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

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 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 O O O O O O O O O

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 last TM injection cultured for 1 . 5 days ; RT - qPCR detection of YY1 mRNA shows ablation in YY1iKO .

O 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 B-CONTROLLED_VAR 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 for Pax7 YY1 on freshly isolated FISCs or ( D ) single myofibers from EDL or ( E ) cryosections from TA showing deletion of YY1 from YY1iKO . Scale bar = 100 µm in ( C ) or 50 µm in ( D - E ) .

O O O O O B-MEASURED_VAR I-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 B-CONTROLLED_VAR 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 from 2 - month - old Ctrl YY1iKO mice were sorted by FACS 3 post - TM injection . 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 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

(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 was performed on injured TA collected from designated times post - CTX injection to visualize degree of regeneration . 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

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

O O O O O B-MEASURED_VAR 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

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

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

(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 ( CSA ) of myofibers in TA 4 after injury was measured . 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 of SCs sorted from TA 3 days after CTX injury of Ctrl YY1iKO mice .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 TA 3 days post - CTX injury . Scale bar = 100 µm . Quantifications of numbers of Pax7 + SCs per are shown on right ( n = 3 mice , each ) .

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

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 TA 3 days post - CTX injury . Scale bar = 100 µm . Quantifications of numbers of MyoD + SCs per are shown on 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 for Pax7 Laminin on TA 4 after CTX injury of Ctrl or YY1iKO mice . Quantifications of numbers of Pax7 + SCs per 100 are shown on right ( n = 3 mice , each ) . Scale bar = 100 μm .

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

(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 was performed on TA 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 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

(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 of above TA 7 days after second injury .

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


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

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


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

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

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

O O O O O B-MEASURED_VAR 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 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 last TM injection ; RT - qPCR detection of YY1 mRNA shows ablation in YY1dKO .

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 B-CONTROLLED_VAR 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 of Ctrl or YY1dKO mice 4 months after TM administration .

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

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


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

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

(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 of Dp isolated from Ctrl or YY1dKO mice 4 months after TM injection .

O O O O O O O O O O B-CONTROLLED_VAR 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 was performed on TA or Dp from Ctrl or YY1dKO mice . Quantifications of numbers of MyHC + per of TA or Dp are shown on right ( n = 3 mice , each ) . Scale bar = 100 µm .

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

(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 was performed on TA or Dp from Ctrl or YY1dKO mice . Scale bar = 100 µm .

O O O B-MEASURED_VAR 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

(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 ) distribution of fiber measured by CSA in TA from 3 pairs of 6 - month - old Ctrl 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 B-CONTROLLED_VAR 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 of SCs sorted from 6 - month - old Ctrl YY1dKO mice ( n = 3 mice , each ) .

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

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


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

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

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

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force . Left : Representative trace of normalized tetanic force . ( Right ) : of force by normalizing with cross - sectional ( CSA ) . ( n = 3 mice , each ) . Error bars represent s . d . ' s of mean . Student ' s t - ( 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 designated time ( 0 , 24 , 48 or 72 hrs ) . YY1 expression was quantified by RT - qPCR . 18S was used as 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 B-MEASURED_VAR 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 of YY1 or IgG control in FACS - isolated SCs cultured for designated time ( 0 , 24 or 48hrs ) . YY1 level is normalized to IgG . numbers indicate normalized level of YY1 relative to that of FISCs .

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

(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 EdU labelled for 8 hrs , followed by immunostaining for Pax7 ( green ) EdU ( red ) . Quantifications of of EdU + are shown on right ( n = 3 mice , each ) . Scale bar = 100 µm .

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 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

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

O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR 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 for Pax7 ( red ) MyoD ( green ) . Quantifications of numbers of each population , Pax7 + MyoD - , Pax7 - MyoD + , or Pax7 + MyoD + , are shown on right ( n = 3 mice , each ) . Scale bar = 50 µm .

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

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

O O O B-MEASURED_VAR 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 B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR 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 numbers of EdU + are shown on 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

(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 EdU labeled for 12 hrs , followed by immunostaining for Pax7 ( green ) EdU ( red ) was performed . Quantifications of of EdU + are shown on right ( n = 3 mice , each ) . Scale bar = 100 µm .

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 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

(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 for Pax7 ( green ) MyoD ( red ) . Scale bar = 50 µm . Quantifications of proportion of MyoD + are shown on right ( n = 3 mice , each ) .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR 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 for Pax7 ( green ) MyoD ( red ) . Scale bar = 50 µm . Quantifications of proportion of MyoD + are shown on 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).


( K ) FACS - isolated SCs from YY1iKO mice were infected with YY1 expressing or control , followed by immunostaining for Pax7 ( green ) MyoD ( red ) 36 hrs post - transfection . Scale bar = 100 µm . Quantifications of numbers of each population , Pax7 + MyoD - , Pax7 - MyoD + , or Pax7 + MyoD + , are shown on right ( n = 3 mice , each ) .

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

(K) FACS-isolated SCs from YY1iKO mice were infected with YY1 expressing or control viruses, followed by immunostaining for Pax7 (green) and MyoD (red) 36 hrs post-transfection. Scale bar = 100 µm. Quantifications of the numbers of each population, Pax7+MyoD-, Pax7-MyoD+, or Pax7+MyoD+, are shown on the right (n=3 mice, each).


( L ) Single EDL myofibers were cultured for 3 days , followed by immunostaining for Myogenin ( red ) MyoD ( green ) . Scale bar = 50 µm . Quantifications of numbers of each population , MyoD + Myog + , MyoD - Myog + , or MyoD + Myog - , are shown on right ( n = 3 mice , each ) .

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

(L) Single EDL myofibers were cultured for 3 days, followed by immunostaining for Myogenin (red) and MyoD (green). Scale bar = 50 µm. Quantifications of the numbers of each population, MyoD+Myog+, MyoD-Myog+, or MyoD+Myog-, are shown on the right (n=3 mice, each).


( M ) FACS - isolated SCs were cultured for 2 days in proliferation medium followed by 2 days in differentiation medium ; degree of differentiation was assessed by IF of MF20 + . Scale bar = 100 µm . Quantifications of numbers of MF20 + per nucleus are shown on right ( n = 3 mice , each ) . Error bars represent s . d . ' s of mean . Student ' s t - ( 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 B-MEASURED_VAR 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 O O O O O O O O O

(M) FACS-isolated SCs were cultured for 2 days in proliferation medium followed by 2 days in differentiation medium; the degree of differentiation was assessed by IF staining of MF20+. Scale bar = 100 µm. Quantifications of the numbers of MF20+ area per nucleus 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.


( A ) - seq was performed with RNAs extracted from ASCs ( FACS purified cultured for 36hrs ) of YY1iKO or Ctrl mice ; scatter plot shows differentially expressed genes with a fold change > = 2 ( red dots ) in YY1iKO vs Ctrl .

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 B-CONTROLLED_VAR O O O

(A) RNA-seq was performed with RNAs extracted from ASCs (FACS purified and cultured for 36hrs) of YY1iKO or Ctrl mice; scatter plot shows differentially expressed genes with a fold change >=2 (red dots) in YY1iKO vs Ctrl.


( C ) Genomic snapshots showing examples of mitochondrial genes ( Uqcr11 , Mrp11 , Apo5o Nme4 ) up - regulated in YY1iKO vs Ctrl .

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

(C) Genomic snapshots showing the examples of mitochondrial genes (Uqcr11, Mrp11, Apo5o and Nme4) up-regulated in YY1iKO vs Ctrl cells.


( D ) GSEA shows oxidative phosphorylation gene set is enriched in YY1iKO vs Ctrl ( p - value < 0 . 0001 ) .

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

(D) GSEA analysis shows oxidative phosphorylation gene set is enriched in YY1iKO vs Ctrl cells (p-value < 0.0001).


( E ) RT - qPCR validation of expression of selected up - regulated mitochondrial genes in YY1iKO vs Ctrl .

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

(E) RT-qPCR validation of the expression of the selected up-regulated mitochondrial genes in YY1iKO vs Ctrl.


( F ) of respiratory chain complexes I - V components in YY1iKO vs Ctrl ASCs were detected using a mitoprofile antibody cocktail . β - actin was used as loading control .

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

(F) Protein levels of the respiratory chain complexes I-V components in YY1iKO vs Ctrl ASCs were detected using a mitoprofile antibody cocktail. β-actin was used as the loading control.


( G ) - seq was performed using RNAs from freshly isolated SCs ( FISCs ) ; scatter plot shows differentially expressed genes with a fold change > = 2 ( red dots ) in YY1iKO vs Ctrl .

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(G) RNA-seq was performed using RNAs from freshly isolated SCs (FISCs); scatter plot shows differentially expressed genes with a fold change >=2 (red dots) in YY1iKO vs Ctrl.


( I ) Genomic snapshots show selected mitochondrial genes ( Cox8b , Nme4 , Slc25a29 , Nnt ) up - regulated in YY1iKO vs Ctrl FISCs .

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(I) Genomic snapshots show the selected mitochondrial genes (Cox8b, Nme4, Slc25a29, Nnt) up-regulated in YY1iKO vs Ctrl FISCs.


( J ) ChIPseq was performed using chromatins from WT ASCs genomic distribution of 2031 YY1 binding peaks is shown .

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(J) ChIPseq was performed using chromatins from WT ASCs and the genomic distribution of 2031 YY1 binding peaks is shown.


( N ) Genomic snapshots of two of above identified mitochondrial genes ( Tomm40I Ndufa13 ) that are bound by YY1 in their TSS ( ChIPseq tracks ) up - regulated by YY1 deletion ( RNAseq tracks ) .

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(N) Genomic snapshots of two of the above identified mitochondrial genes (Tomm40I and Ndufa13) that are bound by YY1 in their TSS (ChIPseq tracks) and up-regulated by YY1 deletion (RNAseq tracks).


( O ) ChIP - qPCR validation of YY1 binding on some mitochondrial genes . Negative control ( NC ) represents a genomic region on Chromosome 11 with no YY1 binding peak identified . Enrichment fold was calculated as amount of amplified from YY1 binding sites normalized to values obtained from IgG control .

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(O) ChIP-qPCR validation of YY1 binding on some mitochondrial genes. Negative control (NC) represents a genomic region on Chromosome 11 with no YY1 binding peak identified. Enrichment fold was calculated as the amount of amplified DNA from YY1 binding sites normalized to values obtained from IgG control.


ChIP - qPCR was performed to show enrichment of Ezh2 ( P ) binding on selected mitochondrial genes .

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ChIP-qPCR was performed to show the enrichment of Ezh2 (P) binding on selected mitochondrial genes.


ChIP - qPCR was performed to show enrichment of Suz12 ( Q ) binding on selected mitochondrial genes .

ChIP-qPCR was performed to show the enrichment of Suz12 (Q) binding on selected mitochondrial genes.


ChIP - qPCR was performed to show enrichment of H3K27 me3 ( R ) binding on selected mitochondrial genes .

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ChIP-qPCR was performed to show the enrichment of H3K27 me3 (R) binding on selected mitochondrial genes.


( S ) A construct to express wild type YY1 ( WT ) , or two mutants lacking binding activity , YY1 ( 1 - 394 ) YY1 ( S365D ) were transfected into YY1iKO ; expression of mitochondrial gene was measured . Error bars represent s . d . ' s of mean . Student ' s t - ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(S) A construct to express wild type YY1(WT), or two mutants lacking DNA binding activity, YY1(1-394) and YY1(S365D) were transfected into YY1iKO cells; the expression of mitochondrial gene was measured. 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.


( A ) An equal number of FACS - isolated SCs from C57 mice were cultured for 24 hrs treated with 0 , 1 , 10 , or 20mM 2 - DG ( glycolytic inhibitor ) or NaN3 ( respiration inhibitor ) for 3 hrs before measurement of ATP production . relative ATP normalized to 0 mM values are plotted . N = 3 mice .

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(A) An equal number of FACS-isolated SCs from C57 mice were cultured for 24 hrs and treated with 0, 1, 10, or 20mM 2-DG (glycolytic inhibitor) or NaN3 (respiration inhibitor) for 3 hrs before measurement of ATP production. The relative ATP levels normalized to the 0 mM values are plotted. N=3 mice.


( B ) SCs from C57 mice were treated with 10mM 2 - DG or 10mM NaN3 for 36 hrs before measurement of proliferation rate by MTS ( n = 3 mice , each ) .

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(B) SCs from C57 mice were treated with 10mM 2-DG or 10mM NaN3 for 36 hrs before measurement of proliferation rate by MTS assay (n=3 mice, each).


( C ) SCs from C57 mice were treated with 10mM 2 - DG for 24 hrs subject to EdU labeling for 6 hrs . of Pax7 + / EdU + over total number of Pax7 + was quantified ( n = 3 mice , each ) .

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(C) SCs from C57 mice were treated with 10mM 2-DG for 24 hrs and subject to EdU labeling for 6 hrs. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified (n=3 mice, each).


( D ) Ctrl YY1iKO ASCs were stained with 60 ug / mL 2 - NBDG for 45 mins ( MFI ) of 2 - NBDG was measured by flow cytometry ( n = 3 mice , each ) .

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(D) Ctrl and YY1iKO ASCs were stained with 60 ug/mL 2-NBDG for 45 mins and the fluorescence intensity (MFI) of 2-NBDG was measured by flow cytometry (n=3 mice, each).


An equal number of Ctrl YY1iKO were cultured for 36h ; basal extracellular acidification rate ( ECAR ) level were measured normalized with numbers . N = 3 mice .

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An equal number of Ctrl and YY1iKO cells were cultured for 36h; basal extracellular acidification rate (ECAR) level were measured and normalized with cell numbers. N=3 mice.


An equal number of Ctrl YY1iKO were cultured for 36h Lactate ( F ) were measured normalized with numbers . N = 3 mice .

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An equal number of Ctrl and YY1iKO cells were cultured for 36h Lactate concentration (F) were measured and normalized with cell numbers. N=3 mice. by


An equal number of Ctrl YY1iKO were cultured for 36h ATP production ( G ) were measured normalized with numbers . N = 3 mice .

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An equal number of Ctrl and YY1iKO cells were cultured for 36h ATP production (G) were measured and normalized with cell numbers. N=3 mice.


( H ) Genomic snapshots depict RNAseq profiles of selected glycolytic genes ( Hk2 , Glut1 , Ldha , Pdk1 , Eno1 ) down - regulated in YY1iKO vs Ctrl ASCs .

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(H) Genomic snapshots depict RNAseq profiles of the selected glycolytic genes (Hk2, Glut1, Ldha, Pdk1, Eno1) down-regulated in YY1iKO vs Ctrl ASCs.


( I ) Expression of selected glycolytic genes was quantified by RT - qPCR . Hsp90ab1 was used as normalization control .

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(I) Expression of the selected glycolytic genes was quantified by RT-qPCR. Hsp90ab1 was used as the normalization control.


( J ) Re - expression of YY1 by transfecting YY1iKO ASCs with a YY1 WT expressing plasmid led to up - regulation of glycolytic genes including Glut1 , Pfkl , Gapdh , Eno1 , Pgam1 , Pkm . Hsp90ab1 was used as normalization control .

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(J) Re-expression of YY1 by transfecting YY1iKO ASCs with a YY1 WT expressing plasmid led to up-regulation of glycolytic genes including Glut1, Pfkl, Gapdh, Eno1, Pgam1, Pkm. Hsp90ab1 was used as the normalization control.


( K ) FACS - isolated SCs from Pax7 - nGFP mice were transfected with Hif1α or control siRNAs EdU labeled for 5 hrs . of MyoD + EdU + over total number of MyoD + was quantified . Scale bar = 100 µm . N = 3 mice .

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(K) FACS-isolated SCs from Pax7-nGFP mice were transfected with Hif1α or control siRNAs and EdU labeled for 5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice.


( L ) expression of glycolytic genes cycle related genes was detected by RT - qPCR in above transfected . β - actin was used as normalization control .

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(L) The expression of glycolytic genes and cell cycle related genes was detected by RT-qPCR in the above transfected cells. β-actin was used as the normalization control.


( M ) Hif1α level was detected by Western blotting in YY1iKO vs Ctrl ASCs . β - actin was used as loading control .

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(M) Hif1α protein level was detected by Western blotting in YY1iKO vs Ctrl ASCs. β-actin was used as the loading control.


( N ) Left : Image of flow cytometric of Hif1α or IgG control in YY1iKO vs Ctrl ASCs . Right : Relative mean ( MFI ) of Hif1α is shown . N = 3 mice .

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(N) Left: Image of flow cytometric analysis of Hif1α protein or IgG control in YY1iKO vs Ctrl ASCs. Right: Relative mean fluorescence intensity (MFI) of Hif1α protein is shown. N=3 mice.


( O ) YY1iKO ASCs were transfected with empty YY1 WT ; relative expression level of Hif1a was detected by above described flow cytometric N = 3 mice . Error bars represent s . d . ' s of mean . Student ' s t - ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(O) YY1iKO ASCs were transfected with empty YY1 WT viruses; the relative expression level of Hif1a protein was detected by the above described flow cytometric analysis N=3 mice. 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.


( A - B ) Ctrl or YY1iKO ASCs were treated with CHX ( Cycloheximide ) for 90 mins Hif1α was measured by flow cytometry . numbers indicate degradation rate after CHX treatment . N = 3 mice . ( C ) relative degradation rate was calculated by of degradation rate in YY1iKO ( 11 . 14 % ± 1 . 71 % ) vs Ctrl ( 5 . 08 % ± 0 . 796 % ) . N = 3 mice .

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(A-B) Ctrl or YY1iKO ASCs were treated with CHX (Cycloheximide) for 90 mins and Hif1α protein was measured by flow cytometry. The numbers indicate the degradation rate after CHX treatment. N=3 mice. (C) The relative degradation rate was calculated by the ratio of degradation rate in YY1iKO (11.14% ± 1.71%) vs Ctrl (5.08% ± 0.796%). N=3 mice.


( D ) Lysates from C2C12 myoblasts ( GM ) were subjected to co - immunoprecipitation ( Co - IP ) assays with anti - YY1 or anti - IgG antibodies blotted with anti - Hif1α or anti - YY1 antibodies .

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(D) Lysates from C2C12 myoblasts (GM) were subjected to co-immunoprecipitation (Co-IP) assays with anti-YY1 or anti-IgG antibodies and blotted with anti-Hif1α or anti-YY1 antibodies.


( E ) above lysates were also IPed with anti - Hif1α or anti - IgG antibodies blotted with anti - YY1 or anti - Hif1α antibodies .

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(E) The above lysates were also IPed with anti-Hif1α or anti-IgG antibodies and blotted with anti-YY1 or anti-Hif1α antibodies.


( F ) ASCs from YY1iKO mice were infected with pRlenti - Hif1α ( P402A / P564A ) ( Hif1α - DM ) to overexpress a non - degradable Hif1α or GFP - expressing . ASCs from Ctrl mice were also infected with GFP . 36 hrs after infection , expression of target glycolytic genes was detected by RT - qPCR . Hsp90ab1 was used as normalization control .

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(F) ASCs from YY1iKO mice were infected with pRlenti-Hif1α (P402A/P564A) (Hif1α-DM) viruses to overexpress a non-degradable Hif1α protein or GFP-expressing viruses. ASCs from Ctrl mice were also infected with the GFP viruses. 36 hrs after infection, the expression of target glycolytic genes was detected by RT-qPCR. Hsp90ab1 was used as the normalization control.


( G ) SCs were isolated from Ctrl or YY1iKO mice infected with above . EdU labeling was then performed for 5 . 5 hrs . of MyoD + EdU + over total number of MyoD + was quantified . Scale bar = 100 µm . N = 3 mice .

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(G) SCs were isolated from Ctrl or YY1iKO mice and infected with the above viruses. EdU labeling was then performed for 5.5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice.


Heat maps indicating gene expression ( Log2 [ FPKM ] ) of pentose cycle enzymes ( H ) in YY1iKO vs Ctrl .

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Heat maps indicating gene expression levels (Log2[FPKM]) of pentose cycle enzymes (H) in YY1iKO vs Ctrl.


Heat maps indicating gene expression ( Log2 [ FPKM ] ) of acid transporter ( I ) in YY1iKO vs Ctrl .

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Heat maps indicating gene expression levels (Log2[FPKM]) of amino acid transporter (I) in YY1iKO vs Ctrl.


( J ) YY1 ( WT ) or YY1 ( S365D ) mutant was expressed in YY1iKO ASCs EdU labeled for 6 hrs . ( K ) Similarly , YY1 ( WT ) or YY1 ( 1 - 394 ) mutant was expressed EdU labeled for 6hrs . A GFP expressing plasmid was used negative control . of Pax7 + / EdU + over total number of Pax7 + was quantified . Error bars represent s . d . ' s of mean . Student ' s t - ( two - sided ) : N . S = non - significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(J) YY1(WT) or YY1(S365D) mutant was expressed in YY1iKO ASCs and EdU labeled for 6 hrs. (K) Similarly, YY1 (WT) or YY1 (1-394) mutant was expressed and EdU labeled for 6hrs. A GFP expressing plasmid was used the negative control. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified. 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.


Human osteosarcoma U2OS were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 6 µM as positive control Human osteosarcoma U2OS stably expressing CALR - GFP H2B - RFP were treated as described above were acquired once per hour for 12 h ( A ) . For one representative experiment among three , mean ± of average of high CALR dots ( normalized to control at each timepoint ) of quadruplicates is shown ( B ) . Values are depicted as under curve ± SD of

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Human osteosarcoma U2OS stably expressing CALR-GFP and H2B-RFP were treated as described above and images were acquired once per hour for 12 h (A). For one representative experiment among three, the mean ± SEM of the average area of high CALR dots (normalized to the control at each timepoint) of quadruplicates is shown (B). Values are depicted as the area under the curve ± SD of triplicates


Human osteosarcoma U2OS were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 6 µM as positive control Treated U2OS stably expressing HMGB1 - GFP H2B - RFP were acquired every hour for 24 h ( D ) . For one representative experiment among three , mean ± of green in nucleus ( normalized to control at each timepoint ) of quadruplicates is depicted ( E ) . For each , speed of release ( difference of HMGB1 green between two time points ) was calculated . Values are depicted as average speed of release ± SD of

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Treated U2OS cells stably expressing HMGB1-GFP and H2B-RFP images were acquired every hour for 24 h (D). For one representative experiment among three, the mean ± SEM of the green fluorescence intensity in the nucleus (normalized to the control at each timepoint) of quadruplicates is depicted (E). For each cell, the speed of nuclear release (difference of HMGB1 nuclear green fluorescence intensity between two time points) was calculated. Values are depicted as the average speed of the nuclear release ± SD of quadruplicates


Human osteosarcoma U2OS were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 6 µM as positive control U2OS were treated for 6 , 12 or 24 h ATP was stained with quinacrine ( G ) . number of quinacrine negative was assessed on distribution of green in MTX versus control conditions . For one representative experiment among three mean ± SD of quadruplicate assessments is

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated for 6, 12 or 24 h and ATP was stained with quinacrine (G). The number of quinacrine negative cells was assessed based on the distribution of cellular green fluorescence intensity in MTX versus control conditions. For one representative experiment among three the mean ± SD of quadruplicate assessments is shown


Human osteosarcoma U2OS were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 6 µM as positive control U2OS wild - type were treated with MTX or DACT as described above for 6 h . Then medium was refreshed 24 h later , type I interferon response was assessed by transferring supernatant on HT29 MX1 - GFP reporter lines for additional 48 h . Human type 1α interferon ( IFNα1 ) was also added on as an additional positive control . were acquired by microscopy number of positive was assessed on distribution of green in IFNα1 versus control conditions ( I ) . of MX1 positive was calculated mean ± of five independent experiments is

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated with MTX or DACT as described above for 6 h. Then medium was refreshed and 24 h later, type I interferon response was assessed by transferring the supernatant on HT29 MX1-GFP reporter cells lines cells for additional 48 h. Human type 1α interferon (IFNα1) was also added on the cells as an additional positive control. Images were acquired by fluorescence microscopy and the number of positive cells was assessed based on the distribution of cellular green fluorescence intensity in IFNα1 versus control conditions (I). The percentage of MX1 positive cells was calculated and the mean ± SEM of five independent experiments is depicted


Human osteosarcoma U2OS were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 6 µM as positive control U2OS wild - type were treated as mentioned above for 6 h then medium was refreshed . Twenty - four hours later , were collected surface - exposed calreticulin ( CALR ) was stained with an antibody specific for CALR . DAPI was used as an exclusion dye were acquired by flow cytometry ( K ) . of CALR + among viable ( DAPI - ) ones are depicted . mean ± of six independent experiments is

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated as mentioned above for 6 h and then medium was refreshed. Twenty-four hours later, cells were collected and surface-exposed calreticulin (CALR) was stained with an antibody specific for CALR. DAPI was used as an exclusion dye and cells were acquired by flow cytometry (K). The percentage of CALR+ cells among viable (DAPI-) ones are depicted. The mean ± SEM of six independent experiments is depicted


Human osteosarcoma U2OS were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 6 µM as positive control U2OS were treated as described above for 24 h of HMGB1 released in supernatant was quantified with an ELISA kit , then normalized to control . mean ± of four independent experiments is shown .

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated as described above for 24 h and the concentration of HMGB1 released in the supernatant was quantified with an ELISA kit, then normalized to control. The mean ± SEM of four independent experiments is shown.


Human osteosarcoma U2OS were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 6 µM as positive control U2OS were treated as described above for 24 h . of secreted ATP in supernatant was quantified with a luciferase - bioluminescence kit . mean ± SD of quadruplicates from one representative among three experiments is depicted .

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS were treated as described above for 24 h. Concentration of secreted ATP in the supernatant was quantified with a luciferase-based bioluminescence kit. The mean ± SD of quadruplicates from one representative among three experiments is depicted.


Human osteosarcoma U2OS were treated with different concentrations of dactinomycin ( DACT ) ( 0 . 25 , 0 . 5 or 1 µM ) for 6 h . Thapsigargin ( THAPS ) at 3 µM was used as a positive control . After fixation , were stained with phospho - eIF2α ( Ser51 ) specific antibody followed by an AlexaFluor - 647 secondary antibody , nuclei were counterstained with Hoechst 33342 phosphorylation was assessed by microscopy . were segmented , analyzed red was measured . are shown for untreated control ( Ctr ) , THAPS DACT at 1

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Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin (DACT) (0.25, 0.5 or 1 µM) for 6 h. Thapsigargin (THAPS) at 3 µM was used as a positive control. After fixation, cells were stained with phospho-eIF2α (Ser51) specific antibody followed by an AlexaFluor-647 secondary antibody, nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy. Images were segmented, analyzed and the red cytoplasmic fluorescence intensity was measured. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS stably expressing ATF4 - reporter were treated as described above for 12 h . expression translocation of ATF4 was assessed by microscopy green was quantified . are shown for untreated control ( Ctr ) , THAPS DACT at 1

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U2OS cells stably expressing ATF4-reporter were treated as described above for 12 h. The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS stably expressing ATF6 - GFP were treated as described above translocation of ATF6 was represented as of versus green . are shown for untreated control ( Ctr ) , THAPS DACT at 1

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U2OS stably expressing ATF6-GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS stably expressing venus in frame with alternatively spliced XBP1 ( sXBP1 ) were treated as described above for 12 h . De novo expressed venus was measured intracellular . are shown for untreated control ( Ctr ) , THAPS DACT at 1

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U2OS cells stably expressing venus in frame with alternatively spliced XBP1 (sXBP1) were treated as described above for 12 h. De novo expressed venus was measured intracellular. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS wild - type knock out for eIF2α kinases 1 , 2 , 3 4 were treated with 3 µM THAPS as a positive control for EIF2AK3 - mediated eIF2α phosphorylation or with 1 µM DACT for 6 h . After fixation , were stained for peIF2α as described above was quantified . are shown for untreated control ( Ctr ) , THAPS DACT at 1

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U2OS wild-type and knock out for eIF2α kinases 1, 2, 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3-mediated eIF2α phosphorylation or with 1 µM DACT for 6 h. After fixation, cells were stained for peIF2α as described above and cytoplasmic intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS CALR - RFP were injected subcutaneously ( s . c ) in flank of nu / nu mice . Tumors were further injected with PBS ( Ctr ) ( n = 5 ) , 0 . 5 mg / kg tunicamycin ( TM ) for 6 h ( n = 3 ) or 0 . 5 mg / kg DACT for 6 h ( n = 4 ) or 24 h ( n = 3 ) . slices were cut stained with a peIF2α antibody followed by an AlexaFluor - 647 secondary antibody counterstained with Hoechst 33342 . Two slices per were imaged for their DAPI , RFP Cy5 signals . Out - of - focused were removed from dataset leading to of following conditions : 8 x Ctr , 5 x TM , 8 x DACT 6 h 6 x for DACT 24 h . PeIF2α was quantified measuring Cy5 in cytoplasm ( K , L ) CALR translocation by measuring coefficient variation ( CV ) of RFP signal in cytoplasm ( M , N ) . Representative of peIF2α are shown for Ctr , TM DACT at 6 h ( K ) , whereas of CALR are shown for Ctr DACT at 24

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

U2OS CALR-RFP were injected subcutaneously (s.c) in the flank of nu/nu mice. Tumors were further injected with PBS (Ctr) (n=5), 0.5 mg/kg tunicamycin (TM) for 6 h (n=3) or 0.5 mg/kg DACT for 6 h (n=4) or 24 h (n=3). Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor-647 secondary antibody and counterstained with Hoechst 33342. Two slices per tumor were imaged for their DAPI, RFP and Cy5 signals. Out-of-focused images were removed from the dataset leading to the analysis of the following conditions: 8 x Ctr, 5 x TM, 8 x DACT 6 h and 6 x for DACT 24 h. PeIF2α was quantified measuring Cy5 intensity in the cytoplasm (K, L) and CALR translocation by measuring the coefficient variation (CV) of the RFP signal in the cytoplasm (M, N). Representative images of peIF2α are shown for Ctr, TM and DACT at 6 h (K), whereas images of CALR are shown for Ctr and DACT at 24 h


Mouse fibrosarcoma MCA205 were stained with CellTracker Orange ( CMTMR ) treated for 24 h with 1 µM dactinomycin ( DACT ) or 500 µM oxaliplatin ( OXA ) as a positive control . Then , untreated or dying MCA205 were co - cultured with differentiated bone marrow - derived dendritic ( BMDCs ) for 4 h at 37 ° C or at 4 ° C . were collected dendritic were stained with CD11c specific antibody before by flow cytometry of CMTMR CD11c double positive among CD11c + are

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Mouse fibrosarcoma MCA205 cells were stained with CellTracker Orange (CMTMR) and treated for 24 h with 1 µM dactinomycin (DACT) or 500 µM oxaliplatin (OXA) as a positive control. Then, untreated or dying MCA205 were co-cultured with differentiated bone marrow-derived dendritic cells (BMDCs) for 4 h at 37 °C or at 4 °C. Cells were collected and dendritic cells were stained with CD11c specific antibody before analysis by flow cytometry The percentage of CMTMR and CD11c double positive cells among all CD11c+ cells are indicated


MCA205 were treated for 24 h with 500 µM oxaliplatin ( OXA ) or with 0 . 25 , 0 . 5 or 1 µM dactinomycin ( DACT ) . Then , untreated or dying MCA205 were co - cultured with BMDCs for 24 h at 37 ° C . As a positive control , BMDCs were co - cultured with 1 μg / mL LPS 100 ng / mL IFNγ . increase in percentages of MHCII + CD86 + among CD11c + was quantified with respect to untreated controls .

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MCA205 cells were treated for 24 h with 500 µM oxaliplatin (OXA) or with 0.25, 0.5 or 1 µM dactinomycin (DACT). Then, untreated or dying MCA205 were co-cultured with BMDCs for 24 h at 37 °C. As a positive control, BMDCs were co-cultured with 1 μg/mL LPS and 100 ng/mL IFNγ. The increase in the percentages of MHCII+ and CD86+ cells among CD11c+ cells was quantified with respect to untreated controls.


1 x 106 mouse fibrosarcoma MCA205 were treated in vitro with 1 µM dactinomycin ( DACT ) . Next , was measured regularly growths of DACT - vaccinated versus Ctr mice are

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1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Next, tumor size was measured regularly and individual tumor growths of DACT-vaccinated versus Ctr mice are depicted


1 x 106 mouse fibrosarcoma MCA205 were treated in vitro with 1 µM dactinomycin ( DACT ) . Overall survival is depicted p - values ( * * * p < 0 . 001 . ) were calculated with a Log - Rank

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1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Overall survival is depicted and p-values (*** p<0.001.) were calculated with a Log-Rank test


3 x 105 mouse fibrosarcoma WEHI 164 were injected subcutaneously ( s . c ) into flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . was assessed regularly growth curves of DACT versus

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3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. Tumor size was assessed regularly and individual tumor growth curves of DACT versus Ctr


3 x 105 mouse fibrosarcoma WEHI 164 were injected subcutaneously ( s . c ) into flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . DACT + anti - CD4 / CD8 versus anti - CD - 4 / anti - CD - 8 Mean for each group was calculated significances were tested using a type II ANOVA Overall survival is depicted p - values were calculated with a Log - Rank Stars indicate p - values of each treatment versus Ctr hashes indicate p - values of DACT + anti - CD - 4 / anti - CD - 8 versus DACT

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

3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. DACT + anti-CD4/CD8 versus anti-CD-4/anti-CD-8 Mean tumor area for each group was calculated and significances were tested using a type II ANOVA test Overall survival is depicted and p-values were calculated with a Log-Rank test Stars indicate the p-values of each treatment versus Ctr and hashes indicate the p-values of the DACT + anti-CD-4/anti-CD-8 versus DACT alone


3 x 105 mouse fibrosarcoma WEHI 164 were injected subcutaneously ( s . c ) into flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . DACT + anti - PD - 1 versus anti - PD - 1 are depicted . Overall survival is depicted p - values were calculated with a Log - Rank Stars indicate p - values of each treatment versus Ctr of DACT + anti - PD - 1 versus anti - PD - 1

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

3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. DACT + anti-PD-1 versus anti-PD-1 are depicted. Overall survival is depicted and p-values were calculated with a Log-Rank test Stars indicate the p-values of each treatment versus Ctr and of DACT + anti-PD-1 versus anti-PD-1 alone


3 x 105 mouse fibrosarcoma WEHI 164 were injected subcutaneously ( s . c ) into flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . Five naïve mice or eleven mice that were cured by treatment with DACT alone or in combination with PD - 1 blockade were ( re ) challenged with WEHI 164 , growths ( I , J ) , as well as overall survival ( * * * p < 0 . 001 , Log - Rank ) ( K ) , were monitored .

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

3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. Five naïve mice or the eleven mice that were cured by treatment with DACT alone or in combination with PD-1 blockade were (re)challenged with WEHI 164 cells, and individual tumor growths (I, J), as well as overall survival (*** p<0.001, Log-Rank test) (K), were monitored.


Mice bearing WEHI 164 sarcomas were treated by systemic ( intraperitoneal , i . p . ) injections of DACT or PBS as a control ( Ctr ) , were excised 9 days later for quantitation of mRNA coding for IFNγ Two independent experiments were conducted with a total of 22 mice in Ctr group 23 mice in DACT - treated group , with each data point indicating one . cycle threshold of qRT - PCR of IFNγ was normalized to one of housekeeping gene peptidylpropyl isomerase A ( Ppia ) in each mouse results are shown normalized with respect to controls as a dot plot depicting mean ± SD . p - value ( * p < 0 . 05 ) was calculated by means of a Student ' s t ( B ) .

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

Mice bearing WEHI 164 sarcomas were treated by systemic (intraperitoneal, i.p.) injections of DACT or PBS as a control (Ctr), and tumor were excised 9 days later for the quantitation of mRNA coding for IFNγ Two independent experiments were conducted with a total of 22 mice in the Ctr group and 23 mice in the DACT-treated group, with each data point indicating one tumor. The cycle threshold of the qRT-PCR of IFNγ was normalized to the one of the housekeeping gene peptidylpropyl isomerase A (Ppia) in each mouse and results are shown normalized with respect to controls as a dot plot depicting mean ± SD . The p-value (* p<0.05) was calculated by means of a Student's t test (B).


Balb / c mice with palpable WEHI 164 sarcomas ( n = 7 mice in Ctr anti - IFNγ groups ; n = 8 mice in DACT DACT + anti - IFNγ groups ) received two injections of DACT - chemotherapy ( 0 . 5 mg / kg ) as well as multiple injections ( 3 times per week ) of neutralizing IFNγ - specific antibody ( C ) . growth curves are shown for mice ( D ) as means ( E ) . Statistical difference between DACT - treated tumors respective controls , which was calculated with a type II ANOVA ( * p < 0 . 05 ) , is lost upon IFNγ neutralization ( E ) . Overall survival of mice is also indicated with statistics to respective controls calculated with a Log - Rank ( * p < 0 . 05 ) ( F ) .

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

Balb/c mice with palpable WEHI 164 sarcomas (n=7 mice in Ctr and anti-IFNγ groups; n=8 mice in DACT and DACT+anti-IFNγ groups) received two injections of DACT-based chemotherapy (0.5 mg/kg) as well as multiple injections (3 times per week) of neutralizing IFNγ-specific antibody (C). Tumor growth curves are shown for individual mice (D) and as means (E). Statistical difference between DACT-treated tumors and respective controls, which was calculated with a type II ANOVA (* p<0.05), is lost upon IFNγ neutralization (E). Overall survival of mice is also indicated with statistics to respective controls calculated with a Log-Rank test (* p<0.05) (F).


Human osteosarcoma U2OS were pre - treated with dactinomycin ( DACT ) , bortezomib ( BTZ ) , daunorubicin ( DAUN ) , docetaxel ( DOC ) , doxorubicin ( DOXO ) , epirubicin ( EPI ) , mitoxantrone ( MTX ) , paclitaxel ( PACL ) , vinblastine ( VB ) vincristine ( VC ) at 0 . 5 , 1 5 µM ; with cisplatin ( CDDP ) at 75 , 150 300 µM , with oxaliplatin ( OXA ) at 250 , 500 1000 µM with crizotinib ( CRIZ ) at 10 , 20 40 µM for 1 . 5 to 2 . 5 h followed by an additional hour of treatment in presence of 100 mM 5 - ethynyl uridine ( EU ) . After fixation were permeabilized EU was stained with an AlexaFluor - 488 - coupled azide . Representative are shown for each treatment ( A ) . EU in nucleus of each condition was ranked between untreated control ( Ctr , 0 % transcription inhibition ) control that was not incubated with EU ( corresponding to 100 % transcription inhibition ) ( B ) . Representative of DACT 1 µM , BTZ 1 µM , CDDP 150 µM , CRIZ 20 µM , DAUN 0 . 5 µM , DOC 1 µM , DOXO 1 µM , EPI 1 µM , MTX 1 µM , OXA 500 µM , PACL 1 µM , VB 1 µM , VC 1 µM are

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

Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h and followed by an additional hour of treatment in the presence of 100 mM 5-ethynyl uridine (EU). After fixation cells were permeabilized and EU was stained with an AlexaFluor-488-coupled azide. Representative images are shown for each treatment (A). The EU intensity in the nucleus of each condition was ranked between the untreated control (Ctr, 0 % transcription inhibition) and the control that was not incubated with EU (corresponding to 100 % transcription inhibition) (B). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown


Human osteosarcoma U2OS were pre - treated with dactinomycin ( DACT ) , bortezomib ( BTZ ) , daunorubicin ( DAUN ) , docetaxel ( DOC ) , doxorubicin ( DOXO ) , epirubicin ( EPI ) , mitoxantrone ( MTX ) , paclitaxel ( PACL ) , vinblastine ( VB ) vincristine ( VC ) at 0 . 5 , 1 5 µM ; with cisplatin ( CDDP ) at 75 , 150 300 µM , with oxaliplatin ( OXA ) at 250 , 500 1000 µM with crizotinib ( CRIZ ) at 10 , 20 40 µM for 1 . 5 to 2 . 5 h were treated for 2 . 5 h before fixation permeabilization . Then , were stained with a rabbit anti - fibrillarin antibody followed by a with an anti - rabbit AlexaFluor - 647 - or AlexaFluor - 546 - coupled secondary antibody as well as with a mouse anti - nucleolin antibody followed by a with an anti - mouse AlexaFluor - 488 - coupled secondary antibody . Then were acquired colocalization between both signals was assessed ( C ) . surface overlap coefficient ( SOC ) was calculated ranked between untreated control ( Ctr ) positive control ( DACT ) Representative of DACT 1 µM , BTZ 1 µM , CDDP 150 µM , CRIZ 20 µM , DAUN 0 . 5 µM , DOC 1 µM , DOXO 1 µM , EPI 1 µM , MTX 1 µM , OXA 500 µM , PACL 1 µM , VB 1 µM , VC 1 µM are

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

Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were treated for 2.5 h and before fixation and permeabilization. Then, cells were stained with a rabbit anti-fibrillarin antibody followed by a staining with an anti-rabbit AlexaFluor-647- or AlexaFluor-546-coupled secondary antibody as well as with a mouse anti-nucleolin antibody followed by a staining with an anti-mouse AlexaFluor-488-coupled secondary antibody. Then images were acquired and colocalization between both signals was assessed (C). The surface overlap coefficient (SOC) was calculated and ranked between the untreated control (Ctr) and the positive control (DACT) Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown


Human osteosarcoma U2OS were pre - treated with dactinomycin ( DACT ) , bortezomib ( BTZ ) , daunorubicin ( DAUN ) , docetaxel ( DOC ) , doxorubicin ( DOXO ) , epirubicin ( EPI ) , mitoxantrone ( MTX ) , paclitaxel ( PACL ) , vinblastine ( VB ) vincristine ( VC ) at 0 . 5 , 1 5 µM ; with cisplatin ( CDDP ) at 75 , 150 300 µM , with oxaliplatin ( OXA ) at 250 , 500 1000 µM with crizotinib ( CRIZ ) at 10 , 20 40 µM for 1 . 5 to 2 . 5 h were pre - treated overnight with aforementioned compounds in complete medium followed by washout treatment pursued in methionine - free medium for 30 min . Afterwards , treatments were continued in methionine - free medium supplemented with 25 µM L - azidohomoalanine ( AHA ) for additional 1 . 5 h . AHA incorporation was detected after fixation , permeabilization blocking by addition of an AlexaFluor - 488 - coupled azide . Then were acquired ( E ) AHA in was ranked between untreated control ( Ctr , 0 % translation inhibition ) untreated control without AHA ( corresponding to 100 % translation inhibition ) ( F ) . Representative of DACT 1 µM , BTZ 1 µM , CDDP 150 µM , CRIZ 20 µM , DAUN 0 . 5 µM , DOC 1 µM , DOXO 1 µM , EPI 1 µM , MTX 1 µM , OXA 500 µM , PACL 1 µM , VB 1 µM , VC 1 µM are

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

Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were pre-treated overnight with the aforementioned compounds in complete medium followed by washout and treatment pursued in methionine-free medium for 30 min. Afterwards, the treatments were continued in methionine-free medium supplemented with 25 µM L-azidohomoalanine (AHA) for additional 1.5 h. AHA incorporation was detected after fixation, permeabilization and blocking by the addition of an AlexaFluor-488-coupled azide. Then images were acquired (E) and AHA intensity in the cells was ranked between the untreated control (Ctr, 0 % translation inhibition) and the untreated control without AHA (corresponding to 100 % translation inhibition) (F). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown


Correlation between transcription measured by EU incorporation measured by fibrillarin nucleolin colocalization is depicted with Pearson correlation coefficient ( R ) p - value ( p ) ( G ) . same parameters are shown for correlation between transcription measured by EU incorporation translation measured with AHA

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

Correlation between the transcription measured by EU incorporation and measured by fibrillarin and nucleolin colocalization is depicted with Pearson correlation coefficient (R) and p-value (p) (G). The same parameters are shown for the correlation between transcription measured by EU incorporation and translation measured with AHA incorporation


U2OS wild - type were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition ICD prediction score Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and ICD prediction score Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition peIF2α expression Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and peIF2α expression Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition CALR exposure Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and CALR exposure Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition ATP decrease Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) vinorelbine ( VR )

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and ATP decrease Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition HMBG1 exodus Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) vinorelbine ( VR )

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and HMBG1 exodus Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition biological ICD score ( F ) previously measured expressed as z - scores ( except for ICD prediction score ) Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and biological ICD score (F) previously measured and expressed as z-scores (except for ICD prediction score)  Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . For assessing translation , were pre - treated with library for 12 h followed by 30 min in methionine free - medium , before addition of azidohomoalanine ( AHA ) . of inhibition was calculated transformed as z - scores . correlations between transcription translation inhibitions Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. For assessing translation, cells were pre-treated with the library for 12 h followed by 30 min in methionine free-medium, before addition of azidohomoalanine (AHA). Percentage of inhibition was calculated and transformed as z-scores. The correlations between transcription and translation inhibitions Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


inhibition of transcription was assessed for negative positive ICD hits identified with predictive algorithm ( Figure 1 ) . U2OS were treated with agents at concentrations corresponding to their IC60 : 1 μM dactinomycin ( DACT ) , 50 μM topotecan , 1 μM becatecarin , 0 . 5 μM trabectedin , 5 μM UCN - 01 , 30 μM mycophenolate mofetil , 30 μM nonoxynol - 9 , 25 μM dactolisib , 2 . 5 μM β - lapachone , 5 μM 5 - fluorodeoxycytidine 2 μM RH - 1 for 1 . 5 h followed by 1 h with EU . of transcription inhibition was calculated coefficient of correlation ( R ) associated p - value ( p ) between of inhibition theoretical ICD score was calculated using Pearson method .

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

The inhibition of transcription was assessed for the negative and positive ICD hits identified with the predictive algorithm (Figure 1). U2OS cells were treated with the agents at concentrations corresponding to their IC60: 1 μM dactinomycin (DACT), 50 μM topotecan, 1 μM becatecarin, 0.5 μM trabectedin, 5 μM UCN-01, 30 μM mycophenolate mofetil, 30 μM nonoxynol-9, 25 μM dactolisib, 2.5 μM β-lapachone, 5 μM 5-fluorodeoxycytidine and 2 μM RH-1 for 1.5 h followed by 1 h with EU. The percentage of transcription inhibition was calculated and the coefficient of correlation (R) and associated p-value (p) between the percentage of inhibition and the theoretical ICD score was calculated using the Pearson method.


Jurkat T were transfected with control , FIP3 . 1 or FIP3 . 2 siRNA oligonucleotides . A : Intracellular distribution of endogenous Rab11 Rac1 was assessed by immunofluorescence . A , E , G : Three - dimensional ( 3D ) confocal were post - treated by deconvolution . A 0 . 4 - µ - thick medial stack is shown . pericentrosomal vesicular compartment is zoomed at bottom right - corner . B , F , H : Population of co - localization volume between Rab11 Rac1 within percentriolar vesicular compartment . Each dot represents one . Horizontal bars represent mean ± , Mann - Whitney was used . are representative of three experiments . Bar , 5

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

Jurkat T cells were transfected with control, FIP3.1 or FIP3.2 siRNA oligonucleotides. A: Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence.A, E, G: Three-dimensional (3D) confocal images were post-treated by deconvolution. A 0.4-µ-thick medial stack is shown. The pericentrosomal vesicular compartment is zoomed at the bottom right-hand corner.B, F, H: Population analysis of the co-localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment. Each dot represents one cell. Horizontal bars represent the mean ± SEM, Mann-Whitney test was used. Images are representative of three experiments. Bar, 5 µm


A - C : JurkatT were transfected with control , FIP3 . 1 or FIP3 . 2 siRNA oligonucleotides . A : Intracellular distribution of endogenous Rab11 Rac1 was assessed by immunofluorescence .

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A-C: JurkatT cells were transfected with control, FIP3.1 or FIP3.2 siRNA oligonucleotides. A: Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence.


C : FIP3 expression was analyzed by Western blot . of bands was performed with ImageJ normalized to a non - specific band of 35 kDa used as loading control .

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C: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control.


E , F : Jurkat were transfected with expression vectors encoding GFP , GFP - FIP3 - WT or GFP - FIP3 - I738E . Endogenous Rac1 was detected by immunofluorescence .

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E, F: Jurkat cells were transfected with expression vectors encoding GFP, GFP-FIP3-WT or GFP-FIP3-I738E. Endogenous Rac1 was detected by immunofluorescence.