Figure 4 ( A ) Immunoblotting of XO8 GSCs and DFCs . ( B ) Immunoblotting analysis of Ub in XO8 GSCs and XO8 GSC - derived cells in the course of differentiation ( P1 - P7 ) . ( C ) Similar to ( B ) except that XO6 GSCs were used . ( D ) Immunoblotting analysis of ubiquitin ligases and proteasomal subunits in XO8 GSCs and XO8 GSC - derived cells in the course of differentiation ( P1 - P7 ) . ( E ) Similar to ( E ) except that XO6 GSCs were used . ( F - I ) In vitro proteasome activity assays were performed as described in Materials and Methods . Bars represent means + S . D . from three independent experiments . For statistical analysis , student t - test ( two - sided , one type ) were used . * P value : ( F ) , 0 . 0039 . ( F - I ) In vitro proteasome activity assays were performed as described in Materials and Methods . Bars represent means + S . D . from three independent experiments . For statistical analysis , student t - test ( two - sided , one type ) were used . * P value : ( F ) , 0 . 0039 ; ( G ) , 0 . 0052 ; ( H ) , 0 . 0009 , ( I ) DFC , 0 . 00032 ; ( I ) - HEK293 , 0 . 0039 ; ( I ) - HeLa : 0 . 00051 . ( J ) XO8 GSCs and XO8 DFCs treated with MG132 as described in Materials and Methods were labeled with annexin V and visualized using fluorescent microscopy . Scale bar , 100 μM . ( K ) Cells from ( J ) were subjected to flow cytometry analysis . Annexin V - positive cell numbers were obtained from three independent experiments and normalized to those of controls cells treated with DMSO . Bars , means + S . D . from three independent experiments . For statistical analysis , student t - test ( two - sided , one type ) were used . * P value : 6 hrs , 0 . 0012 ; 17 hrs , 0 . 0041 . ( L ) Cells treated with 50 nM MG132 were subjected to immunoblotting analysis of apoptotic markers . ( M and N ) Similar to ( L ) except that XO6 and XO10 cells were used .

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

Figure 4(A) Immunoblotting of XO8 GSCs and DFCs.(B) Immunoblotting analysis of Ub in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (C) Similar to (B) except that XO6 GSCs were used.(D) Immunoblotting analysis of ubiquitin ligases and proteasomal subunits in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (E) Similar to (E) except that XO6 GSCs were used.(F-I) In vitro proteasome activity assays were performed as described in Materials and Methods. Bars represent means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: (F), 0.0039.(F-I) In vitro proteasome activity assays were performed as described in Materials and Methods. Bars represent means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: (F), 0.0039; (G), 0.0052; (H), 0.0009, (I) DFC, 0.00032; (I)-HEK293, 0.0039; (I)-HeLa: 0.00051.(J) XO8 GSCs and XO8 DFCs treated with MG132 as described in Materials and Methods were labeled with annexin V and visualized using fluorescent microscopy. Scale bar, 100 μM.(K) Cells from (J) were subjected to flow cytometry analysis. Annexin V-positive cell numbers were obtained from three independent experiments and normalized to those of controls cells treated with DMSO. Bars, means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: 6 hrs, 0.0012; 17 hrs, 0.0041.(L) Cells treated with 50 nM MG132 were subjected to immunoblotting analysis of apoptotic markers.(M and N) Similar to (L) except that XO6 and XO10 cells were used.


Figure 5 ( A ) Real - time PCR analysis of the proteasomal subunits PSMB5 , PSMB6 , and PSMB7 in XO8 GSCs and XO8 DFCs . Shown are means + S . D . values from the three independent experiments following normalization to those of GSCs . For statistical analysis , student t - test ( two - sided , one type ) were used . * * P value = PSMB5 : 0 . 053 , PSMB6 : 0 . 034 , PSMB7 : 0 . 067 . ( B ) Similar to ( A ) except that the cells were treated with 50 nM MG132 for 17 hrs . Shown are means + S . D . values from the three independent experiments following normalization to those of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) were used . * * P value = PSMB5 : 0 . 022 , PSMB6 : 0 . 08 , PSMB7 : 0 . 603 . ( C ) Immunoblotting analysis of proteasomal subunits in XO8 GSCs in comparison with XO8 DFCs at passages 11 and 12 . Note that part of Figure 5C is also shown as the left panel of Figure 4A . ( D ) Same as ( C ) except that the cells were treated with MG132 for 17 hrs . ( E ) Immunoblotting analysis of the NF - κB pathway in XO8 GSCs and XO8 DFCs . ( F ) Fractionation analysis of NF - κB in XO8 GSCs and XO8 DFCs treated with 50 nM MG132 for 17 hrs . PARP1 and tubulin were used as markers for the nucleus and cytoplasm , respectively . ( G ) Immunoblotting analysis of JNK and p38 . ( H ) Cell viability assay was performed as in Fig . 2 with cells treated with MG132 alone or in combination with SB203580 . Shown are means + S . D . values from three independent experiments following normalization to those of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) were used . * * P value = 0 . 724 , 0 . 502 , 0 . 439 for 0 . 5 , 1 . 0 and 5 . 0 μM SB203580 , respectively at 0 . 1 μM MG132 , and 0 . 760 , 0 . 683 , 0 . 498 for 0 . 5 , 1 . 0 and 5 . 0 μM SB203580 , respectively at 0 . 5 μM MG132 . ( I ) Experimental conditions were similar to ( H ) except that SP600129 was used . Shown are means + S . D . values from three independent experiments following normalization to those of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) were used . * P value = 0 . 009 , 0 . 002 , 0 . 07 for 0 . 5 , 1 . 0 and 5 . 0 μM SP600129 , respectively at 0 . 1 μM MG132 , and 0 . 018 , 0 . 025 , 0 . 09 for 0 . 5 , 1 . 0 and 5 . 0 μM SP600129 , respectively at 0 . 5 μM MG132 . ( J ) Immunoblotting analysis of XO8 GSCs treated with 50 nM MG132 alone or in combination with the JNK inhibitor SP600129 .

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Figure 5(A) Real-time PCR analysis of the proteasomal subunits PSMB5, PSMB6, and PSMB7 in XO8 GSCs and XO8 DFCs. Shown are means +S.D. values from the three independent experiments following normalization to those of GSCs. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.053, PSMB6: 0.034, PSMB7: 0.067. (B) Similar to (A) except that the cells were treated with 50 nM MG132 for 17 hrs. Shown are means +S.D. values from the three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.022, PSMB6: 0.08, PSMB7: 0.603.(C) Immunoblotting analysis of proteasomal subunits in XO8 GSCs in comparison with XO8 DFCs at passages 11 and 12. Note that part of Figure 5C is also shown as the left panel of Figure 4A. (D) Same as (C) except that the cells were treated with MG132 for 17 hrs.(E) Immunoblotting analysis of the NF-κB pathway in XO8 GSCs and XO8 DFCs.(F) Fractionation analysis of NF-κB in XO8 GSCs and XO8 DFCs treated with 50 nM MG132 for 17 hrs. PARP1 and tubulin were used as markers for the nucleus and cytoplasm, respectively.(G) Immunoblotting analysis of JNK and p38.(H) Cell viability assay was performed as in Fig. 2 with cells treated with MG132 alone or in combination with SB203580. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value = 0.724, 0.502, 0.439 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.1 μM MG132, and 0.760, 0.683, 0.498 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.5 μM MG132.(I) Experimental conditions were similar to (H) except that SP600129 was used. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. * P value = 0.009, 0.002, 0.07 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.1 μM MG132, and 0.018, 0.025, 0.09 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.5 μM MG132.(J) Immunoblotting analysis of XO8 GSCs treated with 50 nM MG132 alone or in combination with the JNK inhibitor SP600129.


Figure 6 ( A and B ) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours . The expression of genes was assessed by real - time PCR using total RNA extracted from MG132 - treated cells . Mean + S . D . values from the three independent experiments are shown after normalization to that of control cells treated with DMSO . ( A and B ) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours . The expression of genes was assessed by real - time PCR using total RNA extracted from MG132 - treated cells . Mean + S . D . values from the three independent experiments are shown after normalization to that of control cells treated with DMSO . ( C ) XO8 GSCs and XO8 DFCs treated with 50 nM MG132 as described in ( A ) were subjected to immunoblotting . ( D ) XO8 GSCs were treated with MG132 , salinosporamide A , epoxomicin , or bortezomib for 24 hrs . Cell lysates were subjected to immunoblot analysis with anti - PARP - 1 , anti - CHOP , anti - ATF3 , anti - ATF3 , and anti - BiP antibodies . ( E ) XO8 cells were treated with 50 nM MG132 in the absence or presence of 5μM actinomycin D ( ActD ) for 6 h . The level of ATF3 mRNA was measured using semi - quantitative real - time PCR in comparison with β - actin .

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 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 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 6(A and B) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours. The expression of genes was assessed by real-time PCR using total RNA extracted from MG132-treated cells. Mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO.(A and B) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours. The expression of genes was assessed by real-time PCR using total RNA extracted from MG132-treated cells. Mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO.(C) XO8 GSCs and XO8 DFCs treated with 50 nM MG132 as described in (A) were subjected to immunoblotting.(D) XO8 GSCs were treated with MG132, salinosporamide A, epoxomicin, or bortezomib for 24 hrs. Cell lysates were subjected to immunoblot analysis with anti-PARP-1, anti-CHOP, anti-ATF3, anti-ATF3, and anti-BiP antibodies.(E) XO8 cells were treated with 50 nM MG132 in the absence or presence of 5μM actinomycin D (ActD) for 6 h. The level of ATF3 mRNA was measured using semi-quantitative real-time PCR in comparison with β-actin.


Figure 7 ( A ) HEK293 cells were transfected with ATF3 shRNA or vector ( pLKO1 ) , treated with 50 nM MG132 , and then subjected to immunoblotting . ( B ) Individualized XO8 GSCs were transduced with lentiviral shRNAs against ATF3 or control as described in Materials and Methods . After four days , individualized GSCs were treated with 50 nM MG132 , and subjected to immunoblotting . ( C ) XO8 GSCs were transfected with control or CHOP siRNA for 48 hrs and then treated with 50 nM MG132 for 24 hrs . Cell viability was determined using the Trypan blue exclusion assay . Error bars represent the mean ± SD from three independent experiments ( * P < 0 . 05 , n = 3 ) . For statistical analysis , student t - test ( two - sided , one type ) were used . ( D ) Immunoblotting analysis of ( C ) . The cells were collected , lysed , and subjected to immunoblot analysis with indicated antibodies . ( E ) Wild - type and CHOP - / - MEF cells were treated with MG132 for 24 hrs . Cell viability was determined using trypan blue exclusion assay . Error bars represent the mean ± SD from three separate experiments ( * P < 0 . 05 , n = 3 ) . For statistical analysis , student t - test ( two - sided , one type ) were used . ( F ) Cell lysates obtained in ( E ) were subjected to immunoblot analysis with anti - PARP - 1 , anti - CHOP or anti - ATF3 antibody . ( G ) The expression of NOXA was assessed with real - time PCR using total RNA extracted from XO8 GSCs treated with 50 nM MG132 alone or in combination with each inhibitor of ER stress - driven apoptosis ( salubrinal ; 15 µM , STK064652 ; 20 µM , STK047915 ; 20 µM ) . The mean + S . D . values from the three independent experiments are shown after normalization to that of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) was used . * P value : salubrinal , 0 . 0003 ; STK064650 , 0 . 0036 ; STK047915 , 0 . 0019 . ( H ) Similar to ( G ) except that XO8 GSC transduced with lentiviral shRNAs were treated with MG132 . The mean + S . D . values from the three independent experiments are shown after normalization to that of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) was used . * P value : # 1 , 0 . 026 ; # 5 , 0 . 0033 ; # 6 , 0 . 0482 . ( I ) XO8 GSCs were treated with 20 nM MG132 alone or in combination with each inhibitor as described in ( C ) , and subjected to immunoblotting . Sal . : salubrinal .

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 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 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 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 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 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

Figure 7(A) HEK293 cells were transfected with ATF3 shRNA or vector (pLKO1), treated with 50 nM MG132, and then subjected to immunoblotting.(B) Individualized XO8 GSCs were transduced with lentiviral shRNAs against ATF3 or control as described in Materials and Methods. After four days, individualized GSCs were treated with 50 nM MG132, and subjected to immunoblotting.(C) XO8 GSCs were transfected with control or CHOP siRNA for 48 hrs and then treated with 50 nM MG132 for 24 hrs. Cell viability was determined using the Trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments (*P < 0.05, n=3). For statistical analysis, student t-test (two-sided, one type) were used.(D) Immunoblotting analysis of (C). The cells were collected, lysed, and subjected to immunoblot analysis with indicated antibodies.(E) Wild-type and CHOP-/- MEF cells were treated with MG132 for 24 hrs. Cell viability was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from three separate experiments (*P < 0.05, n=3). For statistical analysis, student t-test (two-sided, one type) were used.(F) Cell lysates obtained in (E) were subjected to immunoblot analysis with anti-PARP-1, anti-CHOP or anti-ATF3 antibody.(G) The expression of NOXA was assessed with real-time PCR using total RNA extracted from XO8 GSCs treated with 50 nM MG132 alone or in combination with each inhibitor of ER stress-driven apoptosis (salubrinal; 15 µM, STK064652; 20 µM, STK047915; 20 µM). The mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) was used. * P value: salubrinal, 0.0003; STK064650, 0.0036; STK047915, 0.0019.(H) Similar to (G) except that XO8 GSC transduced with lentiviral shRNAs were treated with MG132. The mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) was used. * P value: #1, 0.026; #5, 0.0033; #6, 0.0482.(I) XO8 GSCs were treated with 20 nM MG132 alone or in combination with each inhibitor as described in (C), and subjected to immunoblotting. Sal.: salubrinal.


Figure 8 ( A and B ) XO8 GSCs were subcutaneously ( s . c . ) injected in the flank of mice . When the tumor volume reached approximately 180 mm3 , GSC - bearing mice were administered intraperitoneally ( i . p . ) with 1 mg / kg MG132 and 10 mg / kg STK alone or in combination on every day for 2 weeks . Tumors were dissected from the subcutaneous regions of nude mice . Inset picture shows the tumor in respective group . Therapeutic effects were monitored using bioluminescence imaging . ( C ) The bar diagram represents the tumor volumes . ( D ) Tumor weights were determined at the end of the study . ( E ) The tumor sizes in each group were determined by histological analysis at day 21 after tumor inoculation . ( F ) XO10 GSC were orthotopically injected into the brains of BALB / c nude mice . STK047915 , MG132 , or both were injected intraperitoneally to GSCs - bearing mice at day 14 after XO10 ( 5 × 105 cells ) inoculation . The PBS was used as a control . Representative photographs of H & amp ; amp ; E staining from each group show tumor growth . Magnification , × 1 .

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Figure 8(A and B) XO8 GSCs were subcutaneously (s.c.) injected in the flank of mice. When the tumor volume reached approximately 180 mm3, GSC-bearing mice were administered intraperitoneally (i.p.) with 1 mg/kg MG132 and 10 mg/kg STK alone or in combination on every day for 2 weeks. Tumors were dissected from the subcutaneous regions of nude mice. Inset picture shows the tumor in respective group. Therapeutic effects were monitored using bioluminescence imaging. (C) The bar diagram represents the tumor volumes. (D) Tumor weights were determined at the end of the study. (E) The tumor sizes in each group were determined by histological analysis at day 21 after tumor inoculation.(F) XO10 GSC were orthotopically injected into the brains of BALB/c nude mice. STK047915, MG132, or both were injected intraperitoneally to GSCs-bearing mice at day 14 after XO10 (5×105 cells) inoculation. The PBS was used as a control. Representative photographs of H&amp;E staining from each group show tumor growth. Magnification, ×1.


Figure 1 ( A ) Top : SERA6 architecture . Positions of the papain - like domain , catalytic triad residues , SUB1 cleavage sites and epitopes for the antibodies used are indicated . Positions of the inserted mTAP or myc3 epitope tags ( between codons Asn886 - Val887 ) introduced in SERA6 - mTAP : loxP parasites are shown . Bottom : time - course analysis of egress by western blot , showing processing of SERA6 - mTAP into multiple protein species ( representative of 2 independent experiments ) . Schizonts were sampled at the indicated times following washout of C2 - mediated arrest . Nomenclature for each protein species was based on apparent molecular mass or predicted composition . Note that the predicted mass of the p40 species is ~ 27 kDa , but its migration on SDS PAGE may be aberrant due to its acidic nature ; the predicted pI of sequence between SUB1 site 1 and the central papain - like domain ( Asp371 - Lys605 ) is ~ 4 . 4 ( see https : / / web . expasy . org / compute _ pi / ) .

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

Figure 1(A) Top: SERA6 architecture. Positions of the papain-like domain, catalytic triad residues, SUB1 cleavage sites and epitopes for the antibodies used are indicated. Positions of the inserted mTAP or myc3 epitope tags (between codons Asn886-Val887) introduced in SERA6-mTAP:loxP parasites are shown. Bottom: time-course analysis of egress by western blot, showing processing of SERA6-mTAP into multiple protein species (representative of 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2-mediated arrest. Nomenclature for each protein species was based on apparent molecular mass or predicted composition. Note that the predicted mass of the p40 species is ~27 kDa, but its migration on SDS PAGE may be aberrant due to its acidic nature; the predicted pI of sequence between SUB1 site 1 and the central papain-like domain (Asp371-Lys605) is ~4.4 (see https://web.expasy.org/compute_pi/).


Figure 2 ( C ) Giemsa - stained thin films and western blot of DMSO - and RAP - treated SERA6 - WT - myc3 [ SERA6 - mTAP : loxP ] and SERA6 - Cys644Ala - myc3 [ SERA6 - mTAP : loxP ] parasites showing that cleavage of host RBC β - spectrin ( red arrowhead ) does not occur unless a functional copy of SERA6 is present , and is tightly associated with egress ( reproducible in 2 independent experiments ) . Schizonts were sampled at the indicated times following washout of C2 . Scale bar , 5 μm . Also see Fig EV2 .

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Figure 2(C) Giemsa-stained thin films and western blot of DMSO- and RAP-treated SERA6-WT-myc3 [SERA6-mTAP:loxP] and SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites showing that cleavage of host RBC β-spectrin (red arrowhead) does not occur unless a functional copy of SERA6 is present, and is tightly associated with egress (reproducible in 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2. Scale bar, 5 μm. Also see Fig EV2.


Figure 3 ( F ) Representative IFA images confirming loss of MSA180 - HA3 expression following RAP - treatment ( reproducible in 2 independent experiments ) . Mature C2 - arrested cycle 0 schizonts were co - stained with anti - HA ( red ) and anti - SERA5 ( green ) . Merged signals include that of the DNA dye 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Scale bar , 5 μm . ( G ) Western blots showing MSA180 is a predominantly soluble PV protein ( representative of 2 independent experiments ) . Mature C2 - arrested MSA180 - HA3 : loxP schizonts were sampled prior to ( Total ) and following saponin fractionation to separate RBC cytosol plus soluble PV proteins ( Supernatant ) from other parasite proteins and membrane - associated proteins ( Pellet ) . The same samples were iprobed with anti - SERA5 and anti - MSP1 ( mAb 89 . 1 ) as positive controls for soluble PV proteins and merozoite surface parasite proteins respectively . The presence of MSA180 p45 - HA3 is likely a post - lysis artefact , perhaps due to SUB1 release during sample manipulation . Note the less efficient saponin - mediated release of MSA180 - HA3 compared to SERA5 , indicating limited membrane association of a fraction of MSA180 . See also Fig EV3 .

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Figure 3(F) Representative IFA images confirming loss of MSA180-HA3 expression following RAP-treatment (reproducible in 2 independent experiments). Mature C2-arrested cycle 0 schizonts were co-stained with anti-HA (red) and anti-SERA5 (green). Merged signals include that of the DNA dye 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 5 μm.(G) Western blots showing MSA180 is a predominantly soluble PV protein (representative of 2 independent experiments). Mature C2-arrested MSA180-HA3:loxP schizonts were sampled prior to (Total) and following saponin fractionation to separate RBC cytosol plus soluble PV proteins (Supernatant) from other parasite proteins and membrane-associated proteins (Pellet). The same samples were iprobed with anti-SERA5 and anti-MSP1 (mAb 89.1) as positive controls for soluble PV proteins and merozoite surface parasite proteins respectively. The presence of MSA180 p45-HA3 is likely a post-lysis artefact, perhaps due to SUB1 release during sample manipulation. Note the less efficient saponin-mediated release of MSA180-HA3 compared to SERA5, indicating limited membrane association of a fraction of MSA180. See also Fig EV3.


Figure 4 ( A ) Replication of DMSO - and RAP - treated MSA180 - HA3 : loxP parasites over three erythrocytic cycles . Parasitaemia values are averages from replicates in different blood sources . The similar growth rate of DMSO - treated parasites and the parental B11 clone shows that the genetic modifications to generate MSA180 - HA3 : loxP parasites did not affect parasite viability . Error bars , ± S . D ( B11 : n = 3 ; DMSO - and RAP - treated MSA180 - HA3 : loxP : n = 6 each ) . ( B ) Stills from simultaneous time - lapse DIC and fluorescence microscopic examination of DMSO - and RAP - treated MSA180 - HA3 : loxP cycle 0 schizonts following removal of C2 - arrest ( representative of 2 independent experiments ) . Note the defect in RBCM rupture and merozoite release in RAP - treated parasites despite normal PVM rupture ( indicated by a sudden increased visibility and motility of merozoites ) and RBCM poration ( green ring indicating access and binding of phalloidin to the host RBC cytoskeleton ) . Scale bars , 5 μm . ( D ) Time - course comparing fate of SERA6 from DMSO - and RAP - treated MSA180 - HA3 : loxP parasites ( representative of 2 independent experiments ) . Schizonts were sampled at the indicated times following removal of C2 - arrest , or arrested with E64 - d ( 50 μM ) . RAP - treated SERA6 - Cys644Ala - myc3 [ SERA6 - mTAP : loxP ] parasites were included in the blot to allow comparison with processing of catalytically - dead SERA6 ( SERA6 - Cys644Ala - myc3 ) . Note the accumulation of SERA6 p65 and poor conversion to SERA6 p40 in the RAP - treated ( ΔMSA180 ) parasites .

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Figure 4(A) Replication of DMSO- and RAP-treated MSA180-HA3:loxP parasites over three erythrocytic cycles. Parasitaemia values are averages from replicates in different blood sources. The similar growth rate of DMSO-treated parasites and the parental B11 clone shows that the genetic modifications to generate MSA180-HA3:loxP parasites did not affect parasite viability. Error bars, ± S.D (B11: n=3; DMSO- and RAP-treated MSA180-HA3:loxP: n=6 each).(B) Stills from simultaneous time-lapse DIC and fluorescence microscopic examination of DMSO- and RAP-treated MSA180-HA3:loxP cycle 0 schizonts following removal of C2-arrest (representative of 2 independent experiments). Note the defect in RBCM rupture and merozoite release in RAP-treated parasites despite normal PVM rupture (indicated by a sudden increased visibility and motility of merozoites) and RBCM poration (green ring indicating access and binding of phalloidin to the host RBC cytoskeleton). Scale bars, 5 μm.(D) Time-course comparing fate of SERA6 from DMSO- and RAP-treated MSA180-HA3:loxP parasites (representative of 2 independent experiments). Schizonts were sampled at the indicated times following removal of C2-arrest, or arrested with E64-d (50 μM). RAP-treated SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites were included in the blot to allow comparison with processing of catalytically-dead SERA6 (SERA6-Cys644Ala-myc3). Note the accumulation of SERA6 p65 and poor conversion to SERA6 p40 in the RAP-treated (ΔMSA180) parasites.


Figure 5 ( A ) Representative IFA images demonstrating the dynamic changes in localisation of SERA6 - mTAP during egress ( reproducible in 4 independent experiments ) . SERA6 - mTAP : loxP schizonts were sampled at the indicated times following removal of C2 - arrest , fixed and co - stained with DAPI ( blue ) , anti - HA ( red ) and anti - ankyrin ( green ; ankyrin is a major component of the RBC cytoskeleton ) . Scale bar , 5 μm . ( C ) Representative IFA images demonstrating localisation of MSA180 - HA3 during egress . MSA180 - HA3 [ B11 ] schizonts were sampled at the indicated times following removal of C2 - arrest , fixed and co - stained with DAPI ( blue ) , anti - HA ( red ) and anti - ankyrin ( green ) . Scale bar , 5 μm . See also Fig EV5 .

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Figure 5(A) Representative IFA images demonstrating the dynamic changes in localisation of SERA6-mTAP during egress (reproducible in 4 independent experiments). SERA6-mTAP:loxP schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green; ankyrin is a major component of the RBC cytoskeleton). Scale bar, 5 μm.(C) Representative IFA images demonstrating localisation of MSA180-HA3 during egress. MSA180-HA3 [B11] schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green). Scale bar, 5 μm. See also Fig EV5.


Figure 6 ( B ) E64 - d , MMV676881 and Compound 31 inhibit RBCM rupture and merozoite release despite normal PVM rupture , indicated by loss of a continuous ring of EXP2 - mNeon signal ( green ) and RBCM poration ( red ring indicating access and binding of phalloidin to the host RBC cytoskeleton ) ( reproducible in 2 independent experiments ) . P . falciparum EXP2 - mNeon schizonts were allowed to reach the point of egress in the presence of C2 ( 1 µM ) , E64 - d ( 10 µM ) , MMV676881 ( 2 µM ) , Compound 31 ( 2 µM ) or DMSO ( 0 . 1 % v / v ; control ) then examined by DIC and fluorescence microscopy . Scale bar , 5 μm . ( C ) Western blot comparing dose - response of E64 - d , MMV676881 and Compound 31 - mediated inhibition of maturation of SERA6 - mTAP from SERA6 - mTAP : loxP parasites ( reproducible in 3 independent experiments ) .

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Figure 6(B) E64-d, MMV676881 and Compound 31 inhibit RBCM rupture and merozoite release despite normal PVM rupture, indicated by loss of a continuous ring of EXP2-mNeon signal (green) and RBCM poration (red ring indicating access and binding of phalloidin to the host RBC cytoskeleton) (reproducible in 2 independent experiments). P. falciparum EXP2-mNeon schizonts were allowed to reach the point of egress in the presence of C2 (1 µM), E64-d (10 µM), MMV676881 (2 µM), Compound 31 (2 µM) or DMSO (0.1% v/v; control) then examined by DIC and fluorescence microscopy. Scale bar, 5 μm.(C) Western blot comparing dose-response of E64-d, MMV676881 and Compound 31-mediated inhibition of maturation of SERA6-mTAP from SERA6-mTAP:loxP parasites (reproducible in 3 independent experiments).


Figure 1E , Immunoblot analysis of cells described in ( D ) , harvested at the indicated time points after G1 / S release . Exemplary blot from three independent experiments . F , DUB activity assay for OTUD6B in asynchronous , G1 / S - or mitotically synchronized and released A549 cells using HA - ubiquitin vinyl - sulfone to isolate active forms of DUBs . Analysis was performed by immunoblotting using the indicated antibodies .

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Figure 1E, Immunoblot analysis of cells described in (D), harvested at the indicated time points after G1/S release. Exemplary blot from three independent experiments.F, DUB activity assay for OTUD6B in asynchronous, G1/S- or mitotically synchronized and released A549 cells using HA-ubiquitin vinyl-sulfone to isolate active forms of DUBs. Analysis was performed by immunoblotting using the indicated antibodies.


Figure 2B , In - vivo ubiquitylation analyses of LIN28B in HEK293T cells in which OTUD6B was silenced by siRNA . Cells were transfected with the indicated siRNAs and overexpression constructs , then treated with MG132 for 3 hrs . Lysis and IP was done under denaturing conditions followed by WB analysis . Data information : For B , exemplary blot of two independent experiments . C , In - vivo ubiquitylation assay of LIN28B in OTUD6B and a catalytically inactive variant of OTUD6B ( OTUD6B - C158A ) overexpressing cells . HEK293T cells were transfected with indicated combinations of FLAG - LIN28B , HA - Ubiquitin , OTUD6B , OTUD6B - C158A and EV control and treated with MG132 for 3 hrs 24 hrs later . Denatured WCE were subjected to FLAG - IP . WCE and IP were analyzed by immunoblotting including a K48 - specific ubiquitin antibody . Exemplary blots from three independent experiments are shown . D , Pull - down of endogenous LIN28B from HEK293T WCE using bacterially purified GST - OTUD6B . Pulldowns and WCE were analyzed by immunoblot using the indicated antibodies . Data information : For D , exemplary blot of two independent experiments . E , Immunoblot analysis of an in - vitro deubiquitylation assay using GST - purified OTUD6B from bacteria and FLAG - purified ubiquitylated LIN28B from HEK293T . FLAG - IP was performed under denaturing conditions from HEK293T WCEs expressing FLAG - LIN28B and HA - Ubiquitin . Purified FLAG - LIN28B was eluted from the beads and incubated with GST or GST - OTUD6B proteins followed by immunoblot analysis . Data information : For E : exemplary blot of two independent experiments .

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Figure 2B, In-vivo ubiquitylation analyses of LIN28B in HEK293T cells in which OTUD6B was silenced by siRNA. Cells were transfected with the indicated siRNAs and overexpression constructs, then treated with MG132 for 3 hrs. Lysis and IP was done under denaturing conditions followed by WB analysis. Data information: For B, exemplary blot of two independent experiments.C, In-vivo ubiquitylation assay of LIN28B in OTUD6B and a catalytically inactive variant of OTUD6B (OTUD6B-C158A) overexpressing cells. HEK293T cells were transfected with indicated combinations of FLAG-LIN28B, HA-Ubiquitin, OTUD6B, OTUD6B-C158A and EV control and treated with MG132 for 3 hrs 24 hrs later. Denatured WCE were subjected to FLAG-IP. WCE and IP were analyzed by immunoblotting including a K48-specific ubiquitin antibody. Exemplary blots from three independent experiments are shown.D, Pull-down of endogenous LIN28B from HEK293T WCE using bacterially purified GST-OTUD6B. Pulldowns and WCE were analyzed by immunoblot using the indicated antibodies. Data information: For D, exemplary blot of two independent experiments.E, Immunoblot analysis of an in-vitro deubiquitylation assay using GST-purified OTUD6B from bacteria and FLAG-purified ubiquitylated LIN28B from HEK293T. FLAG-IP was performed under denaturing conditions from HEK293T WCEs expressing FLAG-LIN28B and HA-Ubiquitin. Purified FLAG-LIN28B was eluted from the beads and incubated with GST or GST-OTUD6B proteins followed by immunoblot analysis. Data information: For E: exemplary blot of two independent experiments.


Figure 3A , Co - IP of FLAG - OTUD6B with endogenous LIN28B from MM1 . S cells synchronized in G1 / S and 12 hrs post release . MM1 . S cells stably expressing FLAG - OTUD6B or FLAG - EV were synchronized at G1 / S and harvested at the indicated time points after release . IPs were preformed and analyzed together with WCE by immunoblotting . Data information : All blots are representations of three independent experiments . B , Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle . RPMI ( B ) cells were synchronized at the G1 / S transition by a double thymidine block or in mitosis by a sequential thymidine / nocodazole block . Synchronized cells were released , harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies . Data information : All blots are representations of three independent experiments . C , Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle . A549 ( C ) cells were synchronized at the G1 / S transition by a double thymidine block or in mitosis by a sequential thymidine / nocodazole block . Synchronized cells were released , harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies . Data information : All blots are representations of three independent experiments . D , Immunoblot analysis of LIN28B protein half - life in G1 / S - synchronized ( upper panel ) and asynchronous ( lower panel ) A549 cells upon OTUD6B depletion . Cells transfected with the respective siRNAs were synchronized in G1 / S - phase or not and treated with cycloheximide ( CHX ) as indicated . Data information : All blots are representations of three independent experiments .

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Figure 3A, Co-IP of FLAG-OTUD6B with endogenous LIN28B from MM1.S cells synchronized in G1/S and 12 hrs post release. MM1.S cells stably expressing FLAG-OTUD6B or FLAG-EV were synchronized at G1/S and harvested at the indicated time points after release. IPs were preformed and analyzed together with WCE by immunoblotting. Data information: All blots are representations of three independent experiments.B, Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle. RPMI (B) cells were synchronized at the G1/S transition by a double thymidine block or in mitosis by a sequential thymidine/nocodazole block. Synchronized cells were released, harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies. Data information: All blots are representations of three independent experiments.C, Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle. A549 (C) cells were synchronized at the G1/S transition by a double thymidine block or in mitosis by a sequential thymidine/nocodazole block. Synchronized cells were released, harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies. Data information: All blots are representations of three independent experiments.D, Immunoblot analysis of LIN28B protein half-life in G1/S-synchronized (upper panel) and asynchronous (lower panel) A549 cells upon OTUD6B depletion. Cells transfected with the respective siRNAs were synchronized in G1/S-phase or not and treated with cycloheximide (CHX) as indicated. Data information: All blots are representations of three independent experiments.


Figure 4D , Immunoblot analysis of WCE obtained from a representative experiment depicted in ( C ) .

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Figure 4D, Immunoblot analysis of WCE obtained from a representative experiment depicted in (C).


Figure 5C , Immunoblot analysis of RPMI8226 , KMS12BM and MM1 . S cells expressing the indicated shRNAs . Depicted are examples of three independent experiments per cell line . Specification of MYC rearrangements : RPMI8226 ( der ( 17 ) t ( ? 8 ; 17 ) ( q21 . 2 ; q25 ) ) , KMS12BM ( der ( 1 ; 8 ) ( q10 ; q10 ) x2 ) , MM1 . S ( der3t ( 3 ; 8 ) ) .

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Figure 5C, Immunoblot analysis of RPMI8226, KMS12BM and MM1.S cells expressing the indicated shRNAs. Depicted are examples of three independent experiments per cell line. Specification of MYC rearrangements: RPMI8226 (der(17)t(?8;17)(q21.2;q25)), KMS12BM (der(1;8)(q10;q10)x2), MM1.S (der3t(3;8)).


Figure 6A , Correlation between MYC and OTUD6B expression in MM patient samples at diagnosis . mRNA expression in primary CD138 + MM cells was quantified by real - time qPCR ( n = 89 patients ) . Data are fit by linear regression ( black line ) ; Pearson r , Pearson correlation coefficient ; P < 0 . 0001 ; by linear regression and Pearson correlation .

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Figure 6A, Correlation between MYC and OTUD6B expression in MM patient samples at diagnosis. mRNA expression in primary CD138+ MM cells was quantified by real-time qPCR (n = 89 patients). Data are fit by linear regression (black line); Pearson r, Pearson correlation coefficient; P < 0.0001; by linear regression and Pearson correlation.


Figure 1F , G Immunoblot analysis of ST3GAL5 in ( F ) NMuMG and ( G ) A549 - VIM - RFP cells treated with vehicle control or TGF - β for the indicated time . Vinculin and GAPDH : loading controls .

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Figure 1F, G Immunoblot analysis of ST3GAL5 in (F) NMuMG and (G) A549-VIM-RFP cells treated with vehicle control or TGF-β for the indicated time. Vinculin and GAPDH: loading controls.


Figure 2Immunoblot analysis of p - SMAD2 and total SMAD2 ( t - SMAD2 ) in NMuMG cells with or without Ugcg deficiency and treated with vehicle control or TGF - β for 1 h . Tubulin : loading control . Immunoblot analysis of E - cadherin and N - cadherin in Ugcg KO NMuMG or control NMuMG cells treated with vehicle control or TGF - β for 48 h . Tubulin : loading control .

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Figure 2Immunoblot analysis of p-SMAD2 and total SMAD2 (t-SMAD2) in NMuMG cells with or without Ugcg deficiency and treated with vehicle control or TGF-β for 1 h. Tubulin: loading control.Immunoblot analysis of E-cadherin and N-cadherin in Ugcg KO NMuMG or control NMuMG cells treated with vehicle control or TGF-β for 48 h. Tubulin: loading control.


Figure 3Immunoblot analysis of p - SMAD and t - SMAD2 in A549 - VIM - RFP cells pretreated with eliglustat for 4 or 6 days and then treated with vehicle control or TGF - β for 1 h . GAPDH : loading control . Quantification of the p - SMAD2 level in A549 - VIM - RFP cells as shown in ( A ) . The levels were normalized to that of GAPDH , and fold changes were then further normalized to the level of p - SMAD2 in control cells without TGF - β treatment . Immunoblot analysis of the epithelial marker E - cadherin and mesenchymal markers N - cadherin and vimentin in A549 - VIM - RFP cells treated with eliglustat for 4 or 6 days and / or with vehicle control and / or TGF - β for 2 days . Tubulin : loading control . mCherry - labeled A549 cells were pretreated with eliglustat ( 2 μM ) for 4 days and were then injected into ducts of Cuvier of zebrafish embryos . Representative images with magnified regions ( outlined with dotted squares ) of extravasated cells were acquired 4 days after injection by confocal microscopy . SB group zebrafish were treated with the inhibitor SB505124 ( 1 μM ) in the egg water together with eliglustat for 4 days after injection with daily refreshment of the treatments . Quantification of the number of extravasated cell clusters from 28 embryos per group .

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Figure 3Immunoblot analysis of p-SMAD and t-SMAD2 in A549-VIM-RFP cells pretreated with eliglustat for 4 or 6 days and then treated with vehicle control or TGF-β for 1 h. GAPDH: loading control. Quantification of the p-SMAD2 level in A549-VIM-RFP cells as shown in (A). The levels were normalized to that of GAPDH, and fold changes were then further normalized to the level of p-SMAD2 in control cells without TGF-β treatment.Immunoblot analysis of the epithelial marker E-cadherin and mesenchymal markers N-cadherin and vimentin in A549-VIM-RFP cells treated with eliglustat for 4 or 6 days and/or with vehicle control and/or TGF-β for 2 days. Tubulin: loading control.mCherry-labeled A549 cells were pretreated with eliglustat (2 μM) for 4 days and were then injected into ducts of Cuvier of zebrafish embryos. Representative images with magnified regions (outlined with dotted squares) of extravasated cells were acquired 4 days after injection by confocal microscopy. SB group zebrafish were treated with the inhibitor SB505124 (1 μM) in the egg water together with eliglustat for 4 days after injection with daily refreshment of the treatments.Quantification of the number of extravasated cell clusters from 28 embryos per group.


Figure 4Lysates from NMuMG control cells ( Cas9 - expressing cells ) and Ugcg KO cells were subjected to sucrose density gradient ultracentrifugation . The expression levels of flotillin - 1 , β1 - integrin , EEA1 , and TβRI in the sucrose gradient fractions were analyzed by immunoblotting . Fractions 3 , 4 , and 5 contained lipid rafts , whereas fractions 10 - 12 corresponded to the non - lipid raft fractions . Quantification of TβRI percentages in lipid raft and non - lipid raft fractions from Ugcg KO NMuMG and control cells . Isolation of lipid rafts from other cellular components in A549 - VIM - RFP cells treated with the UGCG inhibitor eliglustat ( 2 μM ) for 6 days using sucrose density gradient ultracentrifugation and measurement of flotillin - 1 , β1 - integrin , EEA1 , and TβRI levels in the sucrose gradient fractions using immunoblot analysis . Fractions 3 - 6 contained lipid rafts , whereas fractions 10 - 12 corresponded to the non - lipid raft fractions . Quantification of TβRI percentages in lipid raft and non - lipid raft fractions from A549 - VIM - RFP cells treated with eliglustat ( 2 μM ) for 6 days . Ubiquitination of TβRI was detected by immunoprecipitation ( IP ) of Myc - tagged caTβRI from HA - Ub - transfected HEK293T cells with or without eliglustat ( 2 μM ) treatment for 6 days . All groups were treated with MG132 ( 5 μM ) for 6 h . Immunoblot analysis of TβRI expression levels in control and eliglustat ( 2 μM ) - treated A549 - VIM - RFP cells treated with 50 μg / mL CHX for the indicated times . Vinculin : loading control . Quantification of TβRI expression levels with normalization to the t = 0 controls .

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Figure 4Lysates from NMuMG control cells (Cas9-expressing cells) and Ugcg KO cells were subjected to sucrose density gradient ultracentrifugation. The expression levels of flotillin-1, β1-integrin, EEA1, and TβRI in the sucrose gradient fractions were analyzed by immunoblotting. Fractions 3, 4, and 5 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions. Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from Ugcg KO NMuMG and control cells. Isolation of lipid rafts from other cellular components in A549-VIM-RFP cells treated with the UGCG inhibitor eliglustat (2 μM) for 6 days using sucrose density gradient ultracentrifugation and measurement of flotillin-1, β1-integrin, EEA1, and TβRI levels in the sucrose gradient fractions using immunoblot analysis. Fractions 3-6 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions. Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from A549-VIM-RFP cells treated with eliglustat (2 μM) for 6 days.Ubiquitination of TβRI was detected by immunoprecipitation (IP) of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without eliglustat (2 μM) treatment for 6 days. All groups were treated with MG132 (5 μM) for 6 h.Immunoblot analysis of TβRI expression levels in control and eliglustat (2 μM)-treated A549-VIM-RFP cells treated with 50 μg/mL CHX for the indicated times. Vinculin: loading control.   Quantification of TβRI expression levels with normalization to the t=0 controls.


Figure 5Immunoblot analysis of p - SMAD2 , t - SMAD2 , and ST3GAL5 in A549 - VIM - RFP cells with siRNA - mediated ST3GAL5 knockdown or transfection of nontargeting siRNA and treated with vehicle control or TGF - β for 1 h . Vinculin : loading control . Quantification of the p - SMAD2 level in A549 - VIM - RFP cells transfected with nontargeting or ST3GAL5 siRNA and treated with TGF - β as shown in ( B ) . Expression levels of ST3GAL5 , the epithelial marker E - cadherin , and mesenchymal markers , including N - cadherin , vimentin , and SNAIL , in siRNA ST3GAL5 - depleted and nontargeting siRNA - transfected A549 - VIM - RFP cells treated with vehicle control or TGF - β for 48 h . Vinculin : loading control . Immunoblot analysis of p - SMAD2 and t - SMAD2 in A549 - VIM - RFP cells transduced with empty vector ( pLV - EV ) or the ST3GAL5 expression construct and stimulated with vehicle control or TGF - β for 1 h . GAPDH : loading control . Quantification of the p - SMAD2 level in A549 - VIM - RFP cells transduced with pLV - EV or the ST3GAL5 overexpression construct and treated with TGF - β , as shown in ( F ) . Immunoblot analysis of the epithelial marker E - cadherin and mesenchymal markers , including N - cadherin , vimentin , and SNAIL , in A549 - VIM - RFP cells transduced with the pLV - EV control or ST3GAL5 expression construct and treated with vehicle control or TGF - β for 48 h . GAPDH : loading control .

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Figure 5Immunoblot analysis of p-SMAD2, t-SMAD2, and ST3GAL5 in A549-VIM-RFP cells with siRNA-mediated ST3GAL5 knockdown or transfection of nontargeting siRNA and treated with vehicle control or TGF-β for 1 h. Vinculin: loading control.   Quantification of the p-SMAD2 level in A549-VIM-RFP cells transfected with nontargeting or ST3GAL5 siRNA and treated with TGF-β as shown in (B).  Expression levels of ST3GAL5, the epithelial marker E-cadherin, and mesenchymal markers, including N-cadherin, vimentin, and SNAIL, in siRNA ST3GAL5-depleted and nontargeting siRNA-transfected A549-VIM-RFP cells treated with vehicle control or TGF-β for 48 h. Vinculin: loading control.Immunoblot analysis of p-SMAD2 and t-SMAD2 in A549-VIM-RFP cells transduced with empty vector (pLV-EV) or the ST3GAL5 expression construct and stimulated with vehicle control or TGF-β for 1 h. GAPDH: loading control. Quantification of the p-SMAD2 level in A549-VIM-RFP cells transduced with pLV-EV or the ST3GAL5 overexpression construct and treated with TGF-β, as shown in (F).Immunoblot analysis of the epithelial marker E-cadherin and mesenchymal markers, including N-cadherin, vimentin, and SNAIL, in A549-VIM-RFP cells transduced with the pLV-EV control or ST3GAL5 expression construct and treated with vehicle control or TGF-β for 48 h. GAPDH: loading control.


Figure 6HEK293T cells transfected with Myc - tagged caTβRI , ( HA - Ub , and nontargeting siRNA or ST3GAL5 siRNA were collected for IP with an anti - Myc antibody and immunoblot analysis . All groups were treated with MG132 ( 5 μM ) for 6 h . Ubiquitination of TβRI was detected by IP of Myc - tagged caTβRI from HA - Ub - transfected HEK293T cells with or without ST3GAL5 overexpression . All groups were treated with MG132 ( 5 μM ) for 6 h . Western blot analysis of TβRI expression in A549 - VIM - RFP cells transduced with the pLV - EV control or ST3GAL5 expression construct and treated with CHX for the indicated times . Vinculin : loading control . Quantification of TβRI expression in pLV - EV control - and ST3GAL5 - expressing A549 - VIM - RFP cells . The results were normalized to the t = 0 controls .

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Figure 6HEK293T cells transfected with Myc-tagged caTβRI, (HA-Ub, and nontargeting siRNA or ST3GAL5 siRNA were collected for IP with an anti-Myc antibody and immunoblot analysis. All groups were treated with MG132 (5 μM) for 6 h.Ubiquitination of TβRI was detected by IP of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without ST3GAL5 overexpression. All groups were treated with MG132 (5 μM) for 6 h.Western blot analysis of TβRI expression in A549-VIM-RFP cells transduced with the pLV-EV control or ST3GAL5 expression construct and treated with CHX for the indicated times. Vinculin: loading control.Quantification of TβRI expression in pLV-EV control- and ST3GAL5-expressing A549-VIM-RFP cells. The results were normalized to the t=0 controls.


Figure 7In vivo zebrafish extravasation experiments with mCherry - labeled A549 cells with or without ectopic expression of ST3GAL5 . Representative images with magnified regions ( outlined with dotted squares ) of extravasated cells were acquired 4 days after injection by confocal microscopy . The number of extravasated cell clusters was quantified in 30 embryos injected with A549 cells transduced with the pLV - EV control or ST3GAL5 expression construct . Kaplan - Meier survival curves showing the first progression survival of lung cancer patients in a publicly available lung cancer dataset according to ST3GAL5 expression ( n = 982 ) . Box plots of ST3GAL5 gene expression levels in lung cancer tissues and normal tissues in the Bhattacharjee Lung database . The central bands indicate the medium expression values of ST3GAL5 , boxes indicate the expression level ranges of ST3GAL5 . Data are presented as the means ± SDs from the indicated number of tissues . Representative images of ST3GAL5 immunohistochemistry in a human lung tissue microarray including normal and cancer tissues . Wide field and magnified images ( outlined with dotted squares ) are shown . Scatter plot showing the expression of ST3GAL5 in normal and cancerous lung tissues . Each point represents the H - score of a single tissue sample with ST3GAL5 staining ranging from completely absent ( H - score 0 ) to very strong ( H - score 300 ) . H - scores are presented as the means ± SDs ; normal tissues , n = 48 ; cancer tissues , n = 97 ; * * * * , P < 0 . 0001 based on unpaired Student ' s t test .

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Figure 7In vivo zebrafish extravasation experiments with mCherry-labeled A549 cells with or without ectopic expression of ST3GAL5. Representative images with magnified regions (outlined with dotted squares) of extravasated cells were acquired 4 days after injection by confocal microscopy. The number of extravasated cell clusters was quantified in 30 embryos injected with A549 cells transduced with the pLV-EV control or ST3GAL5 expression construct.Kaplan-Meier survival curves showing the first progression survival of lung cancer patients in a publicly available lung cancer dataset according to ST3GAL5 expression (n=982).Box plots of ST3GAL5 gene expression levels in lung cancer tissues and normal tissues in the Bhattacharjee Lung database. The central bands indicate the medium expression values of ST3GAL5, boxes indicate the expression level ranges of ST3GAL5. Data are presented as the means ± SDs from the indicated number of tissues.Representative images of ST3GAL5 immunohistochemistry in a human lung tissue microarray including normal and cancer tissues. Wide field and magnified images (outlined with dotted squares) are shown.Scatter plot showing the expression of ST3GAL5 in normal and cancerous lung tissues. Each point represents the H-score of a single tissue sample with ST3GAL5 staining ranging from completely absent (H-score 0) to very strong (H-score 300). H-scores are presented as the means ± SDs; normal tissues, n=48; cancer tissues, n=97; ****, P < 0.0001 based on unpaired Student's t test.


Figure 1 ( A ) Survival graphs of newborn pups from breeding of Rab11aFl / + ; Rab11b + / - ; Villin - Cre and Rab11aFl / + ; Rab11b + / - ( or Rab11aFl / + ; Rab11b - / - ) mice . The total number of pups for each genotype was labeled next to the corresponding curve . The graph represents over 6 independent litters from three different mating pairs . ( B ) Western blots for Rab11a and Rab11b using small intestinal lysates prepared from adult wild type ( WT , lane 1 ) , Rab11aFl / Fl ; Villin - CreER ( aKO , lane 2 ) 2 days after tamoxifen treatment , Rab11b - / - ( bKO , lane 3 ) , and Rab11aFl / Fl ; Rab11b - / - ; Villin - CreER ( DKO ) mice 1 - day ( lane 4 ) , 2 - day ( lane 5 ) , and 3 - day ( lane 6 ) after tamoxifen treatment . All mice were given a single tamoxifen injection . β - actin was used as loading control . Results represent more than 3 independent experiments . ( C ) H . & E . staining of adult Rab11aFl / Fl ; Rab11b - / - ; Villin - CreER mice , before and after tamoxifen injection . Images represent jejunum tissues collected 1 , 2 , and 3 days following tamoxifen injection . Experiments were repeated over 5 times using independent litters ( n > 10 ) . Scale bars , 50 μ ( D ) Alkaline phosphatase ( AP ) staining was performed on intestinal tissue sections of adult Rab11aFl / Fl ; Rab11b - / - ; Villin - CreER mice that were treated with corn oil or with tamoxifen . Results represent at least 3 independent experiments . Scale bars , 50 μm . ( F ) Alcian blue staining was performed on adult Rab11aFl / Fl ; Rab11b - / - ; Villin - CreER mice before or 1 to 3 days after tamoxifen injection . Results represent at least 3 independent experiments . Scale bars , 100 μm . ( G ) Quantification of the number of alcian blue positive cells per crypt - villus axis in mice of various genotypes . Data represent average values of approximately 10 - 15 different microscopic fields taken from 3 mice per condition . ( H ) Immunofluorescent staining for lysozyme and E - Cad was performed on adult Rab11aFl / Fl ; Rab11b - / - ; Villin - CreER mice before and after tamoxifen injection . Experiments represent 3 independent replicates . Scale bars , 50 μm . ( I ) Quantification of lysozyme fluorescent signal abundance per crypt in mice of various genotypes . Data represent approximately 10 - 15 different microscopic fields taken from 3 mice per condition .

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Figure 1(A) Survival graphs of newborn pups from breeding of Rab11aFl/+;Rab11b+/-;Villin-Cre and Rab11aFl/+;Rab11b+/- (or Rab11aFl/+;Rab11b-/-) mice. The total number of pups for each genotype was labeled next to the corresponding curve. The graph represents over 6 independent litters from three different mating pairs.(B) Western blots for Rab11a and Rab11b using small intestinal lysates prepared from adult wild type (WT, lane 1), Rab11aFl/Fl;Villin-CreER (aKO, lane 2) 2 days after tamoxifen treatment, Rab11b-/- (bKO, lane 3), and Rab11aFl/Fl;Rab11b-/-;Villin-CreER (DKO) mice 1-day (lane 4), 2-day (lane 5), and 3-day (lane 6) after tamoxifen treatment. All mice were given a single tamoxifen injection. β-actin was used as loading control. Results represent more than 3 independent experiments.(C) H. & E. staining of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice, before and after tamoxifen injection. Images represent jejunum tissues collected 1, 2, and 3 days following tamoxifen injection. Experiments were repeated over 5 times using independent litters (n>10). Scale bars, 50 μ(D) Alkaline phosphatase (AP) staining was performed on intestinal tissue sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice that were treated with corn oil or with tamoxifen. Results represent at least 3 independent experiments. Scale bars, 50 μm.(F) Alcian blue staining was performed on adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before or 1 to 3 days after tamoxifen injection. Results represent at least 3 independent experiments. Scale bars, 100 μm. (G) Quantification of the number of alcian blue positive cells per crypt-villus axis in mice of various genotypes. Data represent average values of approximately 10-15 different microscopic fields taken from 3 mice per condition.(H) Immunofluorescent staining for lysozyme and E-Cad was performed on adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (I) Quantification of lysozyme fluorescent signal abundance per crypt in mice of various genotypes. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.


Figure 2 ( B ) Immunohistochemistry for Ki67 staining was performed on intestinal sections of adult Rab11aFl / Fl ; Rab11b - / - ; Villin - CreER mice before and after tamoxifen injection . Experiments represent 3 independent replicates . Scale bars , 50 μm . ( C ) Quantification of ratio of Ki67 + cells over total epithelial cells per crypt - villus axis in DKO mice before and after tamoxifen treatment . Data represent approximately 10 - 15 different microscopic fields taken from 3 mice per condition . ( D ) Immunofluorescent staining for pHH3 and E - Cad was performed on intestinal sections of adult Rab11aFl / Fl ; Rab11b - / - ; Villin - CreER mice before and after tamoxifen injection . Experiments represent 3 independent replicates . Scale bars , 50 μm . ( E ) Quantification of ratio of pHH3 + cells over total IECs per crypt in DKO mice before and after tamoxifen treatment . Data represent approximately 10 - 15 different microscopic fields taken from 3 mice per condition . ( G ) Representative Western blots for Olfm4 and pHH3 in intestinal tissue lysates of DKO mice before and after tamoxifen injection . ( H ) Immunohistochemistry for Olfm4 was performed on intestinal sections of adult Rab11aFl / Fl ; Rab11b - / - ; Villin - CreER mice before and after tamoxifen injection . Experiments represent 3 independent replicates . Scale bars , 50 μm . ( I ) Quantification of Olfm4 + cell number per crypt in DKO mice before and after tamoxifen treatment . Data represent approximately 10 - 15 different microscopic fields taken from 3 mice per condition . ( J ) Immunofluorescent staining for Sox9 and E - Cad in DKO mice before and after tamoxifen treatment . Scale bars , 50 μm . ( K ) Quantification of Sox9 + cell number per crypt in DKO mice before and after tamoxifen treatment . Data represent approximately 10 - 15 different microscopic fields taken from 3 mice per condition . ( L ) Immunohistochemistry for c - Myc was performed on intestinal sections of adult Rab11aFl / Fl ; Rab11b - / - ; Villin - CreER mice before and after tamoxifen injection . Experiments represent 3 independent replicates . Scale bars , 50 μm . ( M ) Quantification of c - Myc + cell number per crypt - villus axis in DKO mice before and after tamoxifen treatment . Data represent approximately 10 - 15 different microscopic fields taken from 3 mice per condition .

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Figure 2(B) Immunohistochemistry for Ki67 staining was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (C) Quantification of ratio of Ki67+ cells over total epithelial cells per crypt-villus axis in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.(D) Immunofluorescent staining for pHH3 and E-Cad was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (E) Quantification of ratio of pHH3+ cells over total IECs per crypt in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.(G) Representative Western blots for Olfm4 and pHH3 in intestinal tissue lysates of DKO mice before and after tamoxifen injection.(H) Immunohistochemistry for Olfm4 was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (I) Quantification of Olfm4+ cell number per crypt in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.(J) Immunofluorescent staining for Sox9 and E-Cad in DKO mice before and after tamoxifen treatment. Scale bars, 50 μm. (K) Quantification of Sox9+ cell number per crypt in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.(L) Immunohistochemistry for c-Myc was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (M) Quantification of c-Myc+ cell number per crypt-villus axis in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.


Figure 3 ( F ) High magnification images were taken from CIdU / IdU labeled WT and DKO mouse crypt regions to visualize double positive cells . White arrows point to shedding CIdU cells . Scale bars , 50 μm . ( G - H ) Quantification of IdU + cells , and shedding CIdU + cells per crypt - villus axis in WT and DKO mice . Data represent 10 - 15 different microscopic fields taken from 3 mice per condition .

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Figure 3(F) High magnification images were taken from CIdU/IdU labeled WT and DKO mouse crypt regions to visualize double positive cells. White arrows point to shedding CIdU cells. Scale bars, 50 μm. (G-H) Quantification of IdU+ cells, and shedding CIdU+ cells per crypt-villus axis in WT and DKO mice. Data represent 10-15 different microscopic fields taken from 3 mice per condition.


Figure 4 ( F ) Co - IP assays were performed in HEK293T cells to validate the association between 3 × Flag - Rab11a , 3 × Flag - Rab11b and endogenous KIF11 . Lane 1 : input lysate ; lane 2 : flow through after co - IP ; lane 3 - 5 : 3 washes ; lane 6 : on beads ; and lane 7 : immunoprecipitation elutes . Cells transfected with 3 × Flag empty vector were uses as a negative control . Experiments repeated over 6 times . ( G ) Cells were transfected with mCherry - tagged WT , Rab11 - S25N , and Rab11 - S20V , fixed and stained for endogenous KIF11 ( grey ) and pericentrin ( green ) . The mCherry signal ( red ) represents direct fluorescent signal visualized under confocal microscope . Arrows denote position of spindle poles defined by pericentrin . Images represent three independent experiments . Scale bars , 10 μ ( H ) Quantification of the percentage of bipolar , monopolar , or multipolar ( 3 or more ) spindles observed in mitotic cells identified from each microscopic field . Data represents 10 - 20 independent fields per condition from 3 experiments . ( I ) Immunofluorescent staining for KIf11 ( green ) and E - Cad ( grey ) in WT and DKO mouse intestines . Yellow arrows point to Kif11 + spindles . Images represents at least 10 images per mouse and 5 mice for each genotype . Scale bars , 50 μm . ( J ) WT and DKO mice were injected with IdU 3 hrs before sacrifice to label new dividing cells . Immunofluorescent staining for KIf11 ( green ) , E - Cad ( grey ) , and IdU ( red ) were performed . Yellow arrows point to abnormal Kif11 localization in DKO cells . Scale bars , 50 μm . ( L - M ) Immunofluorescent staining for KIf11 ( red ) and spindle pole markers γ - tubulin or pericentrin ( green ) in WT and DKO mouse intestines . Images represent 3 mice each genotype . White arrows point to spindle pole . Scale bars , 20 μm , 10 μm .

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Figure 4(F) Co-IP assays were performed in HEK293T cells to validate the association between 3×Flag-Rab11a, 3×Flag-Rab11b and endogenous KIF11. Lane 1: input lysate; lane 2: flow through after co-IP; lane 3-5: 3 washes; lane 6: on beads; and lane 7: immunoprecipitation elutes. Cells transfected with 3×Flag empty vector were uses as a negative control. Experiments repeated over 6 times.(G) Cells were transfected with mCherry-tagged WT, Rab11-S25N, and Rab11-S20V, fixed and stained for endogenous KIF11 (grey) and pericentrin (green). The mCherry signal (red) represents direct fluorescent signal visualized under confocal microscope. Arrows denote position of spindle poles defined by pericentrin. Images represent three independent experiments. Scale bars, 10 μ (H) Quantification of the percentage of bipolar, monopolar, or multipolar (3 or more) spindles observed in mitotic cells identified from each microscopic field. Data represents 10-20 independent fields per condition from 3 experiments.(I) Immunofluorescent staining for KIf11 (green) and E-Cad (grey) in WT and DKO mouse intestines. Yellow arrows point to Kif11+ spindles. Images represents at least 10 images per mouse and 5 mice for each genotype. Scale bars, 50 μm. (J) WT and DKO mice were injected with IdU 3 hrs before sacrifice to label new dividing cells. Immunofluorescent staining for KIf11 (green), E-Cad (grey), and IdU (red) were performed. Yellow arrows point to abnormal Kif11 localization in DKO cells. Scale bars, 50 μm.(L-M) Immunofluorescent staining for KIf11 (red) and spindle pole markers γ-tubulin or pericentrin (green) in WT and DKO mouse intestines. Images represent 3 mice each genotype. White arrows point to spindle pole. Scale bars, 20 μm, 10 μm.


Figure 5 ( B - C ) HEK293T cells were transiently transfected with 3 × Flag - Rab11a ( or 3 × Flag - Rab11b ) and V5 - tagged KIF11 truncates . Lysates were immunoprecipitated by anti - Flag antibody , and probed by an anti - V5 antibody to assess intracellular associations . Cells transfected by V5 empty vector were used as a negative control . ( G ) Quantification of co - immunoprecipitated KIF11 was normalized to input KIF11 from cells treated by different inhibitors . DMSO data was quantified for 5 experimental replicates and all other treatments were quantified for 4 experimental replicates . ( H ) HEK293T cells were transiently transfected with 3 × Flag - Rab11a , treated with nocodazole ( 100 ng / ml ) or vehicle overnight , and lysates were used for Flag immunoprecipitation followed by immunoblot for KIF11 . Quantification of co - immunoprecipitated KIF11 was normalized to input KIF11 from control ( n = 6 ) and nocodazole - treated ( n = 9 ) cells . ( L ) KIF11 co - IP analysis were performed using WT and RAB11 - KD CaCo2 cell lysates . The immunoprecipitates were probed for CLIP1 , ZW10 , CCAR1 , USO1 , and CCNB1 . Experiments represent 2 - 4 replicates for each target . ( N ) Immunofluorescent staining for endogenous Clip1 ( red ) and Kif11 ( green ) in WT and Rab11 DKO mouse intestinal epithelial tissues . n = 4 - 5 mice for each genotype . White arrows point to Kif11 + spindles . Scale bars , 20 μm .

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Figure 5(B-C) HEK293T cells were transiently transfected with 3×Flag-Rab11a (or 3×Flag-Rab11b) and V5-tagged KIF11 truncates. Lysates were immunoprecipitated by anti-Flag antibody, and probed by an anti-V5 antibody to assess intracellular associations. Cells transfected by V5 empty vector were used as a negative control.(G) Quantification of co-immunoprecipitated KIF11 was normalized to input KIF11 from cells treated by different inhibitors. DMSO data was quantified for 5 experimental replicates and all other treatments were quantified for 4 experimental replicates.(H) HEK293T cells were transiently transfected with 3×Flag-Rab11a, treated with nocodazole (100 ng/ml) or vehicle overnight, and lysates were used for Flag immunoprecipitation followed by immunoblot for KIF11. Quantification of co-immunoprecipitated KIF11 was normalized to input KIF11 from control (n=6) and nocodazole-treated (n=9) cells.(L) KIF11 co-IP analysis were performed using WT and RAB11-KD CaCo2 cell lysates. The immunoprecipitates were probed for CLIP1, ZW10, CCAR1, USO1, and CCNB1. Experiments represent 2-4 replicates for each target.(N) Immunofluorescent staining for endogenous Clip1 (red) and Kif11 (green) in WT and Rab11 DKO mouse intestinal epithelial tissues. n=4-5 mice for each genotype. White arrows point to Kif11+ spindles. Scale bars, 20 μm.


Figure 6 ( B ) Propidium iodide ( PI ) staining of WT , BKO , and DKO enteroids 3 days after 4 - OHT treatment ex vivo . Note DKO enteroids had increased PI staining along the epithelial edge . Vehicle treated DKO enteroids were used as controls . Scale bars , 200 μm . ( C ) PI - stained areas were normalized to total enteroid area for each enteroid of different genotypes . Immunofluorescent staining for Kif11 ( red ) and E - cad ( green ) were performed on enteroids of various genotypes , all of which were treated with 4 - OHT . Same experiments were done for DKO enteroids treated with vehicle or 4 - OHT . White arrows point to Kif11 + spindles . Scale bars , 20 μm . ( I - J ) Numbers of pHH3 + or Lyz1 + cells were counted per enteroid of vehicle ( n = 11 for pHH3 ; n = 8 for Lyz1 ) or 4 - OHT treated DKO ( n = 9 for both pHH3 and Lyz1 ) enteroids from biological replicates . ( K - L ) Representative confocal fluorescent images of pHH3 ( red ) or Lyz1 ( red ) stained with E - Cad ( green ) from vehicle or 4 - OHT treated DKO enteroids . Scale bars , 50 μm .

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Figure 6(B) Propidium iodide (PI) staining of WT, BKO, and DKO enteroids 3 days after 4-OHT treatment ex vivo. Note DKO enteroids had increased PI staining along the epithelial edge. Vehicle treated DKO enteroids were used as controls. Scale bars, 200 μm. (C) PI-stained areas were normalized to total enteroid area for each enteroid of different genotypes.Immunofluorescent staining for Kif11 (red) and E-cad (green) were performed on enteroids of various genotypes, all of which were treated with 4-OHT. Same experiments were done for DKO enteroids treated with vehicle or 4-OHT. White arrows point to Kif11+ spindles. Scale bars, 20 μm.(I-J) Numbers of pHH3+ or Lyz1+ cells were counted per enteroid of vehicle (n=11 for pHH3; n=8 for Lyz1) or 4-OHT treated DKO (n=9 for both pHH3 and Lyz1) enteroids from biological replicates.(K-L) Representative confocal fluorescent images of pHH3 (red) or Lyz1 (red) stained with E-Cad (green) from vehicle or 4-OHT treated DKO enteroids. Scale bars, 50 μm.


Figure 7 ( A ) Immunohistochemistry for cleaved caspase 3 ( CC3 ) was performed on TAM - treated enteroids of different genotypes . Black arrows point to CC3 + cells at the epithelial lining of DKO enteroids . Scale bars , 50 μm . Representative apoptotic genes , Anxa1 , Anxa5 , Bcl2l1 which were highly elevated in DKO intestines . n = 3 mice for each genotype . ( I ) Immunofluorescent staining for CC3 ( green ) was performed on WT and Rab11 DKO intestinal epithelial tissues before and after tamoxifen treatment . Scale bars , 50 μm . ( J ) Quantification of CC3 positive cells per crypt - villus axis in WT and Rab11 DKO intestines . Results were obtained from 10 - 15 independent microscopic images taken from 4 - 5 mice for each genotype . ( N ) Immunohistochemistry for NFκB was performed on WT and Rab11 DKO intestinal tissues . ( O ) Quantification of ratio of nuclear NFκB + cell number over total IECs per crypt - villus axis . Data were analyzed from 10 - 15 independent microscopic fields . n = 4 - 5 mice for each genotype . ( P ) Immunofluorescent staining for p53 ( green ) and E - cad ( red ) in WT and Rab11 DKO intestinal tissues . Scale bars , 50 μm , 20 μm .

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Figure 7(A) Immunohistochemistry for cleaved caspase 3 (CC3) was performed on TAM-treated enteroids of different genotypes. Black arrows point to CC3+ cells at the epithelial lining of DKO enteroids. Scale bars, 50 μm.Representative apoptotic genes, Anxa1, Anxa5, Bcl2l1 which were highly elevated in DKO intestines. n=3 mice for each genotype.(I) Immunofluorescent staining for CC3 (green) was performed on WT and Rab11 DKO intestinal epithelial tissues before and after tamoxifen treatment. Scale bars, 50 μm. (J) Quantification of CC3 positive cells per crypt-villus axis in WT and Rab11 DKO intestines. Results were obtained from 10-15 independent microscopic images taken from 4-5 mice for each genotype.(N) Immunohistochemistry for NFκB was performed on WT and Rab11 DKO intestinal tissues. (O) Quantification of ratio of nuclear NFκB+ cell number over total IECs per crypt-villus axis. Data were analyzed from 10-15 independent microscopic fields. n=4-5 mice for each genotype.(P) Immunofluorescent staining for p53 (green) and E-cad (red) in WT and Rab11 DKO intestinal tissues. Scale bars, 50 μm, 20 μm.


Figure 16mA / dA levels ( mol / mol ) of seven stages of Hydractinia development . purple area indicates the background level of 6mA contamination from the digestive enzymes used in the respective experiment . hpf : hours post fertilization . The number of biological replicates used for each sample are indicated by the amount of data points in the graph . Whole - mount immunofluorescence of 6mA from 16 - and 64 - cell stages of Hydractinia . Samples were RNase - treated . . In D , the scale bare is equal to 20 µm .

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Figure 16mA/dA levels (mol/mol) of seven stages of Hydractinia development . purple area indicates the background level of 6mA contamination from the digestive enzymes used in the respective experiment. hpf: hours post fertilization. The number of biological replicates used for each sample are indicated by the amount of data points in the graph.Whole-mount immunofluorescence of 6mA from 16- and 64-cell stages of Hydractinia. Samples were RNase-treated. . In D, the scale bare is equal to 20 µm.


Figure 2Whole - mount immunofluorescence of anti - 6mA in 64 - 128 - cell embryos upon injection of shGFP ( as control ) , shAlkbh1Relative quantification of anti - 6mA signals from immunofluorescence images Fluorescence intensity were normalized to the highest and lowest measured area in shGFP ( n = 135 ) , shAlkbh1 ( n = 168 ) , and rescue ( n = 140 ) , where n = nuclei numbers . Central band shows the mean , the boxes show lower and upper quartiles and whiskers show minimum and maximum data values . Quantification of shAlkbh1 - electroporated embryos showing significantly higher level of 6mA / dA ( P < 0 . 05 ) compared to shGFP electroporated embryos and to wild type embryos at 64 - 128 cell stage .

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Figure 2Whole-mount immunofluorescence of anti-6mA in 64-128-cell embryos upon injection of shGFP (as control), shAlkbh1Relative quantification of anti-6mA signals from immunofluorescence images Fluorescence intensity were normalized to the highest and lowest measured area in shGFP (n=135), shAlkbh1 (n=168), and rescue (n=140), where n = nuclei numbers. Central band shows the mean, the boxes show lower and upper quartiles and whiskers show minimum and maximum data values.Quantification of shAlkbh1-electroporated embryos showing significantly higher level of 6mA/dA (P<0.05) compared to shGFP electroporated embryos and to wild type embryos at 64-128 cell stage.


Figure 3High EU incorporation in 64 - cell but undetectable in 16 - cell embryos of Hydractinia . RNase treatment abolishes the EU signal .

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Figure 3High EU incorporation in 64-cell but undetectable in 16-cell embryos of Hydractinia.RNase treatment abolishes the EU signal.


Figure 4Whole - mount image of EU incorporation signals at 64 cells upon injection with shGFP , shAlkbh1 , rescue , and mutant - rescue solution Relative quantification of EU signals Fluorescence intensity were normalized to the highest and lowest measured area in shGFP ( n = 86 ) , shAlkbh1 ( n = 63 ) , rescue ( n = 64 ) , and mutant - rescue ( n = 66 ) where n = nuclei numbers . Central band shows the mean , the boxes show lower and upper quartiles and whiskers show minimum and maximum data values .

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Figure 4Whole-mount image of EU incorporation signals at 64 cells upon injection with shGFP, shAlkbh1, rescue, and mutant-rescue solution Relative quantification of EU signals Fluorescence intensity were normalized to the highest and lowest measured area in shGFP (n=86), shAlkbh1 (n=63), rescue (n=64), and mutant-rescue (n=66) where n = nuclei numbers. Central band shows the mean, the boxes show lower and upper quartiles and whiskers show minimum and maximum data values.


Figure 5Rapid decline of m6A - marked maternal RNA occurs between the 2 - to 16 - cell stages , analyzed by UHPLC - QQQ of m6A / A ( % in arbitrary unit ) from four Hydractinia developmental stages . Replication stall at 8 - 16 nuclei following hydroxyurea treatment . The control shows a normal number of nuclei at the same developmental stage as treated one . Whole - mount immunofluorescence of anti - 6mA in 32 - cell embryos upon injection of ATP and m6ATP at 20 mM The uninjected control shows a normal level of anti - 6mA at 32 - cell embryos .

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Figure 5Rapid decline of m6A-marked maternal RNA occurs between the 2- to 16-cell stages, analyzed by UHPLC-QQQ of m6A/A (% in arbitrary unit) from four Hydractinia developmental stages.Replication stall at 8-16 nuclei following hydroxyurea treatment. The control shows a normal number of nuclei at the same developmental stage as treated one.Whole-mount immunofluorescence of anti-6mA in 32-cell embryos upon injection of ATP and m6ATP at 20 mM The uninjected control shows a normal level of anti-6mA at 32-cell embryos.


Figure 1B Western blot analysis ( probing with an anti - FLAG mAb ) to detect cleavage of the artificial substrate by wild - type ( WT ) / inactive ( S201A ) GlpG encoded on pBAD33 with / without arabinose ( Ara . ) . rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red , arrows , respectively . Controls , empty pBAD33 ( empty ) C Western blot analysis to detect cleavage of the artificial substrate by wild - type ( WT ) / modified ( S133A or H187A ) Rhom7 encoded on pBAD33 with / without arabinose ( Ara . ) . , rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , and blue arrows , respectively . Controls , empty pBAD33 ( empty ) and wild - type S . sonnei ( Ss ) . D Activity of Rhom7 with / without its 7th TMD and / or C - terminal domain . rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , , and blue arrows , respectively . Controls , empty pBAD33 ( empty ) and wild - type S . sonnei ( Ss ) .

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Figure 1B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of the artificial substrate by wild-type (WT)/inactive (S201A) GlpG encoded on pBAD33 with/without arabinose (Ara.). rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, empty pBAD33 (empty)C Western blot analysis to detect cleavage of the artificial substrate by wild-type (WT)/modified (S133A or H187A) Rhom7 encoded on pBAD33 with/without arabinose (Ara.). , rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).D Activity of Rhom7 with/without its 7th TMD and/or C-terminal domain. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, , and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).


Figure 2C Western blot analysis of candidate substrates reproducibly cleaved by GlpG from the screen . Data information : In ( C rhomboid substrates that are uncleaved , cleaved by GlpG are marked by black , red , arrows , respectively . Controls , inactive enzymes and wild - type S . sonnei ( Ss ) . D Western blot analysis of candidate substrates reproducibly cleaved by Rhom7 from the screen . Data information : In D ) rhomboid substrates that are uncleaved , cleaved by Rhom7 , are marked by black , red , and blue arrows , respectively . Controls , inactive enzymes and wild - type S . sonnei ( Ss ) .

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Figure 2C Western blot analysis of candidate substrates reproducibly cleaved by GlpG from the screen. Data information: In (C rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).D Western blot analysis of candidate substrates reproducibly cleaved by Rhom7 from the screen. Data information: In D) rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, red, and blue arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).