( 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 ) .

O O O B-CELL_TYPE O B-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-ORGANISM O O O O

(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 ) .

O O O O B-GENEPROD B-CELL_TYPE O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O

(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 .

O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O

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 .

O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O

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 .

O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O

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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O

(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 .

O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O

(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 .

O O O O O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

(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 .

O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O

(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 .

O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

(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 .

O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

(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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O

(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 .

O O O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O B-GENEPROD B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O 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-ORGANISM O

(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 .

O O O O O B-CELL_LINE B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O

(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 .

O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O

(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 .

O O O B-CELL_TYPE O B-GENEPROD B-ORGANISM O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O B-ORGANISM O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O

(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 .

O O O B-CELL_TYPE O O O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O

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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O

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 .

O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD B-CELL_TYPE B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) 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

B-ORGANISM B-DISEASE B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM B-DISEASE B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-ORGANISM B-DISEASE B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O

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

B-ORGANISM B-DISEASE B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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

B-ORGANISM B-DISEASE B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O

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

B-ORGANISM B-DISEASE B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O 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-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O

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 .

B-ORGANISM B-DISEASE B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

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 .

B-ORGANISM B-DISEASE B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O

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

B-ORGANISM B-DISEASE B-CELL_LINE O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O

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

B-CELL_LINE O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O

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

B-CELL_LINE B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE 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

B-ORGANISM B-DISEASE B-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_LINE O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE B-GENEPROD O O O B-GENEPROD O O

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 .

B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-CELL_LINE O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O O O O O O O O

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

O O O B-ORGANISM B-DISEASE B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-ORGANISM O

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

O O O B-ORGANISM B-DISEASE B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

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

O O O B-ORGANISM B-DISEASE B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE 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. 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 B-ORGANISM B-DISEASE B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O 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-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE

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 B-ORGANISM B-DISEASE B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD

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 B-ORGANISM B-DISEASE B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY 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 ) .

B-ORGANISM O B-CELL_LINE I-CELL_LINE B-DISEASE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O O B-CELL_LINE I-CELL_LINE B-DISEASE O O O O B-ORGANISM O O O O B-GENEPROD O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O 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-GENEPROD O O O O O O B-EXP_ASSAY O B-ORGANISM O O 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

B-ORGANISM B-DISEASE B-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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

B-ORGANISM B-DISEASE B-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O 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-EXP_ASSAY O O B-ORGANISM O O B-GENEPROD O O O O O O O O B-ORGANISM B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O B-ORGANISM B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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

B-ORGANISM B-DISEASE B-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O 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-EXP_ASSAY O O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-SMALL_MOLECULE

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 )

B-CELL_LINE O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 )

B-CELL_LINE O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 )

B-CELL_LINE O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 )

B-CELL_LINE O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 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 )

B-CELL_LINE O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 )

B-CELL_LINE O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 .

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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

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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.


G : Jurkat were transfected with expression vectors encoding GFP , Rab11WT - GFP , or Rab11Q70L - GFP ( constitutively active ) or Rab11S25N - GFP ( dominant negative ) mutants . Endogenous Rac1 was detected 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 B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O 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: Jurkat cells were transfected with expression vectors encoding GFP, Rab11WT-GFP, or Rab11Q70L-GFP (constitutively active) or Rab11S25N-GFP (dominant negative) mutants. Endogenous Rac1 was detected 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 , B : Effect of FIP3 silencing on Rac1 expression . Jurkat were transfected with control , FIP3 . 1 or FIP3 . 2 siRNA . Three days later were lysed expression of FIP3 , Rac1 β - tubulin analyzed by western blot . of bands corresponding to FIP3 to Rac1 was quantified by ImageJ normalized to β - tubulin band used as loading control . mean ± SD of of band in silenced with respect to controls of three independent experiments is shown .

O O O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

A, B: Effect of FIP3 silencing on Rac1 expression. Jurkat cells were transfected with control, FIP3.1 or FIP3.2 siRNA. Three days later cells were lysed and the expression of FIP3, Rac1 and β-tubulin analyzed by western blot. The intensity of bands corresponding to FIP3 and to Rac1 was quantified by ImageJ and normalized to the β-tubulin band used as loading control. The mean ± SD of the percentage of band intensity in silenced cells with respect to controls of three independent experiments is shown.


B : Rac1 interaction with FIP3 : HEK293T were transfected with GFP , GFP - FIP3 WT or GFP - FIP3I738E expression vectors . 24 h later , were lysed immunoprecipitated with anti - GFP Ab . Immunoprecipitates were analyzed by electrophoresis western blot using anti - GFP Ab ( top panel ) or anti - Rac1 Ab ( bottom panel ) . Total lysates were analyzed ( 100 , 000 ) . position of Rac1 ( 20 kDa ) Ig chain ( 25 kDa ) polypeptides is depicted on right side . Arrowheads at bottom point bands corresponding to Rac . of bands was performed with ImageJ as of with respect to total lysate bands on left , shown below . A representative experiment out of three carried out is shown .

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B: Rac1 interaction with FIP3: HEK293T cells were transfected with GFP, GFP-FIP3 WT or GFP-FIP3I738E expression vectors. 24 h later, cells were lysed and immunoprecipitated with anti-GFP Ab. Immunoprecipitates were analyzed by electrophoresis and western blot using anti-GFP Ab (top panel) or anti-Rac1 Ab (bottom panel). Total cell lysates were analyzed (100,000 cells). The position of Rac1 (20 kDa) and Ig light chain (25 kDa) polypeptides is depicted on the right side. Arrowheads at the bottom point the bands corresponding to Rac. Quantification of bands was performed with ImageJ as the percentage of intensity with respect to the total lysate bands on the left, shown below. A representative experiment out of three carried out is shown.


JurkatT were transfected with expression vectors encoding : A , C : GFP , GFP - FIP3WT , or GFP - FIP3I738E . B , C : Rab11WT - GFP , Rab11Q70L - GFP ( constitutively active ) , or Rab11S25N - GFP ( dominant negative ) .

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JurkatT cells were transfected with expression vectors encoding: A, C: GFP, GFP-FIP3WT, or GFP-FIP3I738E. B, C: Rab11WT-GFP, Rab11Q70L-GFP (constitutively active), or Rab11S25N-GFP (dominant negative).


C : Rab11WT - GFP , Rab11Q70L - GFP ( constitutively active ) , or Rab11S25N - GFP ( dominant negative ) .

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C: Rab11WT-GFP, Rab11Q70L-GFP (constitutively active), or Rab11S25N-GFP (dominant negative).


D , E : or transfected with control , or FIP3 . 1 siRNA . were allowed to form immunological synapses with SEE - pulsed Raji for 30 min . Intracellular distribution of endogenous Rac1 surface distribution of CD3 were detected by immunofluorescence . 3D confocalimages were post - treated by deconvolution . A 0 . 4 - µm - thick medial stack is shown . C , E : Population analyses of amount of Rac1 at immunological synapse relative to total Rac1 . Each dot corresponds to one . Horizontal bars represent mean ± . Mann - Whitney . are representative of three experiments . Bar , 5 µm .

O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O 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-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E: or transfected with control, or FIP3.1 siRNA. Cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min. Intracellular distribution of endogenous Rac1 and surface distribution of CD3 were detected by immunofluorescence. 3D confocalimages were post-treated by deconvolution. A 0.4-µm-thick medial stack is shown. C, E: Population analyses of the amount of Rac1 at the immunological synapse relative to the total cellular Rac1. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.


JurkatT were transfected with either control or FIP3 . 1 siRNA . A : were allowed to form immunological synapses with SEE - pulsed Raji for 30 min endogenous Lck was detected by immunofluorescence . 3D confocalimages were post - treated by deconvolution . A 0 . 4 - µm - thick medial stack is shown .

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JurkatT cells were transfected with either control or FIP3.1 siRNA. A: Cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min and endogenous Lck was detected by immunofluorescence. 3D confocalimages were post-treated by deconvolution. A 0.4-µm-thick medial stack is shown.


B , C : minimal angle formed by axis connecting synapse edges axis connecting mass centers of T APC nuclei was measured , as schematized in B .

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B, C: The minimal angle formed by the axis connecting the synapse edges and the axis connecting the mass centers of the T cell and the APC nuclei was measured, as schematized in B.


D : Transfected Jurkat T were allowed to spread on anti - CD3 - coated coverslips for indicated time , F - actin was detected with FITC - Phalloidin . A single optical section at level of contact surface is shown . E , F : Population analyses of spreading of at level of contact surface . Each dot corresponds to one . Horizontal bars represent mean ± . Mann - Whitney . are representative of three experiments . Bar , 5 µm .

O O O B-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D: Transfected Jurkat T cells were allowed to spread on anti-CD3-coated coverslips for the indicated time, and F-actin was detected with FITC-Phalloidin. A single optical section at the level of the contact surface is shown. E, F: Population analyses of the spreading area of cells at the level of the contact surface. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.


G : 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 .

G: 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.


A , B , C : JurkatT transfected with siRNA control , FIP3 . 1 or FIP3 . 2 were allowed to spread on poly - lysine - coated coverslips for indicated times . Intracellular actin was detected with FITC - coupled Phalloidin . A : A single optical section at level of contact surface is shown . Bar , 5 µm . B : Population analyses of spreading of at level of contact surface . Each dot represents one . Horizontal bars represent mean ± . C : Time course representation of B , showing mean ± .

O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B, C: JurkatT cells transfected with siRNA control, FIP3.1 or FIP3.2 were allowed to spread on poly-lysine-coated coverslips for the indicated times. Intracellular actin was detected with FITC-coupled Phalloidin. A: A single optical section at the level of the contact surface is shown. Bar, 5 µm. B: Population analyses of the spreading area of cells at the level of the contact surface. Each dot represents one cell. Horizontal bars represent the mean ± SEM. C:Time course representation of B, showing mean ± SEM.


D - F : humanCD4T were transfected with control or FIP3 . 1 siRNA . D , E : were allowed to spread on poly - lysine - coated coverslips for 5 min . Intracellular Lck was detected by immunofluoresecence . D : A single optical section at level of contact surface is shown . Bar , 10 µm . E : Population analyses of spreading of at level of contact surface .

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D-F: Primary humanCD4T cells were transfected with control or FIP3.1 siRNA. D, E: Cells were allowed to spread on poly-lysine-coated coverslips for 5 min. Intracellular Lck was detected by immunofluoresecence. D: A single optical section at the level of the contact surface is shown. Bar, 10 µm. E: Population analyses of the spreading area of cells at the level of the contact surface.


F : were stained with CMFDA dye , suspended in a paraformaldehyde solution submitted to centrifugation at 3 , 724 xg onto poly - lysine - coated coverslips . Confocal optical sections separated by 0 . 2 µm were acquired . X - Z were built with ImageJ . maximal sections in ' x ' ( width ) ' z ' ( thickness ) were determined for each ( upper panel ) . Plots represent deformability index ( x / z ) . Each dot corresponds to one . Horizontal bars represent mean ± . Mann - Whitney . are representative of three experiments .

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F: Cells were stained with CMFDA dye, suspended in a paraformaldehyde solution and submitted to centrifugation at 3,724 xg onto poly-lysine-coated coverslips. Confocal optical sections separated by 0.2 µm were acquired. X-Z images were built with ImageJ. The maximal sections in 'x' (width) and 'z' (thickness) were determined for each cell (upper panel). Plots represent the deformability index (x/z). Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments.


Jurkat T were transfected with control or FIP3 . 1 siRNA , then transfected again with empty vector or with expression vectors encoding Myc - tagged - Rac1WT , - Rac1G12V ( constitutively active mutant ) , or - Rac1Rac1T17N ( dominant negative mutant ) . were allowed to spread on poly - lysine - coated coverslips for indicated time spreading of measured . A - C , H : Population analyses of spreading of at level of contact surface . A - C : Time course plots representing mean ± of data shown as in I . D - G : for nucleus ( blue = Dapi ) , F - actin ( green = phalloidin ) Rac1 - Myc ( red = anti - Myc ) is shown . A single optical section at level of contact surface is shown . Bar , 5 µm .

B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD 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 or FIP3.1 siRNA, then transfected again with empty vector or with expression vectors encoding Myc-tagged-Rac1WT, -Rac1G12V (constitutively active mutant), or -Rac1Rac1T17N (dominant negative mutant). Cells were allowed to spread on poly-lysine-coated coverslips for the indicated time and the spreading area of cells measured. A-C, H: Population analyses of the spreading area of cells at the level of the contact surface. A-C: Time course plots representing the mean ± SEM of the data shown as in I. D-G: Staining for nucleus (blue = Dapi), F-actin (green = phalloidin) and Rac1-Myc (red = anti-Myc) is shown. A single optical section at the level of the contact surface is shown. Bar, 5 µm.


H : Dot plots corresponding to measurements at 15 min of spreading : Each dot represents one . Horizontal bars represent mean ± . Mann - Whitney . Data are representative of three experiments .

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H: Dot plots corresponding to measurements at 15 min of spreading: Each dot represents one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Data are representative of three experiments.


A : Jurkat T transfected with control or FIP3 . 1 were treated 1 h with Rac1 inhibitor NSC23766 at 100 µM allowed to spread on poly - lysine - coated coverslips for 15 min . F - actin was detected with FITC - Phalloidin spreading of at contact surface was measured .

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A: Jurkat T cells transfected with control or FIP3.1 were treated 1 h with the Rac1 inhibitor NSC23766 at 100 µM and allowed to spread on poly-lysine-coated coverslips for 15 min. F-actin was detected with FITC-Phalloidin and the spreading area of cells at the contact surface was measured.


B : Transfected were allowed to form immunological synapses with SEE - pulsed Raji for 30 min intracellular distribution of Lck was detected by immunofluorescence . Synapse angle was measured as in figure 4 . Each dot corresponds to one . Horizontal bars represent mean ± . Mann - Whitney .

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B: Transfected cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min and intracellular distribution of Lck was detected by immunofluorescence. Synapse angle was measured as in figure 4. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test.


A : Jurkat T transfected with control or FIP3 . 1 were activated on anti - CD3 - coated plastic plates in presence of soluble anti - CD28 during 16 h . of IL2 released in supernatant was measured by ELISA . Histograms represent mean ± SD of three experiments . Mann - Whitney .

O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

A: Jurkat T cells transfected with control or FIP3.1 were activated on anti-CD3-coated plastic plates in the presence of soluble anti-CD28 during 16 h. Concentration of IL2 released in the supernatant was measured by ELISA. Histograms represent the mean ± SD of three experiments. Mann-Whitney test.


B : T were activated with soluble anti - CD3 Erk1 / 2 phosphorylation was analyzed by Western blot . Graph represents of each band with respect to that of transfected with control siRNA left non - stimulated ( t = 0 ) . each band from Western blot was normalized with respect to of β - actin for each time point . Representative experiment out of three performed .

O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

B: T cells were activated with soluble anti-CD3 and Erk1/2 phosphorylation was analyzed by Western blot. Graph represents the percentage of each band intensity with respect to that of cells transfected with control siRNA and left non-stimulated (t=0). The intensity each band from Western blot was normalized with respect to the intensity of β-actin for each time point. Representative experiment out of three performed.


A . Representative of of PCIF1 in three CRC tissues adjacent noncancerous tissues . Scale bar , 50 μm ( left ) . of PCIF1 expression in normal CRC tissues . Each dot represents one sample ( right ) . Data are mean ± . * * P < 0 . 01 , by Student ' s t - .

O O O O O B-GENEPROD O O B-DISEASE O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative images of IHC staining of PCIF1 in three CRC tissues and adjacent noncancerous tissues. Scale bar, 50 μm (left). Analysis of the PCIF1 protein expression in normal and CRC tissues. Each dot represents one sample (right). Data are the mean ± SEM. **P < 0.01, by Student's t-test.


B . Kaplan - Meier plots of overall survival of 27 CRC with low PCIF1 expression ( 0 - 4 . 5 score , n = 18 ) or high PCIF1 expression ( 4 . 6 - 9 . 0 score , n = 9 ) . Data are mean ± . * * * P < 0 . 001 by Student ' s t - .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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. Kaplan-Meier plots of overall survival of 27 CRC patients with low tumor PCIF1 expression (0-4.5 staining score, n = 18) or high tumor PCIF1 expression (4.6-9.0 staining score, n = 9). Data are the mean ± SEM. ***P < 0.001 by Student's t-test.


C . Western blot of PCIF1 in normal colon ( CCD841CON ) various CRC lines . GAPDH served as a loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE O B-CELL_LINE O O B-DISEASE O O B-GENEPROD O O O O O O

C. Western blot analysis of PCIF1 in normal colon cells (CCD841CON) and various CRC cell lines. GAPDH served as a loading control.


D . qRT - PCR of PCIF1 mRNA in indicated lines . were normalized to GAPDH expression . Data are mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. qRT-PCR analysis of PCIF1 mRNA in the indicated cell lines. Levels were normalized to GAPDH expression. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


E . Representative immunofluorescence of HT29 HCT116 stained for endogenous PCIF1 ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm .

O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE B-EXP_ASSAY O O B-GENEPROD O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

E. Representative immunofluorescence images of HT29 and HCT116 cells stained for endogenous PCIF1 protein (green). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.


F . Western blot of PCIF1 in whole extract ( W ) , ( C ) , ( N ) fractions of HCT116 HT29 . Tubulin lamin A / C served as markers , respectively .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

F. Western blot analysis of PCIF1 in whole extract (W), cytosolic (C), and nuclear (N) fractions of HCT116 and HT29 cells. Tubulin and lamin A/C served as cytosolic and nuclear markers, respectively.


A . Western blot of PCIF1 in HT29 HCT116 depleted of PCIF1 using four different sgRNAs . GAPDH served as a loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O B-GENEPROD O O O O O O

A. Western blot analysis of PCIF1 in HT29 and HCT116 cells depleted of PCIF1 using four different sgRNAs. GAPDH served as a loading control.


Matrigel invasion ( B ) , of indicated control PCIF1 - depleted CRC lines . Representative of are shown at left right , respectively , of each panel . Data are mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Matrigel invasion assay (B), of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


fibronectin adhesion ( C ) , of indicated control PCIF1 - depleted CRC lines . Representative of are shown at left right , respectively , of each panel . Data are mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

fibronectin adhesion assay (C), of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


colony formation ( D ) of indicated control PCIF1 - depleted CRC lines . Representative of are shown at left right , respectively , of each panel . Data are mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

colony formation assay (D) of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.