Figure 3A - F The indicated cells were transfected with siCon , siH19 , empty vector , or pH19 . RNA , cell viability ( as indicated by viable cell numbers ) , and caspase activity ( as a readout for apoptosis ) were analyzed 48 h post - transfection . Combined results from two patient cells in each group are presented . G E2 stimulates H19 expression in endometrial stromal cells . Endometrial stromal cells # 166 and # 98 were stimulated with E2 ( + ) or vehicle ( − ) for 48 h , followed by RT - qPCR analysis of RNA extracted from the cells . Results combining two patient cells are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-F The indicated cells were transfected with siCon, siH19, empty vector, or pH19. RNA, cell viability (as indicated by viable cell numbers), and caspase activity (as a readout for apoptosis) were analyzed 48 h post-transfection. Combined results from two patient cells in each group are presented.G E2 stimulates H19 expression in endometrial stromal cells. Endometrial stromal cells #166 and #98 were stimulated with E2 (+) or vehicle (−) for 48 h, followed by RT-qPCR analysis of RNA extracted from the cells. Results combining two patient cells are shown.


Figure 2 ( A ) Examples of chromosome arms exhibiting features of telomeric semiconservative ( Semi ) replication , conservative ( Consrv ) replication of the entire telomere ( E ) and conservative replication of part of the telomere ( P ) . Idealized chromosome diagrams ( left ) and actual microscopy images ( right ) are shown . D - FISH , denaturing FISH ; ND - CO - FISH ( non - denaturing CO - FISH ) . ( B ) Percentages of chromosome arms exhibiting conservative ( Consrv ) replication of the entire telomere ( E ) , conservative replication of part of the telomere ( P ) and conservative replication of either the entire telomere of part of it ( E + P ) . NE , U2OS cells expressing normal levels of cyclin E ; OE , U2OS cells overexpressing cyclin E for four days . Bars represent means and standard errors of the mean from three independent experiments . Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres : P < 0 . 025 for Consrv ( P ) and P < 0 . 05 for Consrv ( E + P ) , as calculated using the paired t test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D-FISH, denaturing FISH; ND-CO-FISH (non-denaturing CO-FISH).(B) Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P) and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P<0.025 for Consrv (P) and P<0.05 for Consrv (E+P), as calculated using the paired t test.


Figure 3 ( A ) Decrease in the fraction of chromosome arms exhibiting conservative ( Consrv ) DNA replication of the entire telomere length or part of the telomere ( E + P ) in U2OS cells after depletion of POLD3 or POLD4 by siRNA . si Ctrl , control siRNA ; NE , cells expressing normal levels of cyclin E ; OE , cells overexpressing cyclin E for four days . Bars represent means and standard errors of the mean from two independent experiments . For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells . PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P = 0 . 072 ; whereas for PolD4 depletion the decrease was significant at a level of P < 0 . 025 , as calculated using the paired t test . ( B ) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E ( NE ) or overexpressing cyclin E ( OE ) . The cells were transfected with control siRNA ( si Ctrl ) or siRNAs targeting POLD3 or POLD4 and , five days later , fixed and examined for telomere length by Q - FISH . Bars indicate means and standard error of the mean from more than 2 , 600 telomeres examined per condition . The differences between control and PolD3 or PolD4 - depleted cells were significant ( P < 10 - 6 ) for both NE and OE cells , as determined by unpaired t tests . ( C ) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells . The cells were transfected with control siRNA ( si Ctrl ) or siRNAs targeting POLD3 or POLD4 and , five days later , fixed and examined for telomere length by Q - FISH . Bars indicate means and standard error of the mean from more than 2 , 800 telomeres examined per condition . The differences between control and PolD3 or PolD4 - depleted cells were significant ( P < 10 - 6 ) , as determined by unpaired t tests . ( D ) Increased frequency of chromosome end - to - end fusions following POLD3 or POLD4 depletion . U2OS cells expressing normal levels of cyclin E ( NE ) or overexpressing cyclin E for four days ( OE ) were transfected with control siRNA ( siCtrl ) os siRNAs targeting POLD3 or POLD4 . For each condition , 25 metaphases were examined ( representing about 1 , 900 high quality chromosome arms per condition ) ; the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean . For statistical analysis , the number of chromosome end - to - end fusions were compared by chi square tests among the various conditions . Cyclin E overexpression led to a higher number of fusions ( P < 0 . 0005 ) ; PolD3 and PolD4 depletion also led to a higher number of fusions ( P < 0 . 001 and P < 0 . 025 , respectively ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Decrease in the fraction of chromosome arms exhibiting conservative (Consrv) DNA replication of the entire telomere length or part of the telomere (E+P) in U2OS cells after depletion of POLD3 or POLD4 by siRNA. si Ctrl, control siRNA; NE, cells expressing normal levels of cyclin E; OE, cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from two independent experiments. For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells. PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P=0.072; whereas for PolD4 depletion the decrease was significant at a level of P<0.025, as calculated using the paired t test.(B) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E (OE). The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,600 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6) for both NE and OE cells, as determined by unpaired t tests.(C) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells. The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,800 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6), as determined by unpaired t tests.(D) Increased frequency of chromosome end-to-end fusions following POLD3 or POLD4 depletion. U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E for four days (OE) were transfected with control siRNA (siCtrl) os siRNAs targeting POLD3 or POLD4. For each condition, 25 metaphases were examined (representing about 1,900 high quality chromosome arms per condition); the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean. For statistical analysis, the number of chromosome end-to-end fusions were compared by chi square tests among the various conditions. Cyclin E overexpression led to a higher number of fusions (P<0.0005); PolD3 and PolD4 depletion also led to a higher number of fusions (P<0.001 and P<0.025, respectively).


Figure 1 ( A Total PD - L1 , PARP from colon ( Colo - 205 ) , lung ( A549 ) , pancreatic ( PANK1 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 GAPDH is loading controlTotal PD - L1 , PARP from triple negative breast cancer cell ( MDA - MB - 436 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 , BaCa , GAPDH is loading controlC ) Total PD - L1 , PARP , cleaved caspase - 3 from ovarian cell ( Cavo - 3 ) lysates treated with indicated DR5 agonist antibodies named tigatuzumab . GAPDH is loading control ( D ) Total PD - L1 , CD47 , Calreticulin , PARP from MDA - MB - 436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z - VAD . GAPDH is loading control . ( E ) Total PD - L1 western blotting signal from the lysates of OVCAR - 3 ( n = 3 ) and MDA - MB - 436 ( n = 3 ) cells after 6 hrs treatment of 100nM lexa ( DR5 agonist antibody ) was normalized to GAPDH . ( F ) Surface biotinylation of PD - L1 from indicated tumor cells after indicated DR5 agonist treatments . Cleaved caspase - 3 and PARP indicates activation of DR5 signaling . In lane 3 and 4 , KMTR2 was pre - neutralized either with recombinant DR5 ( rDR5 ) or recombinant FOLR1 ( rFOLR1 ) . ( G ) Representative flow cytometry plots of PD - L1 from two different tumor cell lines treated with indicated DR5 antibodies . Secondary alone and IgG1 control are included . ( H ) Relative surface PD - L1 % cells , after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean ( n = 3 - 4 ) . ( J ) Relative surface PD - L1 % cells in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated tumors and horizontal line in samples indicated mean ( n = 2 - 6 ) ( K ) ER ( - ) , PR ( - ) and HER2 ( - ) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD . Cg - Prkdcscid Il2rgtm1Wjl / SzJ mice , following by treatment with IgG1 or KMTR2 ( 50μg , 4 doses ) . Harvested tumors were analyzed for PD - L1 , CD47 and PARP in lysates . ( L ) KMTR2 treated ( 50μg , 4 doses ) UCD52 TNBC PDX tumors were stained for PD - L1 using immunohistochemistry ( IHC ) . ( M ) Total PD - L1 and CD47 blotting analysis from DR5 resistant MDA - MB - 436 cell lines after indicated DR5 agonist treatment ( 50nM , 6hr ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A Total PD-L1, PARP from colon (Colo-205), lung (A549), pancreatic (PANK1) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2 GAPDH is loading controlTotal PD-L1, PARP from triple negative breast cancer cell (MDA-MB-436) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2, BaCa, GAPDH is loading controlC) Total PD-L1, PARP, cleaved caspase-3 from ovarian cell (Cavo-3) lysates treated with indicated DR5 agonist antibodies named tigatuzumab. GAPDH is loading control(D) Total PD-L1, CD47, Calreticulin, PARP from MDA-MB-436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z-VAD. GAPDH is loading control.(E) Total PD-L1 western blotting signal from the lysates of OVCAR-3 (n=3) and MDA-MB-436 (n=3) cells after 6 hrs treatment of 100nM lexa (DR5 agonist antibody) was normalized to GAPDH.(F) Surface biotinylation of PD-L1 from indicated tumor cells after indicated DR5 agonist treatments. Cleaved caspase-3 and PARP indicates activation of DR5 signaling. In lane 3 and 4, KMTR2 was pre-neutralized either with recombinant DR5 (rDR5) or recombinant FOLR1 (rFOLR1).(G) Representative flow cytometry plots of PD-L1 from two different tumor cell lines treated with indicated DR5 antibodies. Secondary alone and IgG1 control are included.(H) Relative surface PD-L1 % cells, after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean (n=3-4).(J) Relative surface PD-L1 % cells in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated tumors and horizontal line in samples indicated mean (n=2-6)(K) ER (-), PR (-) and HER2 (-) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, following by treatment with IgG1 or KMTR2 (50μg, 4 doses). Harvested tumors were analyzed for PD-L1, CD47 and PARP in lysates.(L) KMTR2 treated (50μg, 4 doses) UCD52 TNBC PDX tumors were stained for PD-L1 using immunohistochemistry (IHC).(M) Total PD-L1 and CD47 blotting analysis from DR5 resistant MDA-MB-436 cell lines after indicated DR5 agonist treatment (50nM, 6hr).


Figure 2 ( C ) Luciferase activity of reporter lines from tumor cell - Jurkat cell co - culture assay using MDA - MB - 436 and OVCAR3 cells after treatment ( 50nM ) with indicated DR5 agonists ( tiga : tigatuzumab , AMG : AMG655 , KMTR1 , Lexa : Lexatumumab ) . The background luciferase signal from untreated cells ( due to basal surface PD - L1 ) was subtracted . Various controls treatments ( IgG1 , anti - PD - L1 , anti - EGFR ) are also shown ( n = 3 ) . ( D ) Same as ( C ) except tumor cells were pre - treated with indicated inhibitors for AKT , ERK , mTOR , MEK , STAT3 , PARP - i and NF - Kβ along with DR5 agonist KMTR2 antibody prior to co - culture . MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 GAPDH is loading control in EMDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 phosphorylated p65 , total p65 , cleaved caspase - 3 and PARP . Tubulin is loading control in F . MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 , phosphorylated p65 and PARP . GAPDH is loading control in G . ( H ) Flow cytometry analysis of MDA - MB - 436 cells treated with TNFα ± MG132 ( top ) and indicated DR5 agonist ± MG132 ( bottom ) . ( I ) Total PD - L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132 . ( J ) Flow cytometry surface PD - L1 analysis of OVCAR - 3 cells treated with indicated DR5 agonist ± MG132 . ( K ) MDA - MB - 436 cells were treated with MG132 for indicated times and total PD - L1 from lysates was analyzed . GAPDH is loading control . ( L ) Immunoblotting of DR5 confirming generation of knock out ( DR5 - KO ) cell lines . ( M ) Cell viability analysis of DR5 - KO and DR5 - WT cells ( n = 3 ) . ( N ) DR5 - KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD - L1 specific antibodies . ( O ) TNBC WT and DR5 - KO ( MDA - MB - 436 ) cells were treated with either DR5 agonist or TNFα as indicated . Lysates were analyzed for PD - L1 , S5a and DR5 . GAPDH is loading control . ( P ) OVCAR - 3 and MDA - MB - 436 ( WT and DR5 - KO ) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates . GAPDH is loading control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) Luciferase activity of reporter lines from tumor cell-Jurkat cell co-culture assay using MDA-MB-436 and OVCAR3 cells after treatment (50nM) with indicated DR5 agonists (tiga: tigatuzumab, AMG: AMG655, KMTR1, Lexa: Lexatumumab). The background luciferase signal from untreated cells (due to basal surface PD-L1) was subtracted. Various controls treatments (IgG1, anti-PD-L1, anti-EGFR) are also shown (n=3). (D) Same as (C) except tumor cells were pre-treated with indicated inhibitors for AKT, ERK, mTOR, MEK, STAT3, PARP-i and NF-Kβ along with DR5 agonist KMTR2 antibody prior to co-culture. MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5 GAPDH is loading control in EMDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1 phosphorylated p65, total p65, cleaved caspase-3 and PARP. Tubulin is loading control in F.MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5, phosphorylated p65 and PARP. GAPDH is loading control in G.(H) Flow cytometry analysis of MDA-MB-436 cells treated with TNFα ± MG132 (top) and indicated DR5 agonist ± MG132 (bottom).(I) Total PD-L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132.(J) Flow cytometry surface PD-L1 analysis of OVCAR-3 cells treated with indicated DR5 agonist ± MG132.(K) MDA-MB-436 cells were treated with MG132 for indicated times and total PD-L1 from lysates was analyzed. GAPDH is loading control.(L) Immunoblotting of DR5 confirming generation of knock out (DR5-KO) cell lines.(M) Cell viability analysis of DR5-KO and DR5-WT cells (n=3).(N) DR5-KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD-L1 specific antibodies.(O) TNBC WT and DR5-KO (MDA-MB-436) cells were treated with either DR5 agonist or TNFα as indicated. Lysates were analyzed for PD-L1, S5a and DR5. GAPDH is loading control.(P) OVCAR-3 and MDA-MB-436 (WT and DR5-KO) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates. GAPDH is loading control.


Figure 3ApoEVs isolated after IgG1 and KMTR2 treatment ( OVCAR - 3 ) were blotted against CD63 and PD - L1 in dot blots . ApoEVs isolated after IgG1 and tigatuzumab treatment ( MDA - MB - 436 ) were run on western and blotted against CD63 and PD - L1 . ApoEVs isolated after IgG1 and lexatumumab ( D ) treatment ( MDA - MB - 436 ) were run on western and blotted against CD63 and PD - L1 . ( F ) ApoEVs isolated from DR5 sensitive tumor cells grown in Met - HPG + media were added on to DR5 - KO cells ( growing in regular media ) . After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye . HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met - HPG + ( DR5 - WT ) to DR5 - KO cells . ( G ) ApoEVs isolated from DR5 - WT OVCAR - 3 cells ( KMTR2 treated ) were added on to DR5 - KO ( MDA - MB - 231 ) cells for indicated times ( 24 - 96 hrs ) to analyze PD - L1 transfer kinetics from ApoEVsafter addition of MDA - MB - 436 derived ApoEVs ( treated with combination of KMTR2 + lexatumumab DR5 agonists or IgG1 ) on to DR5 - KO MDA - MB - 231 cells , total lysates were immunoblotted for PD - L1 after 24 hrs . KMTR2 + lexatumumab ( n = 3 ) , IgG1 ( n = 2 ) . ( I ) Left : PD - L1 surface histogram shows direct antibody treatment on DR5 - KO cells . Right : PD - L1 surface histogram from DR5 - KO cells after treatment ( 24 hrs ) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment . ApoEVs treated tumor cells were analyzed in PD - 1 reporter co - culture assays for luciferase signal ( K ) MDA - MB - 436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times . - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor . Lysates were immunoblotted for cleaved caspase - 3 , 8 , PARP , PD - L1 , S5a , ROCK1 and GAPDH ( n = 3 ) . u . c : uncleaved , c : cleaved , n . s : non - specific band ( L ) Relative caspase - 8 and caspase - 3 activity assays caspase - 8 maintained steady activity , while caspase - 3 activity required at least 40 ( + ) mins of DR5 agonist treatment without ZDEVD ( n = 3 ) . ( M ) After indicated treatments with KMTR2 ( DR5 agonist ) and ZDEVD ( caspase - 3 preferred inhibitor ) , cells were allowed to grow 24 hrs , followed by cell viability analysis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3ApoEVs isolated after IgG1 and KMTR2 treatment (OVCAR-3) were blotted against CD63 and PD-L1 in dot blots.ApoEVs isolated after IgG1 and tigatuzumab treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.ApoEVs isolated after IgG1 and lexatumumab (D) treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.(F) ApoEVs isolated from DR5 sensitive tumor cells grown in Met-HPG+ media were added on to DR5-KO cells (growing in regular media). After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye. HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met-HPG+ (DR5-WT) to DR5-KO cells.(G) ApoEVs isolated from DR5-WT OVCAR-3 cells (KMTR2 treated) were added on to DR5-KO (MDA-MB-231) cells for indicated times (24-96 hrs) to analyze PD-L1 transfer kinetics from ApoEVsafter addition of MDA-MB-436 derived ApoEVs (treated with combination of KMTR2+lexatumumab DR5 agonists or IgG1) on to DR5-KO MDA-MB-231 cells, total lysates were immunoblotted for PD-L1 after 24 hrs. KMTR2+lexatumumab (n=3), IgG1 (n=2).(I) Left: PD-L1 surface histogram shows direct antibody treatment on DR5-KO cells. Right: PD-L1 surface histogram from DR5-KO cells after treatment (24 hrs) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment.ApoEVs treated tumor cells were analyzed in PD-1 reporter co-culture assays for luciferase signal(K) MDA-MB-436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times. - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor. Lysates were immunoblotted for cleaved caspase-3, 8, PARP, PD-L1, S5a, ROCK1 and GAPDH (n=3). u.c: uncleaved, c: cleaved, n.s: non-specific band(L) Relative caspase-8 and caspase-3 activity assays caspase-8 maintained steady activity, while caspase-3 activity required at least 40 (+) mins of DR5 agonist treatment without ZDEVD (n=3).(M) After indicated treatments with KMTR2 (DR5 agonist) and ZDEVD (caspase-3 preferred inhibitor) , cells were allowed to grow 24 hrs, followed by cell viability analysis.


Figure 4 ( A KMTR2 , KMTR2 + Z - VAD treated MDA - MB - 436 lysates for indicated early time points were analyzed for caspase - 8 , caspase - 3 , ROCK1 PARP as indicated . B ) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase - 3 , ROCK1 , pMLC , PARP , PD - L1 and CMTM6 etc . as indicated . ( C ) OVCAR3 cells were treated with pMLC activating ionophore A23187 ( positive control ) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1 . Lysates were later analyzed for ROCK1 , pMLC , caspase - 3 and PARP with GAPDH as loading control . Blue arrows indicate cleaved and activated ROCK1 and caspase - 3 . ( D ) MDA - MB - 436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment ( 20nM ) . After 6 hours flow cytometry was used to analyze surface PD - L1 . ( GSK269 : GSK269962A , GSK429 : GSK429286A ) ( E ) MDA - MB - 436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors , error bars indicate SD ( n = 2 ) . ( GSK269 : GSK269962A , GSK429 : GSK429286A ) Cultured MDA - MB - 436 cells were treated with KMTR2 ( IgG1 - Fc ) or IgG1 control ± ROCK1i ( GSK269962 ) for 6 hrs . After 6 hrs , 500nM anti - PD - L1 avelumab ( IgG4 - Fc ) was added to the media for additional 1 hr . Cellular lysates were pulled down with anti - hu - IgG4 specific beads . ( G ) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1 , PD - L1 and CMTM6 . Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples . Blue arrows indicate cleaved and activated ROCK1 . ( H ) Immunoprecipitation assays with anti - PD - L1 ( avelumab ) using OVCAR - 3 cell lines . Blue arrow indicates cleaved and activated ROCK1 . ( J ) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors ( n = 3 ) . ( K ) Tumor regression efficacy of MD5 - 1 ( 50μg ) , GSK269962A ( < 2mg / kg ) and MD5 - 1 + GSK269962A ( 50μg + 2mg / kg ) 4T1 tumors ( n = 4 - 7 ) . 6 - 8 weeks old BALB / c mice bearing 4T1 tumors ( ~ 100mm3 ) were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . GSK269962A ( ROCK1i ) was injected directly into the tumors . Tumor volumes were quantified at indicated days by caliper measurements .

O O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A KMTR2, KMTR2+Z-VAD treated MDA-MB-436 lysates for indicated early time points were analyzed for caspase-8, caspase-3, ROCK1 PARP as indicated.B) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase-3, ROCK1, pMLC, PARP, PD-L1 and CMTM6 etc. as indicated.(C) OVCAR3 cells were treated with pMLC activating ionophore A23187 (positive control) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1. Lysates were later analyzed for ROCK1, pMLC, caspase-3 and PARP with GAPDH as loading control. Blue arrows indicate cleaved and activated ROCK1 and caspase-3.(D) MDA-MB-436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment (20nM). After 6 hours flow cytometry was used to analyze surface PD-L1. (GSK269: GSK269962A, GSK429: GSK429286A)(E) MDA-MB-436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors, error bars indicate SD (n=2). (GSK269: GSK269962A, GSK429: GSK429286A)Cultured MDA-MB-436 cells were treated with KMTR2 (IgG1-Fc) or IgG1 control ± ROCK1i (GSK269962) for 6 hrs. After 6 hrs, 500nM anti-PD-L1 avelumab (IgG4-Fc) was added to the media for additional 1 hr. Cellular lysates were pulled down with anti-hu-IgG4 specific beads. (G) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1, PD-L1 and CMTM6. Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples. Blue arrows indicate cleaved and activated ROCK1.(H) Immunoprecipitation assays with anti-PD-L1 (avelumab) using OVCAR-3 cell lines. Blue arrow indicates cleaved and activated ROCK1.(J) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors (n=3).(K) Tumor regression efficacy of MD5-1 (50μg), GSK269962A (<2mg/kg) and MD5-1+ GSK269962A (50μg + 2mg/kg) 4T1 tumors (n=4-7). 6-8 weeks old BALB/c mice bearing 4T1 tumors (~100mm3) were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). GSK269962A (ROCK1i) was injected directly into the tumors. Tumor volumes were quantified at indicated days by caliper measurements.


Figure 5 ( B - D ) FACs plots confirming expression of human DR5 ( huDR5 ) and Chi - G4S - DR5 in mouse 4T1 cells . Chi - DR5 was not expressed on cell surface ( C ) . ( E ) Cell viability analysis of huDR5 and Chi - G4S - DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 ( n = 3 ) . ( F ) Comparison of tumor growth of grafted huDR5 and Chi - G4S - DR5 stable 4T1 cells in Balb / c mice ( n = 3 ) . ( G ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 150 - 200mm3 ) were treated with 4 doses ( 100μg ) of IgG1 , lexatumumab , lexatumumab + avelumab followed by tumor recovery and surface PD - L1 analysis using flow cytometry ( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . ( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . average tumor weight is shown ( n = 4 ) . ( J - K ) Confirmation of selective CD8 + T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti - PD - L1 avelumab alone or avelumab + anti - CD8a antibody . CD4 + T - cells remained unchanged . ( L ) Average of tumor weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , ROCK1i , lexatumumab + ROCK1i and anti - CD8 + lexatumumab + ROCK1i ( n = 4 - 6 , 50μg Lexatumumab , 2mg / kg ROCK1i , 6 doses ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections . Various treatments were started when tumors were ~ 100mm3 size . ( M ) Average of tumor weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , avelumab , lexatumumab + avelumab and anti - CD8 + lexatumumab + avelumab ( n = 4 - 6 ) . ( N ) 6 - 8 weeks old C57BL / 6 bearing Chi - G4S - DR5 expressing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Various treatments were started when tumors were ~ 100mm3 size . Tumor volumes were quantified at indicated days by caliper measurements . Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . ( O ) KMTR2 ( 8 doses ) antibody used as the DR5 agonist for the experiment . ( P ) Average of tumor weights of data shown in ( O ) . ( Q ) Kaplan - Meier plot depicting the survival of syngeneic Chi - G4S - DR5 4T1 tumor bearing animals injected i . p . with 100μg of indicated antibodies such as IgG1 , KMTR2 and avelumab . Animals were injected with GSK269962A 2mg / kg ( in PBS ) directly into tumors wherever ROCK1i is indicated .

O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B-D) FACs plots confirming expression of human DR5 (huDR5) and Chi-G4S-DR5 in mouse 4T1 cells. Chi-DR5 was not expressed on cell surface (C).(E) Cell viability analysis of huDR5 and Chi-G4S-DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 (n=3).(F) Comparison of tumor growth of grafted huDR5 and Chi-G4S-DR5 stable 4T1 cells in Balb/c mice (n=3).(G) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~150-200mm3) were treated with 4 doses (100μg) of IgG1, lexatumumab, lexatumumab +avelumab followed by tumor recovery and surface PD-L1 analysis using flow cytometry(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg).(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). average tumor weight is shown (n=4).(J-K) Confirmation of selective CD8+ T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti-PD-L1 avelumab alone or avelumab+anti-CD8a antibody. CD4+ T-cells remained unchanged.(L) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, ROCK1i, lexatumumab + ROCK1i and anti-CD8+ lexatumumab + ROCK1i (n=4-6, 50μg Lexatumumab, 2mg/kg ROCK1i, 6 doses). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections. Various treatments were started when tumors were ~100mm3 size.(M) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, avelumab, lexatumumab +avelumab and anti-CD8+ lexatumumab +avelumab (n=4-6).(N) 6-8 weeks old C57BL/6 bearing Chi-G4S-DR5 expressing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Various treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). (O) KMTR2 (8 doses) antibody used as the DR5 agonist for the experiment. (P) Average of tumor weights of data shown in (O). (Q) Kaplan-Meier plot depicting the survival of syngeneic Chi-G4S-DR5 4T1 tumor bearing animals injected i.p. with 100μg of indicated antibodies such as IgG1, KMTR2 and avelumab. Animals were injected with GSK269962A 2mg/kg (in PBS) directly into tumors wherever ROCK1i is indicated.


Figure 6 ( A ) Chi - G4S - DR5 stable 4T1 tumors harboring mice were treated lexatumumab , lexatumumab + ROCK1i , and avelumab + lexatumumab and other controls as indicated . Antibodies were treated i . p at 100μg dose ( 6 total ) , ROCK1i ( in PBS ) was injected directly into tumors at 2mg / kg dose ( 6 total ) . Various treatments were started when tumors were ~ 400mm3 size . Harvested tumors were grouped together and sized matched ( 3 independent sets : n = 2 - 6 tumors in each set ) followed by TIL isolation CD8 / CD45 and CD4 / CD45 expressing cells were measured by flow cytometry . ( B ) Plots showing % of total double positive TILs ( CD8 + CD45 + + CD4 + CD45 + ) in right upper quadrant after combining 3 independent experiments . ( C ) Ratio of CD8 + CD45 + / CD4 + CD45 + isolated TILs from tumors in each indicated treatment ( n = 3 ) . Chi - G4S - DR5 stable MC38 tumors harboring mice were treated ( 6 total doses ) lexatumumab , avelumab , ROCK1i , lexatumumab + ROCKi , and avelumab + lexatumumab and IgG1 control as indicated . Harvested tumors homogenized followed by quantitation . Protein lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , Foxp3 , caspase - 3 and granzyme - b antibody . GAPDH is loading control . Red arrows indicated cleaved caspase - 3 p19 and p17 fragments . ( H ) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5 - 1 and bispecific avelu - MD5 antibody . ( I ) Balb / c mice harboring luciferase stable 4T1 tumors were i . p . injected with indicated antibodies . 50μg dose and mice were imaged after 5 doses . ( J ) 6 - 8 weeks old C57BL / 6 mice bearing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Indicated treatments were started when tumors were ~ 100mm3 size . Tumor volumes were quantified at indicated days by caliper measurements . ( K ) ~ 150 - 200mm3 size MC38 tumor bearing C57LB / 6 mice were treated with indicated MD5 - 1 , avelumab and bispecific antibodies along with control IgG1 , 6 total doses . Harvested tumors were homogenized followed by quantitation . Protein lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , caspase - 3 and granzyme - b antibody . GAPDH is loading control . Red arrows indicated cleaved caspase - 3 p19 and p17 fragments . ( L ) 6 - 8 weeks old C57BL / 6 mice were injected with MC38 cells . When tumors reached ~ 150 - 200mm3 , animals were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day . On day 18 , tumors were harvested , sized matched and pooled by treatment group , exposed to collagenase / DNase and were single cell suspensions enriched for CD8 + cells . Enriched CD8 + T - cells from various treatments were restimulated with anti - CD3 ( OKT3 ) antibody for 4 additional hours . CD8 gated cells were next analyzed for IFN - γ intracellular expression using flow cytometry . ( M ) Percentage of IFN - γ + CD8 + double positive cells from 3 independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 6(A) Chi-G4S-DR5 stable 4T1 tumors harboring mice were treated lexatumumab, lexatumumab +ROCK1i, and avelumab + lexatumumab and other controls as indicated. Antibodies were treated i.p at 100μg dose (6 total), ROCK1i (in PBS) was injected directly into tumors at 2mg/kg dose (6 total). Various treatments were started when tumors were ~400mm3 size. Harvested tumors were grouped together and sized matched (3 independent sets: n=2-6 tumors in each set) followed by TIL isolation CD8/CD45 and CD4/CD45 expressing cells were measured by flow cytometry.(B) Plots showing % of total double positive TILs (CD8+CD45+ + CD4+CD45+) in right upper quadrant after combining 3 independent experiments. (C) Ratio of CD8+CD45+/CD4+CD45+ isolated TILs from tumors in each indicated treatment (n=3).Chi-G4S-DR5 stable MC38 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, ROCK1i, lexatumumab +ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Harvested tumors homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, Foxp3, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.(H) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5-1 and bispecific avelu-MD5 antibody.(I) Balb/c mice harboring luciferase stable 4T1 tumors were i.p. injected with indicated antibodies. 50μg dose and mice were imaged after 5 doses.(J) 6-8 weeks old C57BL/6 mice bearing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Indicated treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.(K) ~150-200mm3 size MC38 tumor bearing C57LB/6 mice were treated with indicated MD5-1, avelumab and bispecific antibodies along with control IgG1, 6 total doses. Harvested tumors were homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.(L) 6-8 weeks old C57BL/6 mice were injected with MC38 cells. When tumors reached ~150-200mm3, animals were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day. On day 18, tumors were harvested, sized matched and pooled by treatment group, exposed to collagenase/DNase and were single cell suspensions enriched for CD8+ cells. Enriched CD8+ T-cells from various treatments were restimulated with anti-CD3 (OKT3) antibody for 4 additional hours. CD8 gated cells were next analyzed for IFN-γ intracellular expression using flow cytometry.(M) Percentage of IFN-γ+CD8+ double positive cells from 3 independent experiments.


Figure 1 ( E and F ) RT - PCR and western blot showing the effect of RNAi - mediated B52 depletion in S2R + cells ( E ) and in larval wing discs ( F ) .

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Figure 1(E and F) RT-PCR and western blot showing the effect of RNAi-mediated B52 depletion in S2R+ cells (E) and in larval wing discs (F).


Figure 2 ( D ) Quantification of Posterior / Total wing area in male flies of the genotypes illustrated in panel C . UAS - GFP was used as a control . Each point represents a single wing , bars represent mean with standard deviation . * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) . Flies were grown at 18 ° C .

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Figure 2(D) Quantification of Posterior/Total wing area in male flies of the genotypes illustrated in panel C. UAS-GFP was used as a control. Each point represents a single wing, bars represent mean with standard deviation. *** p-value<0.001, **** p-value<0.0001 (unpaired two-tailed t-tests). Flies were grown at 18°C.


Figure 3 ( C ) Quantification of Yki reporter genes expression upon overexpression of Yki isoforms in the wing posterior domain ( hh - Gal4 driver ) . For each target gene , relative expression between posterior and anterior domain in the wing discs was quantified by immunostaining using anti - ßgal antibody ( for ex - lacZ and diap1 - lacZ ) or direct visualization of GFP ( ban - sensor , in this case posterior domain was visualized by immunostaining against V5 - tag present in UAS - Yki transgenes ) . In the scatter dot plot , each symbol ( circle , square and triangle ) represents a single wing disc , bars represent mean with SEM . * * p - value < 0 . 01 ; * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) .

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Figure 3(C) Quantification of Yki reporter genes expression upon overexpression of Yki isoforms in the wing posterior domain (hh-Gal4 driver). For each target gene, relative expression between posterior and anterior domain in the wing discs was quantified by immunostaining using anti-ßgal antibody (for ex-lacZ and diap1-lacZ) or direct visualization of GFP (ban-sensor, in this case posterior domain was visualized by immunostaining against V5-tag present in UAS-Yki transgenes). In the scatter dot plot, each symbol (circle, square and triangle) represents a single wing disc, bars represent mean with SEM. ** p-value<0.01; **** p-value<0.0001 (unpaired two-tailed t-tests).


Figure 4 ( C ) Comparison of Yki isoforms binding capabilities by dual - luciferase co - IP . Graphs show the normalized co - IP ( NcoIP ) results for 13 proteins tested against the two Yki isoforms and the negative control ( Flag - Firefly ) . ( D ) Interaction between Sd and Tgi in absence or presence of Yki isoforms . ( E ) Interaction between Sd and Wbp2 in absence or presence of Yki isoforms . ( F ) Interaction between Tgi and Wbp2 in absence or presence of Yki isoforms . In C , D , E and F , bars represent mean and error bars represent SD , n = 4 . p - values : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( unpaired two - tailed t - tests ) . ( G ) Luciferase assay using 3xSd - Luciferase reporter in S2 cells , transfected with single or combination of Yki isoforms . Error bars represent SD , n = 6 . * p - value < 0 . 05 ; * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) . ( H ) Co - overexpression of Yki isoforms using GMR - Gal4 driver provides evidence of competition between Yki isoforms .

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Figure 4(C) Comparison of Yki isoforms binding capabilities by dual-luciferase co-IP. Graphs show the normalized co-IP (NcoIP) results for 13 proteins tested against the two Yki isoforms and the negative control (Flag-Firefly).(D) Interaction between Sd and Tgi in absence or presence of Yki isoforms. (E) Interaction between Sd and Wbp2 in absence or presence of Yki isoforms. (F) Interaction between Tgi and Wbp2 in absence or presence of Yki isoforms. In C, D, E and F, bars represent mean and error bars represent SD, n=4. p-values: *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001 (unpaired two-tailed t-tests). (G) Luciferase assay using 3xSd-Luciferase reporter in S2 cells, transfected with single or combination of Yki isoforms. Error bars represent SD, n=6. * p-value<0.05; **** p-value<0.0001 (unpaired two-tailed t-tests).(H) Co-overexpression of Yki isoforms using GMR-Gal4 driver provides evidence of competition between Yki isoforms.


Figure 5 ( C ) Comparison of posterior / total area ratio of females wings upon depletion of B52 in a yki + ( wild type ) or yki2 - only background . Each point corresponds to a single wing , bars represent mean with SD ( unpaired two - tailed t - tests : * * * * p - value < 0 . 0001 ) . ( E and F ) Quantification of sibling clones size in the wing pouch . In ( D ) , sibling wild - type clones are labelled with 2xGFP and 2xRFP . In ( E ) , unlabelled clones are homozygous for the yki2 - only - A allele whereas sibling clones ( RFP / RFP ) are wild type . In both graphs , clones pairs are sorted according to RFP / RFP clones size .

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Figure 5(C) Comparison of posterior/total area ratio of females wings upon depletion of B52 in a yki+ (wild type) or yki2-only background. Each point corresponds to a single wing, bars represent mean with SD (unpaired two-tailed t-tests: **** p-value <0.0001).(E and F) Quantification of sibling clones size in the wing pouch. In (D), sibling wild-type clones are labelled with 2xGFP and 2xRFP. In (E), unlabelled clones are homozygous for the yki2-only-A allele whereas sibling clones (RFP/RFP) are wild type. In both graphs, clones pairs are sorted according to RFP/RFP clones size.


Figure 6 ( C ) Distribution of wing size and wing asymmetry among the flies analyzed in panel B . Each circle represents the area of a single wing . Each wing was measured in duplicate . The difference between Right ( orange ) and Left ( blue ) sides of a single fly is schematized by a black line .

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Figure 6(C) Distribution of wing size and wing asymmetry among the flies analyzed in panel B. Each circle represents the area of a single wing. Each wing was measured in duplicate. The difference between Right (orange) and Left (blue) sides of a single fly is schematized by a black line.


Figure 2 ( A - C ) qRT - PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages ( A ) , THP1 cells ( B ) and human peripheral blood mononuclear cells ( PBMC ) ( C ) that were left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib ( 0 . 1 , 1 , 10 μM ) . The data are presented as fold induction of Ifnb1 / Gapdh transcripts . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by one - way ANOVA followed by Dunnett ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 . ( D ) MTS detection of the viability of mouse peritoneal macrophages ( PM ) , THP1 and PBMC cells left untreated ( DMSO ) or treated with Palbociclib ( 1 μM , 10 μM ) for 4 h . The data shown are mean ± SEM from n = 3 biological replicates . Statistical significance was determined by one - way ANOVA . ns , not significant . ( E - F ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( E ) and THP1 cells ( F ) stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are representative of n = 3 biological replicates . ( G - H ) qRT - PCR measurement of Il1b ( G ) and Il6 ( H ) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by two - way ANOVA followed by Tukey ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 ; ns , not significant . ( I - J ) qRT - PCR measurement of Il1b ( I ) and Il6 ( J ) transcripts in THP1 cells left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by two - way ANOVA followed by Tukey ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 ; ns , not significant . ( K - L ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( K ) and THP1 ( L ) cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are representative of n = 3 biological replicates . ( M - N ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( M ) and THP1 ( N ) cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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Figure 2(A-C) qRT-PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages (A), THP1 cells (B) and human peripheral blood mononuclear cells (PBMC) (C) that were left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib (0.1, 1, 10 μM). The data are presented as fold induction of Ifnb1/Gapdh transcripts. The data shown are mean±SEM of n=3 biological replicates. Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test. *, P<0.05; ****, P<0.0001.(D) MTS detection of the viability of mouse peritoneal macrophages (PM), THP1 and PBMC cells left untreated (DMSO) or treated with Palbociclib (1 μM, 10 μM) for 4 h. The data shown are mean ± SEM from n=3 biological replicates. Statistical significance was determined by one-way ANOVA. ns, not significant.(E-F) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (E) and THP1 cells (F) stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.(G-H) qRT-PCR measurement of Il1b (G) and Il6 (H) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant. (I-J) qRT-PCR measurement of Il1b (I) and Il6 (J) transcripts in THP1 cells left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant.(K-L) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (K) and THP1 (L) cells stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.(M-N) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (M) and THP1 (N) cells stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


Figure 4 ( A ) A real - time OR - ID assay showing the association - dissociation curves of surface - immobilized Palbociclib with recombinant STING protein . The data shown are representative of n = 3 biological replicates . Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) and the lysates of HEK293T cells transfected with Flag - STING ( C ) The data shown are representative of n = 3 biological replicates . Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) mouse peritoneal macrophages ( D ) The data shown are representative of n = 3 biological replicates . Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) THP1 cells ( E ) . The data shown are representative of n = 3 biological replicates . ( F ) Immunoblotting of the streptavidin precipitates of the mixture of biotin - c - diAMP ( 2 μM ) and recombinant SUNO - tagged STING protein ( 10 μg / mL ) in the presence of DMSO and increasing concentrations of Palbociclib ( 0 , 1 , 10 μM ) . The data shown are representative of n = 3 biological replicates . ( G ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STING and HA - STING in the presence of DMSO and indicated concentrations of Palbociclib . The data shown are representative of n = 3 biological replicates . Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) ( H ) . Scale bar , 25 μm . STING and ERGIC in mouse peritoneal macrophages stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) The quantification data is shown in ( I ) . The data shown are mean ± SD from indicated number of cells from 1 representative of n = 3 biological replicates . Statistical significance was determined by two - tailed Student ' s t - test . * * * * , P < 0 . 0001 . ( J ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STEEP and HA - STING in the presence of DMSO or Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates . ( K ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STING and HA - TBK1 in the presence of DMSO or indicated concentrations of Palbociclib . The data shown are representative of n = 3 biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) A real-time OR-ID assay showing the association-dissociation curves of surface-immobilized Palbociclib with recombinant STING protein. The data shown are representative of n=3 biological replicates.Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with Flag-STING (C) The data shown are representative of n=3 biological replicates.Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) mouse peritoneal macrophages (D) The data shown are representative of n=3 biological replicates.Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) THP1 cells (E). The data shown are representative of n=3 biological replicates.(F) Immunoblotting of the streptavidin precipitates of the mixture of biotin-c-diAMP (2 μM) and recombinant SUNO-tagged STING protein (10 μg/mL) in the presence of DMSO and increasing concentrations of Palbociclib (0, 1, 10 μM). The data shown are representative of n=3 biological replicates.(G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-STING in the presence of DMSO and indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (H). Scale bar, 25 μm.STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (I). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ****, P<0.0001.(J) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STEEP and HA-STING in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.(K) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-TBK1 in the presence of DMSO or indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.


Figure 5 ( C ) Immunoblotting of the streptavidin precipitates of the mixture of biotin - Palbociclib ( 100 μM ) and the lysates of HEK293T cells transfected with corresponding Flag - STING mutants . The data shown are representative of n = 3 biological replicates . ( D ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - STING and Flag - STING or Flag - STING Y167A in the presence of DMSO and Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates . Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) ( E ) . Scale bars , 25 μm . STING and ERGIC in HeLa cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) The quantification data is shown in ( F ) . The data shown are mean ± SD from indicated number of cells from 1 representative of n = 3 biological replicates . Statistical significance was determined by two - tailed Student ' s t - test . * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant . ( G ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - TBK1 and Flag - STING or Flag - STING Y167A . The data shown are representative of n = 3 biological replicates . ( H ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - STING and Flag - STING C91A in the presence of DMSO and Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(C) Immunoblotting of the streptavidin precipitates of the mixture of biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with corresponding Flag-STING mutants. The data shown are representative of n=3 biological replicates.(D) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING or Flag-STING Y167A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (E). Scale bars, 25 μm.STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (F). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ***, P<0.001; ****, P<0.0001; ns, not significant.(G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-TBK1 and Flag-STING or Flag-STING Y167A. The data shown are representative of n=3 biological replicates.(H) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING C91A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


Figure 6 ( B ) The weight of mice left untreated or treated with DSS in the absence or presence of H151 or Palbociclib . Statistical significance between data sets was assessed by one - way ANOVA followed by Tukey ' s multiple comparisons post - hoc test between all groups . Values are means ± SEM , n = 5 mice per group with differences denoted by * , P < 0 . 05 ; ns , not significant . The representative image of colon tissue harvested from mice of indicated groups is shown in ( C ) . The length of colon is analyzed in ( D ) . Statistical significance between data sets was assessed by one - way ANOVA followed by Tukey ' s multiple comparisons post - hoc test between all groups . Values are means ± SEM , n = 5 mice per group with differences denoted by * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant . ( E - F ) Representative H & E staining of the histological changes in colon tissues harvested from mice of indicated groups ( E ) . Scale bar , 200 μm . The quantification of histological score was shown in ( F ) . Mann - Whitney U - test was used for statistical analysis of two independent groups . Values represent means ± SEM , each symbol indicates the value of 1 mouse from n = 5 mice per group . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant . ( G - I ) qRT - PCR measurement of transcripts of Ifnb1 ( G ) , Il6 ( H ) and Il1b ( I ) in mouse colon tissues harvested from mice of indicated groups . The data shown are mean ± SEM of n = 5 mice from 1 of 3 biological replicates with similar results . Each symbol indicates the value of 1 mouse from n = 5 mice per group . Statistical significance was determined by one - way ANOVA test followed by Tukey ' s multiple comparisons post - hoc test between all groups . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * * , P < 0 . 0001 ; ns , not significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) The weight of mice left untreated or treated with DSS in the absence or presence of H151 or Palbociclib. Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n = 5 mice per group with differences denoted by *, P<0.05; ns, not significant.The representative image of colon tissue harvested from mice of indicated groups is shown in (C).The length of colon is analyzed in (D). Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n = 5 mice per group with differences denoted by *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.(E-F) Representative H&E staining of the histological changes in colon tissues harvested from mice of indicated groups (E). Scale bar, 200 μm. The quantification of histological score was shown in (F). Mann-Whitney U-test was used for statistical analysis of two independent groups. Values represent means ± SEM, each symbol indicates the value of 1 mouse from n = 5 mice per group. **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.(G-I) qRT-PCR measurement of transcripts of Ifnb1 (G), Il6 (H) and Il1b (I) in mouse colon tissues harvested from mice of indicated groups. The data shown are mean ± SEM of n=5 mice from 1 of 3 biological replicates with similar results. Each symbol indicates the value of 1 mouse from n = 5 mice per group. Statistical significance was determined by one-way ANOVA test followed by Tukey's multiple comparisons post-hoc test between all groups. *, P<0.05; **, P<0.01; ****, P<0.0001; ns, not significant.


Figure 7 ( B - F ) Representative H & E staining of the histological changes in heart , stomach , tongue and thigh muscle tissues harvested from mice of indicated groups ( B ) . Scale bar , 200 μm . The quantification of histological score was shown in ( C - F ) . Mann - Whitney U - test was used for statistical analysis of two independent groups . Values represent means ± SEM , each symbol indicates the value of 1 mouse from n = 5 mice per group . * , P < 0 . 05 ; * * , P < 0 . 01 ; ns , not significant . ( G - I ) qRT - PCR measurement of transcripts of Ifnb1 ( G ) , Cxcl10 ( H ) and Il6 ( I ) in mouse stomach tissues harvested from mice of indicated groups . The data shown are mean ± SEM of n = 5 mice from 1 of 3 biological replicates with similar results . Each symbol indicates the value of 1 mouse from n = 5 mice per group . Statistical significance was determined by one - way ANOVA test followed by Tukey ' s multiple comparisons post - hoc test between all groups . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B-F) Representative H&E staining of the histological changes in heart, stomach, tongue and thigh muscle tissues harvested from mice of indicated groups (B). Scale bar, 200 μm. The quantification of histological score was shown in (C-F). Mann-Whitney U-test was used for statistical analysis of two independent groups. Values represent means ± SEM, each symbol indicates the value of 1 mouse from n = 5 mice per group. *, P<0.05; **, P<0.01; ns, not significant.(G-I) qRT-PCR measurement of transcripts of Ifnb1(G), Cxcl10 (H) and Il6 (I) in mouse stomach tissues harvested from mice of indicated groups. The data shown are mean ± SEM of n=5 mice from 1 of 3 biological replicates with similar results. Each symbol indicates the value of 1 mouse from n = 5 mice per group. Statistical significance was determined by one-way ANOVA test followed by Tukey's multiple comparisons post-hoc test between all groups. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


Figure 1 ( A ) BMDMs were infected with L . pneumophila JR32 or the legA9 mutant with MOI of 0 . 5 . CFUs were scored at 1 , 24 , 48 , and 72 h . ( B ) BMDMs were infected with JR32 or the legA9 mutant harboring empty PL , or legA9 + legA9 pBC ‐ KS + PL . ( A , B ) Data are presented as means ± SD of at least three independent experiments . Asterisks indicate significant differences , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two ‐ tailed t ‐ test ) . Four female C57BL / 6 mice / group received 1 × 106 of JR32 or legA9 mutant bacteria intratracheally . Lungs were homogenized and plated for CFUs counting at ( C ) 4 or ( D ) 48 h postinfection . ( C , D ) Data are shown as mean + SD of n = 4 and are representative as one of two independent experiments . Asterisks indicate significant differences ( * * p < 0 . 01 two ‐ tailed t ‐ test )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) BMDMs were infected with L. pneumophila JR32 or the legA9 mutant with MOI of 0.5. CFUs were scored at 1, 24, 48, and 72 h. (B) BMDMs were infected with JR32 or the legA9 mutant harboring empty PL, or legA9+ legA9 pBC‐KS+ PL. (A, B) Data are presented as means ± SD of at least three independent experiments. Asterisks indicate significant differences, (*p < 0.05, **p < 0.01, ***p < 0.001; two‐tailed t‐test).Four female C57BL/6 mice/group received 1 × 106 of JR32 or legA9 mutant bacteria intratracheally. Lungs were homogenized and plated for CFUs counting at (C) 4 or (D) 48 h postinfection. (C, D) Data are shown as mean + SD of n = 4 and are representative as one of two independent experiments. Asterisks indicate significant differences (**p < 0.01 two‐tailed t‐test)


Figure 2 ( A ) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria . Nuclei were stained blue with DAPI and L . pneumophila stained green with L . pneumophila antibody . Lyso − tracker red was used to stain acidified lysosomes . White arrows show L . pneumophila colocalization with lysotracker . ( B ) Percent colocalization of L . pneumophila with lyso ‐ tracker . ( C ) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria . LC3 is stained red with LC3 antibody . White arrows show L . pneumophila colocalization with LC3 . ( D ) Percent colocalization of L . pneumophila with LC3 . Hundred bacteria were scored from each coverslips . Images were captured with the 60 × objective and magnified 3 × , scale bar = 10 μm . ( A - D ) Data are presented as mean + SD of n = 3 and are representative of three independent experiments . Asterisks indicate significant differences ( * * p < 0 . 01 , * * * p < 0 . 001 ; two ‐ tailed t ‐ test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria. Nuclei were stained blue with DAPI and L. pneumophila stained green with L. pneumophila antibody. Lyso−tracker red was used to stain acidified lysosomes. White arrows show L. pneumophila colocalization with lysotracker.(B) Percent colocalization of L. pneumophila with lyso‐tracker.(C) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria. LC3 is stained red with LC3 antibody. White arrows show L. pneumophila colocalization with LC3.(D) Percent colocalization of L. pneumophila with LC3. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. (A-D) Data are presented as mean + SD of n = 3 and are representative of three independent experiments. Asterisks indicate significant differences (**p < 0.01, ***p < 0.001; two‐tailed t‐test).


Figure 3Induction of autophagy in macrophages restricts legA9 mutant replication , and rescues colocalization with LC3 and fusion with lysosomes . BMDMs were untreated or pretreated with rapamycin ( Rap ) for 1 h and kept throughout the infection . BMDMs were infected with L . pneumophila JR32 or the leg A9 mutant for ( A ) 1 or ( B ) 24 h with an MOI of 0 . 5 . CFUs were scored at these time points . ( C , D ) Percent colocalization of L . pneumophila with ( C ) lyso ‐ tracker and ( D ) LC3 . Hundred bacteria were scored from each coverslips . ( A - D ) Data are presented as mean + SD of n = 3 and are representative of three independent experiments . Asterisks indicate significant differences ( * * p < 0 . 01 ; two ‐ tailed t ‐ test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Induction of autophagy in macrophages restricts legA9 mutant replication, and rescues colocalization with LC3 and fusion with lysosomes. BMDMs were untreated or pretreated with rapamycin (Rap) for 1 h and kept throughout the infection. BMDMs were infected with L. pneumophila JR32 or the leg A9 mutant for (A) 1 or (B) 24 h with an MOI of 0.5. CFUs were scored at these time points.(C, D) Percent colocalization of L. pneumophila with (C) lyso‐tracker and (D) LC3. Hundred bacteria were scored from each coverslips. (A-D) Data are presented as mean + SD of n = 3 and are representative of three independent experiments. Asterisks indicate significant differences (**p < 0.01; two‐tailed t‐test).


Figure 4 ( A ) BMDMs derived from WT and Atg5 − / − mice were infected with L . pneumophila JR32 or legA9 mutant at an MOI of 0 . 5 . Data are shown as mean ± SD of n = 3 and are representative of two independent experiments . Asterisks indicate significant differences ( * p < 0 . 05 ; two − tailed t − test . ( B ) Western blot showing the absence of Atg 5 in the knockout . Data are representative of three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) BMDMs derived from WT and Atg5−/− mice were infected with L. pneumophila JR32 or legA9 mutant at an MOI of 0.5. Data are shown as mean ± SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05; two−tailed t−test.(B) Western blot showing the absence of Atg 5 in the knockout. Data are representative of three independent experiments.


Figure 5 ( A ) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria . Polyubiquitin is stained red . White arrows show L . pneumophila colocalization with polyubiquitin . ( B ) Percent colocalization of L . pneumophila with polyubiquitin . Hundred bacteria were scored from each coverslips . Images were captured with the 60 × objective and magnified 3 × , scale bar = 10 μm . Data are presented as means + SD of n = 3 and are representative of two independent experiments . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 01 ; two ‐ tailed t ‐ test ) .

O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria. Polyubiquitin is stained red. White arrows show L. pneumophila colocalization with polyubiquitin.(B) Percent colocalization of L. pneumophila with polyubiquitin. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. Data are presented as means + SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, ***p < 0.01; two‐tailed t‐test).


Figure 6 ( A ) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria . P62 is stained red with p62 antibody . White arrows show L . pneumophila colocalization with p62 . ( B ) Percent colocalization of L . pneumophila with p62 . Hundred bacteria were scored from each coverslips . Images were captured with the 60 × objective and magnified 3 × , scale bar = 10 μm . ( C ) BMDMs were infected with JR32 or the legA9 mutant for 6 h and the level of p62 protein was quantified in cell lysates by ELISA ( performed in triplicate and the SD are too small to detect ) . BMDMs were nucleofected with scrambled control ( sictrl ) or siRNA against p62 ( sip 62 ) . Twenty ‐ four hours after nucleofection , murine macrophages were infected with JR32 or legA9 mutant with an MOI of 0 . 5 for 1 h ( D and F ) or 24 h ( E and G ) . ( H ) Western blot analysis showing the downregulation of p62 at 24 and 48 h postnucleofection . ( A - B and D - H ) Data are shown as mean + SD of n = 3 and are representative of three independent experiments . ( C ) Data are representative of one experiment n = 3 . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 01 ; two − tailed t − test ) .

O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria. P62 is stained red with p62 antibody. White arrows show L. pneumophila colocalization with p62.(B) Percent colocalization of L. pneumophila with p62. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm.(C)BMDMs were infected with JR32 or the legA9 mutant for 6 h and the level of p62 protein was quantified in cell lysates by ELISA (performed in triplicate and the SD are too small to detect).BMDMs were nucleofected with scrambled control (sictrl) or siRNA against p62 (sip 62). Twenty‐four hours after nucleofection, murine macrophages were infected with JR32 or legA9 mutant with an MOI of 0.5 for 1 h (D and F) or 24 h (E and G).(H) Western blot analysis showing the downregulation of p62 at 24 and 48 h postnucleofection. (A-B and D-H) Data are shown as mean + SD of n = 3 and are representative of three independent experiments. (C) Data are representative of one experiment n = 3. Asterisks indicate significant differences (*p < 0.05, **p < 0.01; two−tailed t−test).


Figure 7 ( A ) Caspase − 1 KO ( casp − 1 − / − ) macrophages were infected with L . pneumophila JR32 or legA9 with an MOI of 0 . 5 for 1 , 24 , 48 , and 72 h . ( B ) CFUs were measured at the indicated time points . Levels of IL ‐ 1β were detected in supernatants of WT or casp ‐ 1 − / − BMDMs infected with JR32 or the legA9 mutant after 24 h . WT BMDMs were either not treated ( NT ) or infected with L . pneumophila JR32 or legA9 mutant bacteria for 2 h . Activation of casp ‐ 1 or casp ‐ 7 was detected in cell extracts using ( C ) casp ‐ 1 or ( D ) casp ‐ 7 antibodies , respectively . ( A - D ) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Caspase−1 KO (casp−1−/−) macrophages were infected with L. pneumophila JR32 or legA9 with an MOI of 0.5 for 1, 24, 48, and 72 h.(B) CFUs were measured at the indicated time points. Levels of IL‐1β were detected in supernatants of WT or casp‐1−/− BMDMs infected with JR32 or the legA9 mutant after 24 h. WT BMDMs were either not treated (NT) or infected with L. pneumophila JR32 or legA9 mutant bacteria for 2 h.Activation of casp‐1 or casp‐7 was detected in cell extracts using (C) casp‐1 or (D) casp‐7 antibodies, respectively. (A-D) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.


Figure 8 ( A ) BMDMs were not infected ( NT ) or infected with L . pneumophila JR32 or the legA9 mutant at an MOI of 0 . 5 , 1 or 5 for 24 h . Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic ( apoptosis ) histone − associated − DNA fragments ( ELISA ) . ( B ) BMDMs were either not treated ( NT ) or treated with L . pneumophila JR32 or the legA9 mutant at an MOI of 0 . 5 or 5 for 24 h . The fold change in LDH release was measured from the overall population of macrophages . ( A - B ) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) BMDMs were not infected (NT) or infected with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5, 1 or 5 for 24 h. Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic (apoptosis) histone−associated−DNA fragments (ELISA).(B) BMDMs were either not treated (NT) or treated with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5 or 5 for 24 h. The fold change in LDH release was measured from the overall population of macrophages. (A-B) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.


Figure 1 ( A ) Unlabeled or Cy5 - labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase ( dsRNA - RL ) or the GFP ( dsRNA - GFP ) coding sequences visualised on gel stained with ethidium bromide ( EtBr - stained , top ) or by in - gel fluorescence ( bottom ) . ( B ) Gating strategy used in the flow cytometry - based assay . First , cells were selected ( side scatter ( SSC ) vs . forward scatter ( FSC ) ) and gated for singlets ( SSC - Width ( W ) vs . FSC ) to hence exclude doublets or cell aggregates . The single cell gate was then further analysed for DAPI , gating on the live cells ( DAPI negative ) . DsRNA - transfected cells ( Cy5 - positive ) were then gated and the level of GFP expression analysed within that population . Flow cytometry counting beads ( gated on SSC vs . FSC plot ) were added to quantify cell number in each sample . ( C ) mESCs stably expressing GFP ( mESCs - GFP ) were mock transfected or transfected with the indicated Cy5 - labeled dsRNA and the level of GFP in Cy5 + cells was analysed 3 days later and is represented as histograms ( left ) . Right panel , bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control ( D ) mESCs stably expressing d2GFP ( mESCs - d2GFP ) were mock transfected or transfected with the indicated Cy5 - labeled dsRNA . The level of d2GFP was analysed at the indicated timepoints post - transfection and displayed as in ( C ) . ( E ) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5 - labeled dsRNA and d2GFP level was measured as in ( D ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Unlabeled or Cy5-labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase (dsRNA-RL) or the GFP (dsRNA-GFP) coding sequences visualised on gel stained with ethidium bromide (EtBr-stained, top) or by in-gel fluorescence (bottom).(B) Gating strategy used in the flow cytometry-based assay. First, cells were selected (side scatter (SSC) vs. forward scatter (FSC)) and gated for singlets (SSC-Width (W) vs. FSC) to hence exclude doublets or cell aggregates. The single cell gate was then further analysed for DAPI, gating on the live cells (DAPI negative). DsRNA-transfected cells (Cy5-positive) were then gated and the level of GFP expression analysed within that population. Flow cytometry counting beads (gated on SSC vs. FSC plot) were added to quantify cell number in each sample.(C) mESCs stably expressing GFP (mESCs-GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA and the level of GFP in Cy5+cells was analysed 3 days later and is represented as histograms (left). Right panel, bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control (D) mESCs stably expressing d2GFP (mESCs-d2GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA. The level of d2GFP was analysed at the indicated timepoints post-transfection and displayed as in (C).(E) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5-labeled dsRNA and d2GFP level was measured as in (D).