( G ) Primary human PBMCs transfected with YFP vector or YFP - T1Bwere stimulated with 10 μg / ml anti - CD3 plus 10 μg / ml anti - CD28 for the indicated times , then lysed and analyzed by immunoblotting .

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

(G) Primary human PBMCs transfected with YFP vector or YFP-T1Bwere stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 for the indicated times, then lysed and analyzed by immunoblotting.


( H ) Intracellular cytokine staining analysis of TNF - α ( upper ) and IL - 2 ( lower ) in primary human PBMCs transfected and stimulated as in ( F ) for 3 hours ( TNF - α ) and 8 hours ( IL - 2 ) .

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

(H) Intracellular cytokine staining analysis of TNF-α (upper) and IL-2 (lower) in primary human PBMCs transfected and stimulated as in (F) for 3 hours (TNF-α) and 8 hours (IL-2).


( A ) HEK293T cells co - transfected with YFP - p38IP and Flag - vector , Flag - USP3 or Flag - USP4 as indicated were lysed and immunoprecipitated with anti - Flag antibody and immunoblotted with indicated antibodies .

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

(A) HEK293T cells co-transfected with YFP-p38IP and Flag-vector, Flag-USP3 or Flag-USP4 as indicated were lysed and immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.


( B ) HEK293T cells transfected with Flag - USP4 and YFP - vector or YFP - p38IP as indicated were lysed and immunoprecipitated with anti - IgG or anti - TAK1 and then immunoblotted with anti - Flag and anti - TAK1 .

(B) HEK293T cells transfected with Flag-USP4 and YFP-vector or YFP-p38IP as indicated were lysed and immunoprecipitated with anti-IgG or anti-TAK1 and then immunoblotted with anti-Flag and anti-TAK1.


( C ) HEK293T cells transfected with plasmids as indicated were lysed and immunoprecipitated with anti - TAK1 , followed by in vivo ubiquitination assay of TAK1 as described in methods .

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

(C) HEK293T cells transfected with plasmids as indicated were lysed and immunoprecipitated with anti-TAK1, followed by in vivo ubiquitination assay of TAK1 as described in methods.


( D ) In vitro deubiquitination assay of TAK1 as described in methods .

(D) In vitro deubiquitination assay of TAK1 as described in methods.


( E ) sh - NC and sh - p38IP Jurkat E6 . 1 cells transfected with si - NC , si - p38IP , Flag or Flag - USP4 as indicated were stimulated with anti - CD3 / CD28 for 0 and 15 min at 48 hours post transfection , and then lysed and immunoprecipitated with anti - TAK1 under stringent conditions and immunoblotted with anti - ub . Statistical analysis of p - p38 levels was shown below the blot . * * P < 0 . 01 ; n . s . , not significant ( two - tailed unpaired t - test , mean and s . e . m . ) .

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-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 O O O O O O O O O O

(E) sh-NC and sh-p38IP Jurkat E6.1 cells transfected with si-NC, si-p38IP, Flag or Flag-USP4 as indicated were stimulated with anti-CD3/CD28 for 0 and 15 min at 48 hours post transfection, and then lysed and immunoprecipitated with anti-TAK1 under stringent conditions and immunoblotted with anti-ub. Statistical analysis of p-p38 levels was shown below the blot. **P < 0.01; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).


( F ) Jurkat E6 . 1 T cells were stimulated with anti - CD3 / CD28 for the indicated times . The cell lysates were immunoprecipitated with anti - p38IP or anti - TAK1 and then immunoblotted with the indicated antibodies .

(F) Jurkat E6.1 T cells were stimulated with anti-CD3/CD28 for the indicated times. The cell lysates were immunoprecipitated with anti-p38IP or anti-TAK1 and then immunoblotted with the indicated antibodies.


( G ) sh - NC and sh - p38IP Jurkat E6 . 1 cells were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for the indicated times , and then lysed and immunoprecipitated with anti - TAK1 and immunoblotted with anti - USP4 and anti - TAK1 .

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

(G) sh-NC and sh-p38IP Jurkat E6.1 cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times, and then lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-USP4 and anti-TAK1.


( H ) Mapping the USP4 binding domain of p38IP .

(H) Mapping the USP4 binding domain of p38IP.


( F - K ' ) ( F ' , G ' , H ' , I ' ) are the magnified images of the square region outlined in ( F , G , H , I ) . ( F - K ) are merged images , ( F ' - K ' ) are single channel images of ( F - K ) depicting the presence of NBs ( Mira + ) . Clones ( green ) are outlined by dotted lines . All images in this figure , and all following figures are single confocal sections . ( F - F ' and G - G ' ) No Mira + NBs are recovered in deep sections of control or UAS - dpn clones at 16 hr after clone induction . Quantified in E . ( H - H ' and I - I ' ) At 24 hr after clone induction , Mira + NBs are recovered in UAS - dpn but not control clones . Quantified in E . ( J - J ' and K - K ' ) At 72h after clone induction , numerous Mira + NBs are recovered in UAS - dpn but not in control clones . Quantified in E .

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 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 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-K') (F', G', H', I') are the magnified images of the square region outlined in (F, G, H, I). (F-K) are merged images, (F'-K') are single channel images of (F-K) depicting the presence of NBs (Mira+). Clones (green) are outlined by dotted lines. All images in this figure, and all following figures are single confocal sections. (F-F' and G-G') No Mira+ NBs are recovered in deep sections of control or UAS-dpn clones at 16 hr after clone induction. Quantified in E. (H-H' and I-I') At 24 hr after clone induction, Mira+ NBs are recovered in UAS-dpn but not control clones. Quantified in E. (J-J'and K-K') At 72h after clone induction, numerous Mira+ NBs are recovered in UAS-dpn but not in control clones. Quantified in E.


( C - L ' ) Representative images of the deep medulla neuronal layer or NB superficial layer in the larval optic lobe , in which UAS - dpn or control are driven in clones by hs flp ( marked by GFP and outlined ) and stained with the stem cell marker , Miranda ( Mira , magenta ) , and various temporal transcription factors ( tTFs ) ( grey ) . ( C - G ' ) In superficial sections of control clones , Mira + superficial NBs express the tTF : Hth , Ey , Slp , D , Tll in concentric patterns . Quantified in M . ( H - L ' ) In deep sections of the NB clone induced via UAS - dpn , Mira + NBs do not express early tTF Hth or late tTF Tll , but are positive for mid tTFs Ey , Slp and D . Quantified in M .

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 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

(C-L') Representative images of the deep medulla neuronal layer or NB superficial layer in the larval optic lobe, in which UAS-dpn or control are driven in clones by hs flp (marked by GFP and outlined) and stained with the stem cell marker, Miranda (Mira, magenta), and various temporal transcription factors (tTFs) (grey). (C-G') In superficial sections of control clones, Mira+ superficial NBs express the tTF: Hth, Ey, Slp, D, Tll in concentric patterns. Quantified in M. (H- L') In deep sections of the NB clone induced via UAS-dpn, Mira+ NBs do not express early tTF Hth or late tTF Tll, but are positive for mid tTFs Ey, Slp and D. Quantified in M.


( M ) Quantification of volume of cells that express a specific tTF as % of total Mira + NB volume within a clone . Hth ( Control n = 5 , m = 31 . 86 ± 3 . 89 , UAS - dpn n = 6 , m = 7 . 779 ± 1 . 212 ) , Ey ( Control n = 7 , m = 26 . 05 ± 5 . 302 , UAS - dpn n = 5 , m = 64 . 08 ± 4 . 936 ) , Slp ( Control n = 7 , m = 34 . 87 ± 2 . 753 , UAS - dpn n = 9 , m = 64 . 72 ± 6 . 045 ) , D ( Control n = 4 , m = 34 . 57 ± 15 . 49 , UAS - dpn n = 10 , m = 45 . 2 ± 6 . 339 ) , Tll ( Control , n = 5 m = 15 . 65 ± 4 . 036 , UAS - dpn n = 6 , m = 3 . 584 , ± 1 . 87 ) .

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 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 O O O O O O O O O O O O O O O O O O O O O

(M) Quantification of volume of cells that express a specific tTF as % of total Mira+ NB volume within a clone. Hth (Control n= 5, m= 31.86 ±3.89, UAS-dpn n= 6, m= 7.779 ±1.212), Ey (Control n= 7, m= 26.05 ±5.302, UAS-dpn n= 5, m= 64.08 ±4.936), Slp (Control n= 7, m= 34.87 ±2.753, UAS-dpn n= 9, m= 64.72 ±6.045), D (Control n= 4, m= 34.57 ±15.49, UAS-dpn n= 10, m= 45.2 ±6.339), Tll (Control, n= 5 m= 15.65 ±4.036, UAS-dpn n= 6, m= 3.584, ±1.87).


( B ) Quantification of % of Slp + cells in Mira + cells in control and UAS - dpn clones ( expressed as the ratio between Slp + volume and total Mira + volume within a clone ) . 24 hr ( Control n = 7 , m = 14 . 25 ± 2 . 696 , UAS - dpn n = 9 , m = 60 . 66 ± 5 . 384 ) , 48 hr ( Control n = 3 , m = 6 . 096 ± 1 . 509 , UAS - dpn n = 8 , m = 43 . 64 ± 3 . 398 ) , 72 hr ( Control n = 6 , m = 35 . 13 ± 3 . 242 , UAS - dpn n = 9 , m = 64 . 72 ± 6 . 045 ) , 96 hr ( Control n = 6 , m = 29 . 85 ± 4 . 951 , UAS - dpn n = 10 , m = 58 . 2 ± 6 . 603 ) .

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 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 O O O O

(B) Quantification of % of Slp+ cells in Mira+ cells in control and UAS-dpn clones (expressed as the ratio between Slp+ volume and total Mira+ volume within a clone). 24 hr (Control n= 7, m= 14.25 ±2.696, UAS-dpn n= 9, m= 60.66 ±5.384), 48 hr (Control n= 3, m= 6.096 ±1.509, UAS-dpn n= 8, m= 43.64 ±3.398), 72 hr (Control n= 6, m= 35.13 ±3.242, UAS-dpn n= 9, m= 64.72 ±6.045), 96 hr (Control n= 6, m= 29.85 ±4.951, UAS-dpn n= 10, m= 58.2 ± 6.603).


( C - F ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which UAS - dpn or control are driven by hs flp ( marked by GFP and outlined ) and dissected and stained with the stem cell marker , Miranda ( Mira , magenta ) , and Sloppy - paired ( Slp , grey ) . At 24h and 48h after clone induction , Mira + NBs express mid tTF Slp compared to superficial NB control , quantified in B . ( C ' ' , D ' ' , E ' ' , F ' ' ) are the magnified images of the square region outlined in ( C ' , D ' , E ' , F ' ) , respectively .

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 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

(C-F'') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which UAS-dpn or control are driven by hs flp (marked by GFP and outlined) and dissected and stained with the stem cell marker, Miranda (Mira, magenta), and Sloppy-paired (Slp, grey). At 24h and 48h after clone induction, Mira+ NBs express mid tTF Slp compared to superficial NB control, quantified in B. (C'', D'', E'', F'') are the magnified images of the square region outlined in (C', D', E', F'), respectively.


( G - J ' ) At 96h after clone induction , Mira + NBs express mid tTF Slp and do not express late tTF Tll , compared to control , quantified in B and K . ( K ) Quantification of % of Tll + cells in Mira + cells in control and UAS - dpn clones ( expressed as the ratio between Tll + volume and total Mira + volume within a clone ) . Control n = 4 , m = 48 . 34 ± 5 . 956 , UAS - dpn n = 5 , m = 4 . 805 ± 2 . 375 .

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 O O O O O O O O O O O O O O O O O O O

(G-J') At 96h after clone induction, Mira+ NBs express mid tTF Slp and do not express late tTF Tll, compared to control, quantified in B and K. (K) Quantification of % of Tll+ cells in Mira+ cells in control and UAS-dpn clones (expressed as the ratio between Tll+ volume and total Mira+ volume within a clone). Control n= 4, m= 48.34 ±5.956, UAS-dpn n= 5, m= 4.805 ±2.375. D


Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe , in which UAS - dpn or control ( UAS - luciferase ) are driven by eyR16F10 - GAL4 and stained with the stem cell marker , Miranda ( Mira , magenta ) , ( B - E ) Mira + NBs are recovered in superficial sections of control under eyR16F10 - GAL4 expression at 6 hr APF and 10 hr APF but not from16 hr APF onwards , quantified in J . ( F - I ) Mira + NBs are recovered in deep sections of UAS - dpn driven by eyR16F10 - GAL4 at 6 hr APF , 10 hr APF , and 16 hr APF but not at 24 hr APF , quantified in J . Arrow in H points to the NBs . ( J ) Quantification of % Mira + cells within eyR16F10 - GAL4 expression domain in control and UAS - dpn ( calculated as the ratio of Mira + cell volume as a percentage of total eyR16F10 - GAL4 domain volume ) . 6 hr APF ( Control , n = 3 , m = 14 . 82 ± 3 . 521 , UAS - dpn , n = 6 , m = 18 . 13 ± 1 . 46 ) , 10 hr APF ( Control n = 4 , m = 11 . 65 ± 1 . 605 , UAS - dpn n = 5 , m = 19 . 11 ± 4 . 609 ) , 16 hr APF ( Control n = 4 , m = 0 . 8897 ± 0 . 239 , UAS - dpn n = 7 , m = 16 . 64 ± 3 . 065 ) , 24 hr APF ( Control n = 4 m = 1 . 174 , ± 0 . 7226 , UAS - dpn n = 6 , m = 0 . 8518 ± 0 . 4849 ) .

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 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 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 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

Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe, in which UAS-dpn or control (UAS-luciferase) are driven by eyR16F10-GAL4 and stained with the stem cell marker, Miranda (Mira, magenta), (B-E) Mira+ NBs are recovered in superficial sections of control under eyR16F10-GAL4 expression at 6 hr APF and 10 hr APF but not from16 hr APF onwards, quantified in J. (F-I) Mira+ NBs are recovered in deep sections of UAS-dpn driven by eyR16F10-GAL4 at 6 hr APF, 10 hr APF, and 16 hr APF but not at 24 hr APF, quantified in J. Arrow in H points to the NBs. (J) Quantification of % Mira+ cells within eyR16F10-GAL4 expression domain in control and UAS-dpn (calculated as the ratio of Mira+ cell volume as a percentage of total eyR16F10-GAL4 domain volume). 6 hr APF (Control, n= 3, m= 14.82 ±3.521, UAS-dpn, n= 6, m= 18.13 ±1.46), 10 hr APF (Control n= 4, m= 11.65 ±1.605, UAS-dpn n= 5, m= 19.11 ±4.609), 16 hr APF (Control n= 4, m= 0.8897 ±0.239, UAS-dpn n= 7, m= 16.64 ±3.065), 24 hr APF (Control n= 4 m= 1.174, ±0.7226, UAS-dpn n= 6, m= 0.8518 ±0.4849).


Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe , in which UAS - dpn or control ( UAS - luciferase ) are driven by ok107 - GAL4 ( outlined ) and stained with the stem cell marker , Miranda ( Mira , magenta ) , and mid - temporal progeny marker , Toy - GFP ( grey ) . ( K - L ' ) More Toy + progeny ( grey ) are present within the ok107 - GAL4 domain ( green , outlined ) in UAS - dpn compared to control .

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 O O O O O O O O O O O O O O O

Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe, in which UAS-dpn or control (UAS-luciferase) are driven by ok107-GAL4 (outlined) and stained with the stem cell marker, Miranda (Mira, magenta), and mid-temporal progeny marker, Toy-GFP (grey). (K-L') More Toy+ progeny (grey) are present within the ok107-GAL4 domain (green, outlined) in UAS-dpn compared to control.


( O - R ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which heat shock induced UAS - dpn or control ( UAS - luciferase ) clones are stained with the stem cell marker , Mira ( magenta ) , late neuronal marker Tll ( grey ) or glial marker Repo ( grey ) . Arrow points towards the small band of Tll + neurons in control clones . ( O - P ' ) At 72 hr after clone induction , there is less Tll expression within UAS - dpn clones than control clone , quantified in S . ( Q - R ' ) At 72 hr after clone induction , there is less Repo expression within UAS - dpn clones than control clones , quantified in T .

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 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-R') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which heat shock induced UAS-dpn or control (UAS-luciferase) clones are stained with the stem cell marker, Mira (magenta), late neuronal marker Tll (grey) or glial marker Repo (grey). Arrow points towards the small band of Tll+ neurons in control clones. (O-P') At 72 hr after clone induction, there is less Tll expression within UAS-dpn clones than control clone, quantified in S. (Q-R') At 72 hr after clone induction, there is less Repo expression within UAS-dpn clones than control clones, quantified in T.


Representative images of deep medulla neuronal layer in the larval optic lobe , in which UAS - luc ; UAS - dpn and UAS - D ; UAS - dpn are expressed in clones by hs flp , and stained with the stem cell marker , Miranda ( Mira , magenta ) , and / or Slp , Tll or Repo ( grey ) . ( A - B ' ' ) At 72 hr after clone induction , Mira + NBs within UAS - luc ; UAS - dpn clones express Slp ; Mira + NBs within UAS - D ; UAS - dpn clones do not express Slp , quantified in I . ( C - D ' ) Representative images of deep medulla neuronal layer in the larval optic lobe , in which UAS - dpn and UAS - D ; UAS - dpn are expressed in the ok107 - GAL4 expression domain , and stained with the stem cell marker , Mira ( magenta ) , and mid - temporal progeny marker , Toy - GFP ( grey ) . Fewer Toy + cells were recovered within the ok107 - GAL4 domain ( outlined ) in UAS - D ; UAS - dpn compared to UAS - dpn , quantified in E . ( E ) Quantification of % Toy + cells within ok107 - GAL4 expression domain in UAS - dpn and UAS - D ; UAS - dpn ( calculated as the ratio of Toy + cell volume as a percentage of total ok107 - GAL4 domain volume ) . UAS - dpn n = 9 , m = 22 . 78 ± 4 . 174 , UAS - D ; UAS - dpn n = 5 , m = 14 . 42 ± 2 . 394 .

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 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 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 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

Representative images of deep medulla neuronal layer in the larval optic lobe, in which UAS-luc; UAS-dpn and UAS-D; UAS-dpn are expressed in clones by hs flp, and stained with the stem cell marker, Miranda (Mira, magenta), and/or Slp, Tll or Repo (grey). (A-B'') At 72 hr after clone induction, Mira+ NBs within UAS-luc; UAS-dpn clones express Slp; Mira+ NBs within UAS-D; UAS-dpn clones do not express Slp, quantified in I. (C-D') Representative images of deep medulla neuronal layer in the larval optic lobe, in which UAS-dpn and UAS-D; UAS-dpn are expressed in the ok107-GAL4 expression domain, and stained with the stem cell marker, Mira (magenta), and mid-temporal progeny marker, Toy-GFP (grey). Fewer Toy+ cells were recovered within the ok107-GAL4 domain (outlined) in UAS-D; UAS-dpn compared to UAS-dpn, quantified in E. (E) Quantification of % Toy+ cells within ok107-GAL4 expression domain in UAS-dpn and UAS-D; UAS-dpn (calculated as the ratio of Toy+ cell volume as a percentage of total ok107-GAL4 domain volume). UAS-dpn n= 9, m= 22.78 ±4.174, UAS-D; UAS-dpn n= 5, m= 14.42 ±2.394.


( J - K ) Quantification of % Tll + or Repo + cells within UAS - dpn clones compared to UAS - D ; UAS - dpn clones ( calculated as the ratio of Tll + or Repo + cell volume as a percentage of total clone volume ) . Tll ( UAS - dpn n = 7 , m = 0 . 7198 ± 0 . 2389 , UAS - D ; UAS - dpn n = 9 , m = 35 . 22 ± 4 . 917 ) , Repo ( UAS - dpn n = 7 m = 1 . 488 ± 0 . 2368 , UAS - D ; UAS - dpn n = 9 , m = 14 . 13 ± 2 . 672 ) .

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 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

(J-K) Quantification of % Tll+ or Repo+ cells within UAS-dpn clones compared to UAS-D; UAS-dpn clones (calculated as the ratio of Tll+ or Repo+ cell volume as a percentage of total clone volume). Tll (UAS-dpn n= 7, m= 0.7198 ±0.2389, UAS-D; UAS-dpn n= 9, m= 35.22 ±4.917), Repo (UAS-dpn n= 7 m= 1.488 ±0.2368, UAS-D; UAS-dpn n= 9, m= 14.13 ±2.672).


( L - M ) Mira + NBs are present in deep sections of UAS - dpn driven by eyR16F10 - GAL4 at 126h but are absent in UAS - D ; UAS - dpn , quantified in N . ( N ) Quantification of % Mira cells within eyR16F10 - GAL4 expression domain in UAS - dpn and UAS - D ; UAS - dpn ( calculated as the ratio of Mira + cell volume as a percentage of total eyR16F10 - GAL4 domain volume ) . UAS - dpn n = 6 , m = 18 . 13 ± 1 . 46 , UAS - D ; UAS - dpn n = 8 , m = 1 . 859 ± 0 . 4215 .

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 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

(L-M) Mira+ NBs are present in deep sections of UAS-dpn driven by eyR16F10-GAL4 at 126h but are absent in UAS-D; UAS-dpn, quantified in N. (N) Quantification of % Mira cells within eyR16F10-GAL4 expression domain in UAS-dpn and UAS-D; UAS-dpn (calculated as the ratio of Mira+ cell volume as a percentage of total eyR16F10-GAL4 domain volume). UAS-dpn n= 6, m= 18.13 ±1.46, UAS-D; UAS-dpn n= 8, m= 1.859 ±0.4215. D


( A - C ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which control ( UAS - luc ) and UAS - dpn and UAS - D ; UAS - dpn are expressed in hs flp clones ( 72 hr after clone induction ) . Miranda ( Mira , magenta ) and EdU ( grey ) . Despite increased number of Mira + cells in UAS - dpn compared to control , each NB underwent slower cell cycle progression , as indicated by EdU + incorporation . Quantified in E . This slow cell cycle speed was not rescued in UAS - D ; UAS - dpn clones . Quantified with UAS - luc ; UAS - dpn in F .

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 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

(A-C'') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-luc) and UAS-dpn and UAS-D; UAS-dpn are expressed in hs flp clones (72 hr after clone induction). Miranda (Mira, magenta) and EdU (grey). Despite increased number of Mira+ cells in UAS-dpn compared to control, each NB underwent slower cell cycle progression, as indicated by EdU+ incorporation. Quantified in E. This slow cell cycle speed was not rescued in UAS-D; UAS-dpn clones. Quantified with UAS-luc; UAS-dpn in F.


( G - H ' ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , where UAS - E2f1 ; UAS - CycE ; UAS - dpn or UAS - dpn are expressed in the ok107 - GAL4 domain ( outlined ) , and stained with the stem cell marker , Mira ( magenta ) , mid - tTF , Slp ( grey ) and late progeny marker , Repo ( Cyan ) . Quantified in I - K . UAS - dpn NBs express Slp and do not create Repo + progeny , whereas UAS - E2f1 ; UAS - CycE ; UAS - dpn NBs do not express Slp and create Repo + progeny .

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 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-H''') Representative images of the deep medulla neuronal layer in the larval optic lobe, where UAS- E2f1; UAS- CycE; UAS-dpn or UAS-dpn are expressed in the ok107-GAL4 domain (outlined), and stained with the stem cell marker, Mira (magenta), mid-tTF, Slp (grey) and late progeny marker, Repo (Cyan). Quantified in I-K. UAS-dpn NBs express Slp and do not create Repo+ progeny, whereas UAS- E2f1; UAS- CycE; UAS-dpn NBs do not express Slp and create Repo+ progeny.


( B - D ' ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which control ( UAS - luc ) , UAS - NACT , and UAS - D ; UAS - NACT are driven by eyR16F10 - GAL4 ( outlined ) . Miranda ( Mira , magenta ) , Sloppy paired ( Slp , grey ) , Tailless ( Tll , cyan ) . UAS - NACT NBs express Slp and not Tll . UAS - D ; UAS - NACT NBs express Slp and Tll , quantified in E - H .

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 O O O O O O O O O O O O O O

(B-D''') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-luc), UAS-NACT, and UAS-D; UAS-NACT are driven by eyR16F10-GAL4 (outlined). Miranda (Mira, magenta), Sloppy paired (Slp, grey), Tailless (Tll, cyan). UAS-NACT NBs express Slp and not Tll. UAS-D; UAS-NACT NBs express Slp and Tll, quantified in E-H.


( J - K ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which UAS - NACT and UAS - NACT ; UAS - dpn are driven by ok107 - GAL4 ( marked by GFP and outlined ) and stained with the stem cell marker , Mira ( magenta ) , and mid - temporal progeny marker , Toy - GFP ( grey ) . Fewer cells express Toy + in UAS - NACT ; UAS - dpn compared to UAS - NACT , quantified in I .

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 O O O O O O

(J-K') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which UAS-NACT and UAS-NACT; UAS-dpn are driven by ok107-GAL4 (marked by GFP and outlined) and stained with the stem cell marker, Mira (magenta), and mid-temporal progeny marker, Toy-GFP (grey). Fewer cells express Toy+ in UAS-NACT; UAS-dpn compared to UAS-NACT, quantified in I.


( A - C ' ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which control ( UAS - lacz ) , lola - Ri , and UAS - D ; lola - Ri are driven by eyR16F10 - GAL4 ( outlined ) . Miranda ( Mira , magenta ) , Sloppy - paired ( Slp , grey ) , Twin of eyeless ( Toy , cyan ) . lola - Ri induces ectopic Mira + NBs in the deep medulla layers , compared to control , quantified in E .

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 O O O O O O O O O O

(A-C''') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-lacz), lola-Ri, and UAS-D; lola-Ri are driven by eyR16F10-GAL4 (outlined). Miranda (Mira, magenta), Sloppy-paired (Slp, grey), Twin of eyeless (Toy, cyan). lola-Ri induces ectopic Mira+ NBs in the deep medulla layers, compared to control, quantified in E.


( H - J ' ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which control ( UAS - lacz ) , lola - Ri , and UAS - D ; lola - Ri are driven by eyR16F10 - GAL4 ( outlined ) . Miranda ( Mira , magenta ) , Tailless ( Tll , grey ) , Reversed polarity ( Repo , cyan ) . lola - Ri NBs express less Tll compared to control . There are fewer Tll + and Repo + progeny in the eyR16F10 - GAL4 domain in lola - Ri compared to control . UAS - D ; lola - Ri NBs express more Tll as well as more Tll + and Repo + progeny in eyR16F10 - GAL4 domain compared to lola - Ri alone , quantified in K - L .

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 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

(H-J''') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-lacz), lola-Ri, and UAS-D; lola-Ri are driven by eyR16F10-GAL4 (outlined). Miranda (Mira, magenta), Tailless (Tll, grey), Reversed polarity (Repo, cyan). lola-Ri NBs express less Tll compared to control. There are fewer Tll+ and Repo+ progeny in the eyR16F10-GAL4 domain in lola-Ri compared to control. UAS-D; lola-Ri NBs express more Tll as well as more Tll+ and Repo+ progeny in eyR16F10-GAL4 domain compared to lola-Ri alone, quantified in K-L.


b ) Volcano plot of contrast MPX vs healthy controls ; α < = 0 . 015 and | logFC | > = 1 . 35 were used for selection of regulated proteins .

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) Volcano plot of contrast MPX vs healthy controls; α <= 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins.


d ) Boxplots illustrating key proteins that differ between patients with MPX and controls ( p - values and fdr for corresponding contrast MPX vs Control are provided in brackets ) : TTR ( p - value = 9E - 10 , fdr = 2E - 7 ) , LBP ( p - value = 2E - 4 , fdr = 2E - 3 ) , APOC1 ( p - value = 5E - 8 , fdr = 3E - 6 ) , and C9 ( p - value = 2E - 8 , fdr = 2E - 6 ) . Here , as usual , the central bar marks the median ( second quartile ) , the bottom edge of the box marks the first quartile , the top edge of the box marks the third quartile , and the bottom and top whiskers mark the minimum and maximum values that are not outliers . The specific values of the protein expressions are also shown . Provided p - values are obtained from moderated statistics implemented in limma , dfrs were calculated according to Benjamini - Hochberg .

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 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 O O O O O O O O O

d) Boxplots illustrating key proteins that differ between patients with MPX and controls (p-values and fdr for corresponding contrast MPX vs Control are provided in brackets): TTR (p-value = 9E-10, fdr = 2E-7), LBP (p-value = 2E-4, fdr = 2E-3), APOC1 (p-value = 5E-8, fdr = 3E-6), and C9 (p-value = 2E-8, fdr = 2E-6). Here, as usual, the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided p-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg.


e ) Correlation between MPX severity ( NSkin lesions ) and protein expression ( y - axis ) . One MPX patient had an unclear additional skin condition ( not a pure case of MPX ) and therefore was excluded from the regression analysis that compares the number of skin lesions with the proteome ; however , the proteome of this patient was largely in agreement with those of the other MPX cases As a measure of MPX severity , the log2 ( 1 + NLesions / 15 ) was used . Here NLesions is the number of lesions . R2 shows squared correlation coefficient . MPX patients are colored orange , control patients green .

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 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

e) Correlation between MPX severity (NSkin lesions) and protein expression (y-axis). One MPX patient had an unclear additional skin condition (not a pure case of MPX) and therefore was excluded from the regression analysis that compares the number of skin lesions with the proteome; however, the proteome of this patient was largely in agreement with those of the other MPX cases As a measure of MPX severity, the log2(1 + NLesions / 15) was used. Here NLesions is the number of lesions. R2 shows squared correlation coefficient. MPX patients are colored orange, control patients green.


a ) Heatmap displaying hierarchical clustering using differentially regulated proteins between patients with MPX , COVID - 19 , and controls .

O O O O O O O O O O O O O O O O O O O O O O

a) Heatmap displaying hierarchical clustering using differentially regulated proteins between patients with MPX, COVID-19, and controls.


b ) Scatterplot of log fold - change ( logFC ) for contrast MPX vs control ( C1 , x - axis ) and logFC for contrast COVID - 19 vs control ( C2 , y - axis ) . Only the central part of the cloud is shown here . Three truncated dots ( APOC1 , CRP , and SAA1 ) are shown in the lower left and upper right corner . Differentially abundant ( Regulated ; ' Reg ' ) proteins are color coded , with the red color corresponding to 37 proteins differentially abundant in both MPX vs control ( C1 ) and COVID - 19 vs control ( C2 ) , the orange color corresponding to proteins specifically changed in MPX vs control ( C1 ) only ( 16 proteins ) , and the green color corresponding to proteins responding to both MPX vs control ( C1 ) and MPX vs COVID - 19 ( C3 ) ( 3 proteins ) . There are no intersections between COVID - 19 vs control ( C2 ) and MPX vs COVID - 19 ( C3 ) . The blue color corresponds to proteins responding to MPX vs COVID - 19 ( C3 ) , but not in MPX vs control ( C1 ) ( 11 proteins ) , and the pink color to proteins responding to COVID - 19 vs control ( C2 ) only ( 19 proteins ) . The red dotted line shows a linear regression through the red dots , i . e . proteins differentially abundant in MPX vs control ( C1 ) and COVID - 19 vs control ( C2 ) . Note that orange and pink points have the same direction of regulation in both MPXV vs control ( C1 ) and COVID - 19 vs control ( C2 ) . Only green and blue dots ( except three proteins : ADIPOQ , GPLD1 , and IGHV1 - 2 ) have opposite directions in C1 and in C2 .

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 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 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 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) Scatterplot of log fold-change (logFC) for contrast MPX vs control (C1, x-axis) and logFC for contrast COVID-19 vs control (C2, y- axis). Only the central part of the cloud is shown here. Three truncated dots (APOC1, CRP, and SAA1) are shown in the lower left and upper right corner. Differentially abundant (Regulated; 'Reg') proteins are color coded, with the red color corresponding to 37 proteins differentially abundant in both MPX vs control (C1) and COVID-19 vs control (C2), the orange color corresponding to proteins specifically changed in MPX vs control (C1) only (16 proteins), and the green color corresponding to proteins responding to both MPX vs control (C1) and MPX vs COVID-19 (C3) (3 proteins). There are no intersections between COVID-19 vs control (C2) and MPX vs COVID-19 (C3). The blue color corresponds to proteins responding to MPX vs COVID-19 (C3), but not in MPX vs control (C1) (11 proteins), and the pink color to proteins responding to COVID-19 vs control (C2) only (19 proteins). The red dotted line shows a linear regression through the red dots, i.e. proteins differentially abundant in MPX vs control (C1) and COVID-19 vs control (C2). Note that orange and pink points have the same direction of regulation in both MPXV vs control (C1) and COVID-19 vs control (C2). Only green and blue dots (except three proteins: ADIPOQ, GPLD1, and IGHV1-2) have opposite directions in C1 and in C2.


d ) Differentially regulated proteins of patients with MPX and COVID - 19 ( Volcano Plot ) ; α ≤ 0 . 015 and | logFC | > = 1 . 35 were used for selection of regulated proteins . The chosen significance level ensured that fdr for this contrast was below 22 % .

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

d) Differentially regulated proteins of patients with MPX and COVID-19 (Volcano Plot); α ≤ 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins. The chosen significance level ensured that fdr for this contrast was below 22%.


e ) Key proteins that differ between patients with MPX and COVID - 19 ( Boxplots ) ( p - values and fdr for corresponding contrast MPX vs COVID - 19 are provided in brackets ) : LCP1 ( p - value = 3E - 4 , fdr = 3E - 2 ) , LDHB ( p - value = 2E - 3 , fdr = 6E - 2 ) , CFHR1 ( p - value = 2E - 4 , fdr = 3E - 2 ) . Here the central bar marks the median ( second quartile ) , the bottom edge of the box marks the first quartile , the top edge of the box marks the third quartile , and the bottom and top whiskers mark the minimum and maximum values that are not outliers . The specific values of the protein expressions are also shown . Provided p - values are obtained from moderated statistics implemented in limma , dfrs were calculated according to Benjamini - Hochberg .

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e) Key proteins that differ between patients with MPX and COVID-19 (Boxplots) (p-values and fdr for corresponding contrast MPX vs COVID-19 are provided in brackets): LCP1 (p-value = 3E-4, fdr = 3E-2), LDHB (p-value = 2E-3, fdr = 6E-2), CFHR1 (p-value = 2E-4, fdr = 3E-2). Here the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided p-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg.


f ) Top 8 proteins of an SVM - trained model discriminating between healthy controls ( n = 15 ) and MPX cases ( n = 6 ) ( top ) or COVID - 19 ( n = 10 ) and MPX ( n = 6 ) cases ( bottom ) . Means of the relative coefficients over a 5 - fold cross - validation are shown . Error bars denote the standard deviations . Red denotes positive , blue denotes negative coefficients . The AUC was calculated based on withheld samples that were not used for training the model .

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f) Top 8 proteins of an SVM-trained model discriminating between healthy controls (n=15) and MPX cases (n=6) (top) or COVID-19 (n=10) and MPX (n=6) cases (bottom). Means of the relative coefficients over a 5-fold cross-validation are shown. Error bars denote the standard deviations. Red denotes positive, blue denotes negative coefficients. The AUC was calculated based on withheld samples that were not used for training the model.


b ) Hierarchical clustering using differentially regulated proteins between patients with MPX and controls ( Heatmap ) ; p < 0 . 05 with Mann - Whitney U test with FDR - based multiple testing correction .

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b) Hierarchical clustering using differentially regulated proteins between patients with MPX and controls (Heatmap); p < 0.05 with Mann-Whitney U test with FDR-based multiple testing correction.


c ) Key proteins that differ between patients with MPX and controls , and COVID - 19 ( Boxplots ) . Dashed blue lines indicate the lowest detected peptide concentration from calibration curves . Statistics based on Mann - Whitney U test with multiple testing corrections . For boxplots , median is indicated by a solid line , hinges show the 25th and 75th percentiles , whiskers show values that , at maximum , are within 1 . 5 times the interquartile range .

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c) Key proteins that differ between patients with MPX and controls, and COVID-19 (Boxplots). Dashed blue lines indicate the lowest detected peptide concentration from calibration curves. Statistics based on Mann-Whitney U test with multiple testing corrections. For boxplots, median is indicated by a solid line, hinges show the 25th and 75th percentiles, whiskers show values that, at maximum, are within 1.5 times the interquartile range.


d ) Top 15 peptides of an SVM - trained model discriminating between healthy controls ( n = 15 ) and MPX cases ( n = 6 ) . Means of the relative coefficients over a 5 - fold cross - validation are shown . Error bars denote the standard deviations . Red denotes positive , blue denotes negative coefficients . The AUC was calculated based on withheld samples that were not used for training the model .

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d) Top 15 peptides of an SVM-trained model discriminating between healthy controls (n=15) and MPX cases (n=6). Means of the relative coefficients over a 5-fold cross-validation are shown. Error bars denote the standard deviations. Red denotes positive, blue denotes negative coefficients. The AUC was calculated based on withheld samples that were not used for training the model.


A PTAR1 protein expression in rat tissues . Proteins extracted from various rat tissues ( 15 μg each ) were analyzed by immunoblotting with antibodies against PTAR1 .

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A PTAR1 protein expression in rat tissues. Proteins extracted from various rat tissues (15 μg each) were analyzed by immunoblotting with antibodies against PTAR1.


B Identification of RabGGTβ as a binding protein of PTAR1 . Anti - FLAG immunoprecipitates from control HeLa S3 cells or FLAG - PTAR1 - expressing HeLa S3 cells were analyzed by SDS - PAGE and silver staining . The 35 kDa protein was identified as RabGGTβ by mass spectrometry . The asterisk denotes a degradation product of FLAG - PTAR1 .

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B Identification of RabGGTβ as a binding protein of PTAR1. Anti-FLAG immunoprecipitates from control HeLa S3 cells or FLAG-PTAR1-expressing HeLa S3 cells were analyzed by SDS-PAGE and silver staining. The 35 kDa protein was identified as RabGGTβ by mass spectrometry. The asterisk denotes a degradation product of FLAG-PTAR1.


C Co - immunoprecipitation of endogenous PTAR1 and RabGGTβ from rat brain and thymus cytosol .

C Co-immunoprecipitation of endogenous PTAR1 and RabGGTβ from rat brain and thymus cytosol.


D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography . Column fractions were analyzed by immunoblotting with antibodies against RabGGTα , PTAR1 , and RabGGTβ . RabGGTβ eluted in two peaks ( peak A and peak B ) .

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D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography. Column fractions were analyzed by immunoblotting with antibodies against RabGGTα, PTAR1, and RabGGTβ. RabGGTβ eluted in two peaks (peak A and peak B).


E Co - immunodepletion of PTAR1 and RabGGTβ by anti - PTAR1 IgG . PTAR1 was immunodepleted from the Mono Q peak A fraction using anti - PTAR1 IgG - bound protein A beads , and the supernatant was analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ . Normal rabbit IgG and anti - PTAR1 IgG preabsorbed with the antigen peptide were used as controls .

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E Co-immunodepletion of PTAR1 and RabGGTβ by anti-PTAR1 IgG. PTAR1 was immunodepleted from the Mono Q peak A fraction using anti-PTAR1 IgG-bound protein A beads, and the supernatant was analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ. Normal rabbit IgG and anti-PTAR1 IgG preabsorbed with the antigen peptide were used as controls.


F Fractionation of the Mono Q peak A fraction by Superdex 200 gel filtration chromatography . Column fractions were analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ .

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F Fractionation of the Mono Q peak A fraction by Superdex 200 gel filtration chromatography. Column fractions were analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ.


G Gel filtration chromatography of the recombinant PTAR1 - RabGGTβ complex purified from Sf9 insect cells on a Superdex 200 column . Inset shows SDS - PAGE and Coomassie staining analysis of the purified complex .

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G Gel filtration chromatography of the recombinant PTAR1-RabGGTβ complex purified from Sf9 insect cells on a Superdex 200 column. Inset shows SDS-PAGE and Coomassie staining analysis of the purified complex.


C Identification of Ykt6 as a protein biotin - geranylated by GGTase - III . Cytosolic proteins extracted from statin - treated HeLa S3 cells were applied to GST or GST - GGTase - III affinity columns . Bound proteins were eluted and incubated with recombinant GGTase - III and BGPP . Reaction products were separated by SDS - PAGE , transferred to a nitrocellulose membrane , and probed with horseradish peroxidase ( HRP ) - labeled avidin to detect biotinylated proteins . Mass spectrometry identified the 25 kDa protein as Ykt6 .

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C Identification of Ykt6 as a protein biotin-geranylated by GGTase-III. Cytosolic proteins extracted from statin-treated HeLa S3 cells were applied to GST or GST-GGTase-III affinity columns. Bound proteins were eluted and incubated with recombinant GGTase-III and BGPP. Reaction products were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with horseradish peroxidase (HRP)-labeled avidin to detect biotinylated proteins. Mass spectrometry identified the 25 kDa protein as Ykt6.


D Biotin - geranylation of recombinant Ykt6 by GGTase - III . Bacterially produced recombinant untagged Ykt6 ( left ) was incubated with buffer , GGTase - III , or RabGGTase in the absence or presence of BGPP for 30 min at 37ºC . Biotin - geranylated Ykt6 was

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D Biotin-geranylation of recombinant Ykt6 by GGTase-III. Bacterially produced recombinant untagged Ykt6 (left) was incubated with buffer, GGTase-III, or RabGGTase in the absence or presence of BGPP for 30 min at 37ºC. Biotin-geranylated Ykt6 was detected


C Farnesylation of recombinant Ykt6 by FTase . Recombinant Ykt6 ( 5 μM ) was incubated with FTase ( 0 or 50 nM ) and 3H - FPP ( 1 μM ; ~ 3 , 500 dpm / pmol ) at 37ºC . Reactions were stopped at the indicated time points , and the amount of 3H - farnesyl transferred to Ykt6 was quantified by scintillation counting ( mean ± SEM , n = 3 ) .

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C Farnesylation of recombinant Ykt6 by FTase. Recombinant Ykt6 (5 μM) was incubated with FTase (0 or 50 nM) and 3H-FPP (1 μM; ~3,500 dpm/pmol) at 37ºC. Reactions were stopped at the indicated time points, and the amount of 3H-farnesyl transferred to Ykt6 was quantified by scintillation counting (mean ± SEM, n = 3).


D Geranylgeranylation of recombinant Ykt6 by GGTase - III . Recombinant Ykt6 ( 5 μM ) was incubated with increasing concentrations of GGTase - III ( 0 - 100 nM ) and 3H - GGPP ( 1 μM ; ~ 3 , 000 dpm / pmol ) for the indicated times , and the amount of 3H - geranylgeranyl transferred to Ykt6 was quantified ( mean ± SEM , n = 3 ) .

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D Geranylgeranylation of recombinant Ykt6 by GGTase-III. Recombinant Ykt6 (5 μM) was incubated with increasing concentrations of GGTase-III (0-100 nM) and 3H-GGPP (1 μM; ~3,000 dpm/pmol) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).


E Geranylgeranylation of Cys to Ser mutants of Ykt6 by GGTase - III . WT and mutant Ykt6 proteins ( 5 μM each ) were incubated with GGTase - III ( 100 nM ) and 3H - GGPP ( 1 μM ) for the indicated times , and the amount of 3H - geranylgeranyl transferred to Ykt6 was quantified ( mean ± SEM , n = 3 ) .

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E Geranylgeranylation of Cys to Ser mutants of Ykt6 by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).


G Mass spectra of prenylated Ykt6 peptides . Recombinant Ykt6 was farnesylated by FTase ( FTase ) , further processed by RCE1 and ICMT ( RCE1 / ICMT ) , and finally geranylgeranylated by GGTase - III ( GGTase - III ) . After purification to homogeneity , the prenylated Ykt6 proteins were digested with chymotrypsin ( left ) or V8 protease ( right ) and analyzed by MALDI - TOF mass spectrometry . The calculated m / z values for the C - terminal peptide ions are shown in parentheses . The peaks labeled with an asterisk correspond to amino acids 14 - 38 of Ykt6 ( AKVVLLKAAYDVSSFSFFQRSSVQE ; m / z 2806 . 48 ) .

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G Mass spectra of prenylated Ykt6 peptides. Recombinant Ykt6 was farnesylated by FTase (FTase), further processed by RCE1 and ICMT (RCE1/ICMT), and finally geranylgeranylated by GGTase-III (GGTase-III). After purification to homogeneity, the prenylated Ykt6 proteins were digested with chymotrypsin (left) or V8 protease (right) and analyzed by MALDI-TOF mass spectrometry. The calculated m/z values for the C-terminal peptide ions are shown in parentheses. The peaks labeled with an asterisk correspond to amino acids 14-38 of Ykt6 (AKVVLLKAAYDVSSFSFFQRSSVQE; m/z 2806.48).


Effects of C - terminal processing and Cys195 farnesylation on Cys194 geranylgeranylation by GGTase - III . Purified recombinant unprenyl Ykt6 , Cys195 - farnesyl Ykt6 , and Cys195 - farnesyl Ykt6ΔAIM ( 5 μM each ) were incubated with GGTase - III ( 100 nM ) and 3H - GGPP ( 1 μM ) for the indicated times , and the amount of 3H - geranylgeranyl transferred to Ykt6 was quantified . ( mean ± SEM , n = 3 ) .

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Effects of C-terminal processing and Cys195 farnesylation on Cys194 geranylgeranylation by GGTase-III. Purified recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, and Cys195-farnesyl Ykt6ΔAIM (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified. (mean ± SEM, n = 3).


I Gel filtration chromatography of purified unprenyl Ykt6 , Cys195 - farnesyl Ykt6ΔAIM , and Cys194 / 195 - diprenyl Ykt6ΔAIM ( 135 μg each ) on a Superdex 200 column . Dotted lines indicate the elution profile of unprenyl Ykt6 for comparison . The prenylated Ykt6 proteins were eluted slightly later than the unprenylated Ykt6 , suggesting more compact and stable folds .

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I Gel filtration chromatography of purified unprenyl Ykt6, Cys195-farnesyl Ykt6ΔAIM, and Cys194/195-diprenyl Ykt6ΔAIM (135 μg each) on a Superdex 200 column. Dotted lines indicate the elution profile of unprenyl Ykt6 for comparison. The prenylated Ykt6 proteins were eluted slightly later than the unprenylated Ykt6, suggesting more compact and stable folds.


E Geranylgeranylation activity of WT GGTase - III and the indicated mutants ( 100 nM each ) . Cys195 - farnesyl Ykt6ΔAIM ( 1 μM ) and 3H - GGPP ( 1 μM ) were used as substrates ( mean ± SEM , n = 3 ) .

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E Geranylgeranylation activity of WT GGTase-III and the indicated mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).


F Kinetic analysis of WT GGTase - III and the site 1 or site 2 mutant ( 100 nM each ) using increasing concentrations of Cys195 - farnesyl Ykt6ΔAIM ( mean ± SEM , n = 3 for WT and Y306A , n = 1 for V48A / V50A ) .

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F Kinetic analysis of WT GGTase-III and the site 1 or site 2 mutant (100 nM each) using increasing concentrations of Cys195-farnesyl Ykt6ΔAIM (mean ± SEM, n = 3 for WT and Y306A, n = 1 for V48A/V50A).


Geranylgeranylation of unprenyl WT Ykt6 and the site 1 or site 2 mutants by GGTase - III . WT and mutant Ykt6 proteins ( 5 μM each ) were incubated with GGTase - III ( 100 nM ) and 3H - GGPP ( 1 μM ) , and the amount of 3H - geranylgeranyl transferred to Ykt6 was quantified ( mean ± SEM , n = 3 ) .

Geranylgeranylation of unprenyl WT Ykt6 and the site 1 or site 2 mutants by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM), and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).


H Geranylgeranylation activity of WT GGTase - III and tunnel mutants ( 100 nM each ) . Cys195 - farnesyl Ykt6ΔAIM ( 1 μM ) and 3H - GGPP ( 1 μM ) were used as substrates ( mean ± SEM , n = 3 ) .

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H Geranylgeranylation activity of WT GGTase-III and tunnel mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).


I GGPP binding to GGTase - III . WT GGTase - III and tunnel mutants ( 1 μM each ) were incubated with 3H - GGPP ( 2 μM ) at 4ºC for 10 min . After desalting , enzyme - bound 3H - GGPP was quantified by scintillation counting ( mean ± SEM , n = 3 ) .

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I GGPP binding to GGTase-III. WT GGTase-III and tunnel mutants (1 μM each) were incubated with 3H-GGPP (2 μM) at 4ºC for 10 min. After desalting, enzyme-bound 3H-GGPP was quantified by scintillation counting (mean ± SEM, n = 3).


A Immunoblot of cell lysates from WT HAP1 cells and PTAR1 KO HAP1 cells using anti - Ykt6 antibody . Note the slight difference in the gel mobility of Ykt6 ( arrows ) .

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A Immunoblot of cell lysates from WT HAP1 cells and PTAR1 KO HAP1 cells using anti-Ykt6 antibody. Note the slight difference in the gel mobility of Ykt6 (arrows).


B Prenylation status of rat brain Ykt6 . Recombinant unprenyl Ykt6 , Cys195 - farnesyl Ykt6 , Cys194 / 195 - diprenyl Ykt6 samples ( 1 ng each ) , and rat brain cytosol were electrophoresed on a polyacrylamide gel using deoxycholate ( DOC ) - containing buffer and analyzed by immunoblotting with anti - Ykt6 antibody ( DOC - PAGE ; upper panel ) . The same samples were analyzed by conventional SDS - PAGE and immunoblotting with anti - Ykt6 antibody ( SDS - PAGE ; lower panel ) .

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B Prenylation status of rat brain Ykt6. Recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, Cys194/195-diprenyl Ykt6 samples (1 ng each), and rat brain cytosol were electrophoresed on a polyacrylamide gel using deoxycholate (DOC)-containing buffer and analyzed by immunoblotting with anti-Ykt6 antibody (DOC-PAGE; upper panel). The same samples were analyzed by conventional SDS-PAGE and immunoblotting with anti-Ykt6 antibody (SDS-PAGE; lower panel).


C Prenylation status of Ykt6 in WT and PTAR1 KO cells . Recombinant Ykt6 samples and cell lysates of WT cells , PTAR1 KO cells , and PTAR1 KO cells stably expressing PTAR1 ( KO + PTAR1 ) were separated by DOC - PAGE ( upper ) or SDS - PAGE ( lower ) , and analyzed by immunoblotting with anti - Ykt6 antibody .

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C Prenylation status of Ykt6 in WT and PTAR1 KO cells. Recombinant Ykt6 samples and cell lysates of WT cells, PTAR1 KO cells, and PTAR1 KO cells stably expressing PTAR1 (KO + PTAR1) were separated by DOC-PAGE (upper) or SDS-PAGE (lower), and analyzed by immunoblotting with anti-Ykt6 antibody.


D In vitro reconstitution of Ykt6 double prenylation . Dialyzed cytosol prepared from PTAR1 KO HAP1 cells was incubated at 37ºC for the indicated times with recombinant GGTase - III ( 100 nM ) or RabGGTase ( 100 nM ) in the absence or presence of GGPP ( 10 μM ) . After incubation , reaction products were separated by DOC - PAGE ( upper ) or SDS - PAGE ( lower ) , and analyzed by immunoblotting with anti - Ykt6 antibody .

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D In vitro reconstitution of Ykt6 double prenylation. Dialyzed cytosol prepared from PTAR1 KO HAP1 cells was incubated at 37ºC for the indicated times with recombinant GGTase-III (100 nM) or RabGGTase (100 nM) in the absence or presence of GGPP (10 μM). After incubation, reaction products were separated by DOC-PAGE (upper) or SDS-PAGE (lower), and analyzed by immunoblotting with anti-Ykt6 antibody.


A Representative confocal images of the Golgi apparatus in WT or PTAR1 KO HeLa cells . Cells were co - immunostained for GM130 ( a cis - Golgi marker ) and golgin - 97 ( a trans - Golgi marker ) . Images were deconvoluted using Huygens software . The lower panels show magnified images of the boxed region of the upper panels . Scale bars , 5 μm .

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A Representative confocal images of the Golgi apparatus in WT or PTAR1 KO HeLa cells. Cells were co-immunostained for GM130 (a cis-Golgi marker) and golgin-97 (a trans-Golgi marker). Images were deconvoluted using Huygens software. The lower panels show magnified images of the boxed region of the upper panels. Scale bars, 5 μm.


B Electron micrographs of the Golgi apparatus in WT or PTAR1 KO HAP1 cells . Arrows indicate Golgi stacks . Arrowheads indicate examples of unfused vesicles accumulated around the swollen Golgi cisternae in PTAR1 KO cells . Scale bars , 500 nm .

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B Electron micrographs of the Golgi apparatus in WT or PTAR1 KO HAP1 cells. Arrows indicate Golgi stacks. Arrowheads indicate examples of unfused vesicles accumulated around the swollen Golgi cisternae in PTAR1 KO cells. Scale bars, 500 nm.


Defect in intra - Golgi trafficking in PTAR1 KO cells . ( C ) VSVG - GFP expressing WT and PTAR1 KO HeLa cells were cultured at 40ºC and then shifted to 32ºC . VSVG - GFP fluorescence images taken at the indicated times after the temperature shift are shown . The right panel shows quantification of VSVG - GFP fluorescence in the Golgi region after the temperature shift ( mean ± SEM , n = 6 ) .

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Defect in intra-Golgi trafficking in PTAR1 KO cells. (C) VSVG-GFP expressing WT and PTAR1 KO HeLa cells were cultured at 40ºC and then shifted to 32ºC. VSVG-GFP fluorescence images taken at the indicated times after the temperature shift are shown. The right panel shows quantification of VSVG-GFP fluorescence in the Golgi region after the temperature shift (mean ± SEM, n = 6).


Defect in intra - Golgi trafficking in PTAR1 KO cells . ( D ) VSVG - GFP expressing WT HeLa cells , PTAR1 KO HeLa cells , and PTAR1 KO HeLa cells stably expressing PTAR1 ( KO + PTAR1 ) were cultured at 40ºC and then shifted to 32ºC . At the indicated time points , cell surface proteins were biotinylated using sulfo - NHS - LC - biotin . Biotinylated VSVG - GFP was purified from cell lysates using avidin agarose and analyzed by immunoblotting with anti - GFP antibody . The right panel shows quantification of the cell surface biotinylated VSVG - GFP ( means ± SEM , n = 3 ) . Data were analyzed by one - way ANOVA with Dunnett ' s post - hoc test . * p < 0 . 05 , * * * p < 0 . 001 .

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Defect in intra-Golgi trafficking in PTAR1 KO cells. (D) VSVG-GFP expressing WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were cultured at 40ºC and then shifted to 32ºC. At the indicated time points, cell surface proteins were biotinylated using sulfo-NHS-LC-biotin. Biotinylated VSVG-GFP was purified from cell lysates using avidin agarose and analyzed by immunoblotting with anti-GFP antibody. The right panel shows quantification of the cell surface biotinylated VSVG-GFP (means ± SEM, n = 3). Data were analyzed by one-way ANOVA with Dunnett's post-hoc test. *p < 0.05, ***p < 0.001.


E Analysis of LAMP1 glycosylation . Cell lysates of WT HeLa cells , PTAR1 KO HeLa cells , and PTAR1 KO HeLa cells stably expressing PTAR1 ( KO + PTAR1 ) were analyzed by immunoblotting with anti - LAMP1 antibody ( upper ) . Sialylated LAMP1 was precipitated from the cell lysates using Maackia amurensis leucoagglutinin ( MAL ) agarose and analyzed by immunoblotting ( lower ) .

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

E Analysis of LAMP1 glycosylation. Cell lysates of WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were analyzed by immunoblotting with anti-LAMP1 antibody (upper). Sialylated LAMP1 was precipitated from the cell lysates using Maackia amurensis leucoagglutinin (MAL) agarose and analyzed by immunoblotting (lower).


F Defect in the Golgi SNARE assembly in PTAR1 KO cells . The Golgi SNARE complex was immunoprecipitated from NEM - treated WT HAP1 cells , PTAR1 KO HAP1 cells , and PTAR1 KO HAP1 cells stably expressing PTAR1 ( KO + PTAR1 ) using control mouse IgG , anti - GS28 IgG , or anti - syntaxin 5 IgG . Immunoprecipitates were analyzed by immunoblotting with antibodies against syntaxin 5 , GS28 , GS15 , and Ykt6 . Syntaxin 5 has two isoforms with different translation initiation sites . Inputs were 20 % ( syntaxin 5 , GS28 , and GS15 ) and 2 % ( Ykt6 ) . The data shown are representative of three independent experiments with similar results .

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 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 Defect in the Golgi SNARE assembly in PTAR1 KO cells. The Golgi SNARE complex was immunoprecipitated from NEM-treated WT HAP1 cells, PTAR1 KO HAP1 cells, and PTAR1 KO HAP1 cells stably expressing PTAR1 (KO + PTAR1) using control mouse IgG, anti-GS28 IgG, or anti-syntaxin 5 IgG. Immunoprecipitates were analyzed by immunoblotting with antibodies against syntaxin 5, GS28, GS15, and Ykt6. Syntaxin 5 has two isoforms with different translation initiation sites. Inputs were 20% (syntaxin 5, GS28, and GS15) and 2% (Ykt6). The data shown are representative of three independent experiments with similar results.


( B - C ) Immunofluorescent ( IF ) staining of Pax7 and YY1 was performed on cryosections of myotome or limb bud from Ctrl or YY1cKO mice to confirm the ablation of YY1 in Pax7 expressing progenitors . Scale bar = 100 µm .

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-C) Immunofluorescent (IF) staining of Pax7 and YY1 was performed on cryosections of myotome or limb bud from Ctrl or YY1cKO mice to confirm the ablation of YY1 in Pax7 expressing progenitors. Scale bar = 100 µm.


( D ) Representative images of Ctrl , Heterozygous ( Het ) and YY1cKO newborn ( P0 ) mice or embryos at E18 . 5 day .

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) Representative images of Ctrl, Heterozygous (Het) and YY1cKO newborn (P0) mice or embryos at E18.5 day.


( E ) Excised lungs from P0 Ctrl or YY1cKO pups were placed in a beaker for sinking test . The lung from YY1cKO pup sank whereas Ctrl floated .

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

(E) Excised lungs from P0 Ctrl or YY1cKO pups were placed in a beaker for sinking test. The lung from YY1cKO pup sank whereas Ctrl floated.


( F ) P0 lungs were stained with Hematoxylin and Eosin ( H & E ) and representative images are shown . Scale bar = 100 µm . ( G ) Representative images of diaphragms ( Dps ) isolated from P0 Ctrl or YY1cKO littermates .

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

(F) P0 lungs were stained with Hematoxylin and Eosin (H&E) and representative images are shown. Scale bar = 100 µm. (G) Representative images of diaphragms (Dps) isolated from P0 Ctrl or YY1cKO littermates.


( H ) The above Dps were used for H & E or Immunohistochemistry ( IHC ) staining for MyHC . Representative images from 3 pairs of littermates are shown . Scale bar = 50 µm .

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

(H) The above Dps were used for H&E or Immunohistochemistry (IHC) staining for MyHC. Representative images from 3 pairs of littermates are shown. Scale bar = 50 µm.


The above harvested Dps were subjected to IF staining for MyHC together with Laminin . Scale bar = 50 µm . Quantifications of positively stained fibers per area are shown on the right ( n = 3 mice , each ) .

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

The above harvested Dps were subjected to IF staining for MyHC together with Laminin. Scale bar = 50 µm. Quantifications of positively stained fibers per area are shown on the right (n=3 mice, each).


The above harvested Dps were subjected to IF staining for Troponin T ( TnT ) together with Laminin . Scale bar = 50 µm . Quantifications of positively stained fibers per area are shown on the right ( n = 3 mice , each ) .

The above harvested Dps were subjected to IF staining for Troponin T (TnT) together with Laminin. Scale bar = 50 µm. Quantifications of positively stained fibers per area are shown on the right (n=3 mice, each).


( K ) Representative images of H & E staining of hind limb muscles isolated from Ctrl or YY1cKO embryos at E18 . 5 day . Scale bar = 400 µm ( left ) or 50 µm ( right ) .

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

(K) Representative images of H&E staining of hind limb muscles isolated from Ctrl or YY1cKO embryos at E18.5 day. Scale bar = 400 µm (left) or 50 µm (right).


The above harvested limb muscles were subjected to IF staining for MyHC together with Laminin . Scale bar = 400 µm ( left ) or 50 µm ( right ) . Quantifications of the numbers of MyHC + fibers per area are shown on the right ( n = 3 mice , each ) .

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

The above harvested limb muscles were subjected to IF staining for MyHC together with Laminin. Scale bar = 400 µm (left) or 50 µm (right). Quantifications of the numbers of MyHC+ fibers per area are shown on the right (n=3 mice, each).


The above harvested limb muscles were subjected to IF staining for TnT together with Laminin . Scale bar = 400 µm ( left ) or 50 µm ( right ) . Quantifications of the numbers of TnT + fibers per area are shown on the right ( n = 3 mice , each ) .

The above harvested limb muscles were subjected to IF staining for TnT together with Laminin. Scale bar = 400 µm (left) or 50 µm (right). Quantifications of the numbers of TnT+ fibers per area are shown on the right (n=3 mice, each).


The above harvested limb ( N ) muscles were subjected to IF staining for Pax7 and Laminin . Scale bar = 100 µm ( N ) Quantification of the numbers of Pax7 + muscle progenitor cells per area are shown on the right ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 O O O O O O O O O O O

The above harvested limb (N) muscles were subjected to IF staining for Pax7 and Laminin. Scale bar = 100 µm (N) Quantification of the numbers of Pax7+ muscle progenitor cells per area are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.


The above harvested diaphragm ( O ) muscles were subjected to IF staining for Pax7 and Laminin . Scale bar = 50 µm ( O ) . Quantification of the numbers of Pax7 + muscle progenitor cells per area are shown on the right ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

The above harvested diaphragm (O) muscles were subjected to IF staining for Pax7 and Laminin. Scale bar = 50 µm (O). Quantification of the numbers of Pax7+ muscle progenitor cells per area are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01 ***P<0.001.


( B ) SCs were FACS sorted 3 days after the last TM injection and cultured for 1 . 5 days ; RT - qPCR detection of YY1 mRNA shows the ablation in YY1iKO cells .

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) SCs were FACS sorted 3 days after the last TM injection and cultured for 1.5 days; RT-qPCR detection of YY1 mRNA shows the ablation in YY1iKO cells.


( C ) IF staining for Pax7 and YY1 on freshly isolated FISCs or ( D ) single myofibers from EDL muscles or ( E ) cryosections from TA muscles showing the deletion of YY1 protein from YY1iKO cells . Scale bar = 100 µm in ( C ) or 50 µm in ( D - E ) .

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

(C) IF staining for Pax7 and YY1 on freshly isolated FISCs or (D) single myofibers from EDL muscles or (E) cryosections from TA muscles showing the deletion of YY1 protein from YY1iKO cells. Scale bar =100 µm in (C) or 50 µm in (D-E).


( F ) Representative FACS plots . About 100 , 000 cells from 2 - month - old Ctrl and YY1iKO mice were sorted by FACS 3 weeks post - TM injection . The percentage of SCs was shown . ( n = 3 mice , each ) .

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

(F) Representative FACS plots. About 100,000 cells from 2-month-old Ctrl and YY1iKO mice were sorted by FACS 3 weeks post-TM injection. The percentage of SCs was shown. (n=3 mice, each).


( G ) H & E staining was performed on the injured TA muscles collected from the designated times post - CTX injection to visualize the degree of regeneration . Scale bar = 100 µm .

(G) H&E staining was performed on the injured TA muscles collected from the designated times post-CTX injection to visualize the degree of regeneration. Scale bar = 100 µm.


( H ) IF staining for eMyHC on the TA muscles 5 days post - CTX injury . Quantifications of the number of eMyHC + fibers are shown on the right ( n = 3 , each ) . Scale bar = 50 µm .

(H) IF staining for eMyHC on the TA muscles 5 days post-CTX injury. Quantifications of the number of eMyHC+ fibers are shown on the right (n=3, each). Scale bar = 50 µm.


( I ) Left : representative images of TA muscles isolated from Ctrl or YY1iKO mice 60 days post - CTX injury . Right : Masson ' s Trichrome staining of the above muscles to visualize the degree of fibrosis . Scale bar = 100 µm .

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

(I) Left: representative images of TA muscles isolated from Ctrl or YY1iKO mice 60 days post-CTX injury. Right: Masson's Trichrome staining of the above muscles to visualize the degree of fibrosis. Scale bar = 100 µm.


( J ) Cross - section area ( CSA ) of individual myofibers in TA muscles 4 weeks after injury was measured . The distribution for CSA was calculated ( n = 3 mice ) .

(J) Cross-section area (CSA) of individual myofibers in TA muscles 4 weeks after injury was measured. The distribution for CSA was calculated (n=3 mice).


( K ) Representative FACS plots showing the percentage of SCs sorted from TA muscles 3 days after CTX injury of Ctrl and YY1iKO mice .

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

(K) Representative FACS plots showing the percentage of SCs sorted from TA muscles 3 days after CTX injury of Ctrl and YY1iKO mice.


Immunostaining for Pax7 together with Laminin was performed on the TA muscles 3 days post - CTX injury . Scale bar = 100 µm . Quantifications of the numbers of Pax7 + SCs per area are shown on the right ( n = 3 mice , each ) .

Immunostaining for Pax7 together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of Pax7+ SCs per area are shown on the right (n=3 mice, each).


Immunostaining for MyoD together with Laminin was performed on the TA muscles 3 days post - CTX injury . Scale bar = 100 µm . Quantifications of the numbers of MyoD + SCs per area are shown on the right ( n = 3 mice , each ) .

Immunostaining for MyoD together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of MyoD+ SCs per area are shown on the right (n=3 mice, each).


( N ) IF staining for Pax7 and Laminin on TA muscles 4 weeks after CTX injury of Ctrl or YY1iKO mice . Quantifications of the numbers of Pax7 + SCs per 100 fibers are shown on the right ( n = 3 mice , each ) . Scale bar = 100 μm .

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

(N) IF staining for Pax7 and Laminin on TA muscles 4 weeks after CTX injury of Ctrl or YY1iKO mice. Quantifications of the numbers of Pax7+ SCs per 100 fibers are shown on the right (n=3 mice, each). Scale bar = 100 μm.


( P ) H & E staining was performed on TA muscles of Ctrl or YY1iKO mice 7 day after second CTX injury . Scale bar = 400 μm ( left ) or 100 μm ( right ) .

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

(P) H&E staining was performed on TA muscles of Ctrl or YY1iKO mice 7 day after second CTX injury. Scale bar = 400 μm (left) or 100 μm (right).


( Q ) Representative images of above TA muscles 7 days after second injury .

(Q) Representative images of above TA muscles 7 days after second injury.


( R ) Quantifications of the weight above TA muscles from three pairs of littermate mice .

(R) Quantifications of the weight above TA muscles from three pairs of littermate mice.


( S ) IF staining for eMyHC and Laminin on TA muscles 7 days after the second CTX injury of Ctrl or YY1iKO mice . Quantifications of the number of eMyHC + fibers per area are shown on the right ( n = 3 mice , each ) . Scale bar = 100 μm .

(S) IF staining for eMyHC and Laminin on TA muscles 7 days after the second CTX injury of Ctrl or YY1iKO mice. Quantifications of the number of eMyHC+ fibers per area are shown on the right (n=3 mice, each). Scale bar = 100 μm.


( T ) IF staining for Pax7 and Laminin on the above muscles and the quantifications of the number of Pax7 + SCs per area are shown on the right ( n = 3 mice , each ) . Scale bar = 100 μm . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 O O O O O O O O O O O O

(T) IF staining for Pax7 and Laminin on the above muscles and the quantifications of the number of Pax7+ SCs per area are shown on the right (n=3 mice, each). Scale bar = 100 μm. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.


( B ) SCs were FACS sorted 4 months after the last TM injection ; RT - qPCR detection of YY1 mRNA shows the ablation in YY1dKO cells .

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) SCs were FACS sorted 4 months after the last TM injection; RT-qPCR detection of YY1 mRNA shows the ablation in YY1dKO cells.


( C ) Representative images of Ctrl or YY1dKO mice 4 months after the TM administration .

(C) Representative images of Ctrl or YY1dKO mice 4 months after the TM administration.


( D ) Representative images of limb muscles ( Tibialis anterior , TA ; Gastrocnemius , Gas ; Quadruple , Quad ) from Ctrl or YY1dKO mice . ( E ) Quantifications of muscle weight from three pairs of littermate mice .

(D) Representative images of limb muscles (Tibialis anterior, TA; Gastrocnemius, Gas; Quadruple, Quad) from Ctrl or YY1dKO mice. (E) Quantifications of muscle weight from three pairs of littermate mice.


( F ) Representative images of Dp muscles isolated from Ctrl or YY1dKO mice 4 months after the TM injection .

O O O O O O O O O O O O O O O O O O O O O

(F) Representative images of Dp muscles isolated from Ctrl or YY1dKO mice 4 months after the TM injection.


( G ) H & E or ( H ) MyHC staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice . Quantifications of the numbers of MyHC + fibers per area of TA or Dp muscles are shown on the right ( n = 3 mice , each ) . Scale bar = 100 µm .

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

(G) H&E or (H) MyHC staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice. Quantifications of the numbers of MyHC+ fibers per area of TA or Dp muscles are shown on the right (n=3 mice, each). Scale bar = 100 µm.


( I ) Collagen1 or ( J ) Masson ' s Trichrome staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice . Scale bar = 100 µm .

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) Collagen1 or (J) Masson's Trichrome staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice. Scale bar = 100 µm.


( K ) The distribution of fiber size measured by CSA in TA muscles from 3 pairs of 6 - month - old Ctrl and YY1dKO mice was calculated ( n = 3 mice , each ) .

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

(K) The distribution of fiber size measured by CSA in TA muscles from 3 pairs of 6-month-old Ctrl and YY1dKO mice was calculated (n=3 mice, each).


( L ) Representative FACS plots showing the percentage of SCs sorted from 6 - month - old Ctrl and YY1dKO mice ( n = 3 mice , each ) .

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

(L) Representative FACS plots showing the percentage of SCs sorted from 6-month-old Ctrl and YY1dKO mice (n=3 mice, each).


( M ) IF staining for Pax7 and Laminin on TA muscles of Ctrl or YY1dKO mice . Quantifications of the numbers of Pax7 + SCs per 100 fibers are shown on the right ( n = 3 mice , each ) .

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

(M) IF staining for Pax7 and Laminin on TA muscles of Ctrl or YY1dKO mice. Quantifications of the numbers of Pax7+ SCs per 100 fibers are shown on the right (n=3 mice, each).


EDL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force . Left : Representative trace images of normalized tetanic force . ( Right ) : quantification of the force by normalizing with cross - sectional area ( CSA ) . ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 O O O O O O O O O O O O O O

EDL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force. Left: Representative trace images of normalized tetanic force. (Right): quantification of the force by normalizing with cross-sectional area (CSA). (n = 3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.


SOL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force . Left : Representative trace images of normalized tetanic force . ( Right ) : quantification of the force by normalizing with cross - sectional area ( CSA ) . ( n = 3 mice , each ) . Error bars represent s . d . ' s of the mean . Student ' s t - test ( two - sided ) : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

SOL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force. Left: Representative trace images of normalized tetanic force. (Right): quantification of the force by normalizing with cross-sectional area (CSA). (n = 3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.


( A ) FACS - isolated SCs from Pax7 - nGFP mice were cultured for the designated time ( 0 , 24 , 48 or 72 hrs ) . YY1 expression was quantified by RT - qPCR . 18S was used as the normalization control .

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

(A) FACS-isolated SCs from Pax7-nGFP mice were cultured for the designated time (0, 24, 48 or 72 hrs). YY1 expression was quantified by RT-qPCR. 18S was used as the normalization control.


( B ) Flow cytometric analysis of YY1 protein or IgG control in FACS - isolated SCs cultured for the designated time ( 0 , 24 or 48hrs ) . YY1 protein level is normalized to IgG levels . The numbers indicate the normalized level of YY1 protein relative to that of FISCs .

(B) Flow cytometric analysis of YY1 protein or IgG control in FACS-isolated SCs cultured for the designated time (0, 24 or 48hrs). YY1 protein level is normalized to IgG levels. The numbers indicate the normalized level of YY1 protein relative to that of FISCs.


( C ) An equal number of FACS - isolated SCs from Ctrl or YY1iKO mice were cultured for 48 hrs and EdU labelled for 8 hrs , followed by immunostaining for Pax7 ( green ) and EdU ( red ) . Quantifications of the percentage of EdU + cells are shown on the right ( n = 3 mice , each ) . Scale bar = 100 µm .

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

(C) An equal number of FACS-isolated SCs from Ctrl or YY1iKO mice were cultured for 48 hrs and EdU labelled for 8 hrs, followed by immunostaining for Pax7 (green) and EdU (red). Quantifications of the percentage of EdU+ cells are shown on the right (n=3 mice, each). Scale bar = 100 µm.


( D ) The above cells were also IF stained for Pax7 ( red ) and MyoD ( green ) . Quantifications of each population , Pax7 + MyoD + , or Pax7 - MyoD + , are shown on the right ( n = 3 , each ) . Scale bar = 100 µm .

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

(D) The above cells were also IF stained for Pax7 (red) and MyoD (green). Quantifications of each population, Pax7+MyoD+, or Pax7-MyoD+, are shown on the right (n=3, each). Scale bar = 100 µm.


( E ) Single EDL myofibers were cultured for 48 hrs before staining for Pax7 ( red ) and MyoD ( green ) . Quantifications of the numbers of each population , Pax7 + MyoD - , Pax7 - MyoD + , or Pax7 + MyoD + , are shown on the right ( n = 3 mice , each ) . Scale bar = 50 µm .

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

(E) Single EDL myofibers were cultured for 48 hrs before staining for Pax7 (red) and MyoD (green). Quantifications of the numbers of each population, Pax7+MyoD-, Pax7-MyoD+, or Pax7+MyoD+, are shown on the right (n=3 mice, each). Scale bar = 50 µm.


( F ) Pax7 ( green ) and Ki67 ( red ) staining was performed on the above single EDL myofibers . Scale bar = 50 µm . Quantifications of the numbers of each population , Pax7 + Ki67 - , Pax7 - Ki67 + , or Pax7 + Ki67 + , are shown on the right ( n = 3 mice , each ) .

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

(F) Pax7 (green) and Ki67 (red) staining was performed on the above single EDL myofibers. Scale bar = 50 µm. Quantifications of the numbers of each population, Pax7+Ki67-, Pax7-Ki67+, or Pax7+Ki67+, are shown on the right (n= 3 mice, each).


( G ) SCs expansion in vivo was determined by EdU labeling for 12 hrs 2 . 5 days after CTX injury . Quantifications of the numbers of EdU + cells are shown on the right ( n = 3 mice , each ) . Scale bar = 100 µm .

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

(G) SCs expansion in vivo was determined by EdU labeling for 12 hrs 2.5 days after CTX injury. Quantifications of the numbers of EdU+ cells are shown on the right (n=3 mice, each). Scale bar = 100 µm.


( H ) An equal number of FACS - isolated SCs from Ctrl or YY1iKO mice were cultured for 24hrs and EdU labeled for 12 hrs , followed by immunostaining for Pax7 ( green ) and EdU ( red ) was performed . Quantifications of the percentage of EdU + cells are shown on the right ( n = 3 mice , each ) . Scale bar = 100 µm .

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

(H) An equal number of FACS-isolated SCs from Ctrl or YY1iKO mice were cultured for 24hrs and EdU labeled for 12 hrs, followed by immunostaining for Pax7 (green) and EdU (red) was performed. Quantifications of the percentage of EdU+ cells are shown on the right (n=3 mice, each). Scale bar = 100 µm.


Single EDL myofibers were cultured for 12 ( I ) hrs in growth media before staining for Pax7 ( green ) and MyoD ( red ) . Scale bar = 50 µm . Quantifications of the proportion of MyoD + cells are shown on the right ( n = 3 mice , each ) .

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

Single EDL myofibers were cultured for 12 (I) hrs in growth media before staining for Pax7 (green) and MyoD (red). Scale bar = 50 µm. Quantifications of the proportion of MyoD+ cells are shown on the right (n=3 mice, each).


Single EDL myofibers were cultured for 24 ( J ) hrs in growth media before staining for Pax7 ( green ) and MyoD ( red ) . Scale bar = 50 µm . Quantifications of the proportion of MyoD + cells are shown on the right ( n = 3 mice , each ) .

Single EDL myofibers were cultured for 24 (J) hrs in growth media before staining for Pax7 (green) and MyoD (red). Scale bar = 50 µm. Quantifications of the proportion of MyoD+ cells are shown on the right (n=3 mice, each).