Figure 5 ( A ) 293 / GFP - LC3 cells were pre ‐ treated with 10 μM anisomycin for 30 min before incubation in either full medium or EBSS . Cell lysates were analysed by SDS - PAGE for GFP - LC3 lipidation using an anti ‐ LC3 antibody . The membrane was also probed with anti ‐ β tubulin . The GFP - LC3 lipidation was quantified as the amount of GFP - LC3II / GFP - LC3I ( data are represented as mean ± s . e . m . of triplicates , P = 0 . 0091 , Student ' s t ‐ test ) . ( B ) HEK293A cells labelled with [ 14C ] valine , as described in Material and Methods section , were pretreated with 10 μM anisomycin for 30 min before incubation in either full medium , EBSS , or EBSS with leupeptin for 2 h . Cells were then analysed for long ‐ lived protein degradation ( data are represented as mean ± s . e . m . of triplicates , representative of two experiments , EBSS untreated versus anisomycin treated ( P = 0 . 0363 ) ; EBSS untreated versus leupeptin treated ( P = 0 . 0001 ) , Student ' s t ‐ test ) . ( C ) HEK293 cells were transfected with control siRNA or siRNA for p38IP . After 24 ‐ h incubation , control or p38IP ‐ depleted cells were pre ‐ treated with anisomycin , followed by a 2 ‐ h incubation with full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin . Cell lysates were analysed with anti ‐ p38α or anti ‐ LC3 antibodies . LC3II / LC3I levels were quantified and presented normalized to control , untreated cells incubated in EBSS . Data are representative of two experiments . ( D ) HEK293A cells transiently transfected with HA ‐ p38IP and RFP ‐ mAtg9 were treated with anisomycin for 30 min and mAtg9 was immunoprecipitated . 5 % of the lysates or the immunoprecipitates were probed with anti ‐ HA and anti ‐ mAtg9 antibodies ( data is representative of 3 experiments , * P = 0 . 0332 ) .

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

Figure 5(A) 293/GFP-LC3 cells were pre‐treated with 10 μM anisomycin for 30 min before incubation in either full medium or EBSS. Cell lysates were analysed by SDS-PAGE for GFP-LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐β tubulin. The GFP-LC3 lipidation was quantified as the amount of GFP-LC3II/GFP-LC3I (data are represented as mean±s.e.m. of triplicates, P=0.0091, Student's t‐test).(B) HEK293A cells labelled with [14C]valine, as described in Material and Methods section, were pretreated with 10 μM anisomycin for 30 min before incubation in either full medium, EBSS, or EBSS with leupeptin for 2 h. Cells were then analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS untreated versus anisomycin treated (P=0.0363); EBSS untreated versus leupeptin treated (P=0.0001), Student's t‐test).(C) HEK293 cells were transfected with control siRNA or siRNA for p38IP. After 24‐h incubation, control or p38IP‐depleted cells were pre‐treated with anisomycin, followed by a 2‐h incubation with full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin. Cell lysates were analysed with anti‐p38α or anti‐LC3 antibodies. LC3II/LC3I levels were quantified and presented normalized to control, untreated cells incubated in EBSS. Data are representative of two experiments.(D) HEK293A cells transiently transfected with HA‐p38IP and RFP‐mAtg9 were treated with anisomycin for 30 min and mAtg9 was immunoprecipitated. 5% of the lysates or the immunoprecipitates were probed with anti‐HA and anti‐mAtg9 antibodies (data is representative of 3 experiments, *P=0.0332).


Figure 6 ( A ) HEK293A cells were transfected with RFP - mAtg9 and Flag - p38 ( lanes 2 and 7 ) , RFP - mAtg9 , Flag - p38 and HA - p38IP ( lanes 3 and 8 ) , or RFP - mAtg9 and HA - p38IP ( lanes 4 and 9 ) . RFP - mAtg9 was immunoprecipitated with an anti ‐ mAtg9 antibody , the co ‐ immunoprecipitated HA - p38IP was detected with an anti ‐ HA antibody , and co ‐ immunoprecipitated Flag - p38 was detected with an anti ‐ Flag antibody . RFP - mAtg9 was detected with an anti ‐ Atg9 antibody . ( B ) HEK293A cells were transfected with RFP - Atg9 and HA - p38IP ( lanes 1 and 5 ) , Flag - Flag , Flag - p38 , and HA - p38IP ( lanes 2 and 6 ) , or Flag - p38 and HA - p38IP ( lanes 3 and 7 ) . HA - p38IP was immunoprecipitated with an anti ‐ HA antibody , the co ‐ immunoprecipitated RFP - Atg9 was detected with an anti ‐ Atg9 antibody , and co ‐ immunoprecipitated Flag - p38 was detected with an anti ‐ p38α antibody . HA - p38IP was detected with an anti ‐ HA antibody . ( C ) HEK293A cells were transfected with Flag - p38α for 24 h . Cells were incubated in either full medium , EBSS , or EBSS plus leupeptin for 2 h , lysed , and analysed for endogenous LC3 lipidation using an anti ‐ LC3 antibody , and immunoblotted with anti ‐ Flag antibody . LC3 lipidation was quantified as the amount of LC3II / LC3I ( data are represented as mean ± s . e . m . of triplicates , n = 2 experiments , EBSS control versus EBSS with Flag ‐ p38α , * * * P = 0 . 0026 ) .

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 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 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 6(A) HEK293A cells were transfected with RFP-mAtg9 and Flag-p38 (lanes 2 and 7), RFP-mAtg9, Flag-p38 and HA-p38IP (lanes 3 and 8), or RFP-mAtg9 and HA-p38IP (lanes 4 and 9). RFP-mAtg9 was immunoprecipitated with an anti‐mAtg9 antibody, the co‐immunoprecipitated HA-p38IP was detected with an anti‐HA antibody, and co‐immunoprecipitated Flag-p38 was detected with an anti‐Flag antibody. RFP-mAtg9 was detected with an anti‐Atg9 antibody.(B) HEK293A cells were transfected with RFP-Atg9 and HA-p38IP (lanes 1 and 5), Flag-Flag, Flag-p38, and HA-p38IP (lanes 2 and 6), or Flag-p38 and HA-p38IP (lanes 3 and 7). HA-p38IP was immunoprecipitated with an anti‐HA antibody, the co‐immunoprecipitated RFP-Atg9 was detected with an anti‐Atg9 antibody, and co‐immunoprecipitated Flag-p38 was detected with an anti‐p38α antibody. HA-p38IP was detected with an anti‐HA antibody.(C) HEK293A cells were transfected with Flag-p38α for 24 h. Cells were incubated in either full medium, EBSS, or EBSS plus leupeptin for 2 h, lysed, and analysed for endogenous LC3 lipidation using an anti‐LC3 antibody, and immunoblotted with anti‐Flag antibody. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean±s.e.m. of triplicates, n=2 experiments, EBSS control versus EBSS with Flag‐p38α, ***P=0.0026).


Figure 7 ( A ) 293 / GFP - LC3 cells were transfected with control or p38α siRNA . At 72 h after transfection , cells were incubated in either full medium , EBSS , or EBSS with leupeptin for 2 h , then fixed and visualized by confocal microscopy . Bars = 5 μm ( data are represented as mean ± s . e . m . n = 60 cells , EBSS control versus p38α siRNA ( * * * P = 0 . 0001 ) ; EBSS with leupeptin control versus p38α siRNA ( * * * P = 0 . 0001 ) , Student ' s t ‐ test ) . ( B ) HEK293A cells were transfected with control or p38α siRNA . At 72 h after transfection , cells were incubated in either full medium , EBSS , or EBSS containing leupeptin for 2 h . Cells lysates were analysed for LC3 lipidation using an anti ‐ LC3 antibody . The membrane was also probed with anti ‐ actin and anti ‐ p38α antibodies . LC3 lipidation was quantified as the amount of LC3II / LC3I ( data are represented as mean ± s . e . m . n = 5 , full medium control versus p38α siRNA ( * * P = 0 . 0056 ) ; EBSS control versus p38α siRNA ( * * P = 0 . 048 ) ; EBSS with Leu control versus p38α siRNA ( * * P = 0 . 04 ) , Student ' s t ‐ test ) . ( C ) HEK293A cells were transfected with control or p38α siRNA . At 72 h after transfection , cells were incubated in either full medium or EBSS for 2 h , then fixed and immunostained to detect endogenous mAtg9 localization . The percentage of cells with dispersed mAtg9 was quantified as in Figure 2E ( data are represented as mean ± s . e . m . n = 200 cells , full medium control versus p38α siRNA ( * * P = 0 . 0027 ) ; EBSS control versus p38α siRNA ( * P = 0 . 0459 ) ; Student ' s t ‐ test ) . Bars = 5 μm ( D ) HEK293A cells were transfected with siRNA for p38α , homogenized and subjected to centrifugation , and the resulting post ‐ nuclear supernatant ( PNS ) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol as in Figure 2A . Equal protein amounts were resolved by SDS - PAGE and immunoblotted with anti ‐ MPR , anti ‐ HA , and anti ‐ p38α antibodies . Data are representative of four experiments . ( E ) 72 h cells were transfected with control , p38α siRNA or both p38α siRNA , and p38IP siRNA . At 72 h after transfection , cells were incubated in either full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin for 2 h . Cells lysates were analysed for LC3 lipidation using an anti ‐ LC3 antibody . The membrane was also probed with anti ‐ p38α . LC3 lipidation was quantified as the amount of LC3II / LC3I . Data are representative of two 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 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 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 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 O O O O O O O O O O O O O O O O O

Figure 7(A) 293/GFP-LC3 cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS with leupeptin for 2 h, then fixed and visualized by confocal microscopy. Bars=5 μm (data are represented as mean±s.e.m. n=60 cells, EBSS control versus p38α siRNA (***P=0.0001); EBSS with leupeptin control versus p38α siRNA (***P=0.0001), Student's t‐test).(B) HEK293A cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS containing leupeptin for 2 h. Cells lysates were analysed for LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐actin and anti‐p38α antibodies. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean ±s.e.m. n=5, full medium control versus p38α siRNA (**P=0.0056); EBSS control versus p38α siRNA (**P=0.048); EBSS with Leu control versus p38α siRNA (**P=0.04), Student's t‐test).(C) HEK293A cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium or EBSS for 2 h, then fixed and immunostained to detect endogenous mAtg9 localization. The percentage of cells with dispersed mAtg9 was quantified as in Figure 2E (data are represented as mean±s.e.m. n=200 cells, full medium control versus p38α siRNA (**P=0.0027); EBSS control versus p38α siRNA (*P=0.0459); Student's t‐test). Bars=5 μm(D) HEK293A cells were transfected with siRNA for p38α, homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol as in Figure 2A. Equal protein amounts were resolved by SDS-PAGE and immunoblotted with anti‐MPR, anti‐HA, and anti‐p38α antibodies. Data are representative of four experiments.(E) 72 h cells were transfected with control, p38α siRNA or both p38α siRNA, and p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells lysates were analysed for LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐p38α. LC3 lipidation was quantified as the amount of LC3II/LC3I. Data are representative of two experiments.


Figure 1 ( D - G ) Cell death mechanisms of C3H BMDMs transfected with miR - 342 - 3p mimic ( D ) , or B6 BMDMs transfected with miR - 342 - 3p inhibitor ( F ) , followed by Mtb infection for 36 hours . Cell viabilities of C3H BMDMs transfected with miR - 342 - 3p mimic ( E ) , or B6 BMDMs transfected with miR - 342 - 3p inhibitor ( G ) , followed by stimulation with Mtb or z - VAD ( 20 μM ) for 24 hours . Data are shown as the mean ± s . e . m . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(D-G) Cell death mechanisms of C3H BMDMs transfected with miR-342-3p mimic (D), or B6 BMDMs transfected with miR-342-3p inhibitor (F), followed by Mtb infection for 36 hours. Cell viabilities of C3H BMDMs transfected with miR-342-3p mimic (E), or B6 BMDMs transfected with miR-342-3p inhibitor (G), followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


Figure 2 ( A , B ) Survival of Mir342 + / + C3H mice and their wild - type littermate controls ( C3H ) ( n = 10 ) ( A ) , or Mir342 - / - B6 mice and their wild - type littermate controls ( B6 ) ( n = 10 ) ( B ) after aerosol infection with around 400 CFU Mtb . Data are shown as the Kaplan - Meier curves . ( G , H ) Secretion of cytokines TNF - α , IL - 1 , IL - 6 and CXCL15 in BMDMs obtained from Mir342 + / + C3H and their littermates ( C3H ) ( G ) , or from Mir342 - / - B6 and their littermates ( B6 ) ( H ) after Mtb stimulation , was detected by ELISA . Data are shown as the mean ± s . e . m . 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 O O O O O O O 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

Figure 2(A, B) Survival of Mir342+/+C3H mice and their wild-type littermate controls (C3H) (n=10) (A), or Mir342-/-B6 mice and their wild-type littermate controls (B6) (n=10) (B) after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.(G, H) Secretion of cytokines TNF-α, IL-1, IL-6 and CXCL15 in BMDMs obtained from Mir342+/+C3H and their littermates (C3H) (G), or from Mir342-/-B6 and their littermates (B6) (H) after Mtb stimulation, was detected by ELISA. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


Figure 3 ( C , D ) MiR - 342 - 3p mimic or inhibitor was transfected to NIH - 3T3 fibroblasts . After 48 hours , cells were collected for qRT - PCR ( C , data are shown as the mean ± s . e . m . of n = 3 biological replicates ) and western blotting ( D , representative blots from n = 3 biological replicates are shown ) to detect SOCS6 expression . ( F - H ) C3H BMDMs were transfected with miR - 342 - 3p mimic or SOCS6 - overexpressing lentivirus , followed by stimulation with Mtb or z - VAD ( 20 μM ) for 24 hours . Cell death mechanisms ( F ) , cell viabilities ( G ) and Mtb growth rates ( H ) were analyzed respectively . Data are shown as the mean ± s . e . m . 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 O O O O O O O O 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

Figure 3(C, D) MiR-342-3p mimic or inhibitor was transfected to NIH-3T3 fibroblasts. After 48 hours, cells were collected for qRT-PCR (C, data are shown as the mean ± s.e.m. of n = 3 biological replicates) and western blotting (D, representative blots from n = 3 biological replicates are shown) to detect SOCS6 expression.(F-H) C3H BMDMs were transfected with miR-342-3p mimic or SOCS6-overexpressing lentivirus, followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Cell death mechanisms (F), cell viabilities (G) and Mtb growth rates (H) were analyzed respectively. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


Figure 4 ( A , B ) Survival of Mir342 - / - B6 and Mir342 - / - B6 mice supplemented with Socs6 siRNA ( n = 10 ) ( A ) , or Mir342 + / + C3H and Mir342 + / + C3H mice supplemented with SOCS6 - overexpressing vector ( n = 10 ) ( B ) , after aerosol infection with around 400 CFU Mtb . Data are shown as the Kaplan - Meier curves .

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

Figure 4(A, B) Survival of Mir342-/-B6 and Mir342-/-B6 mice supplemented with Socs6 siRNA (n=10) (A), or Mir342+/+C3H and Mir342+/+C3H mice supplemented with SOCS6-overexpressing vector (n=10) (B), after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.


Figure 5 ( C - F ) Relative expressions of chemokines CCL5 , CXCL10 , ICAM1 ( C ) , and Caspase 3 , Caspase 7 , Caspase 8 ( F ) in Mtb - stimulated Socs6 + / + or Socs6 - / - BMDMs were detected by qRT - PCR . Data are shown as the mean ± s . e . m . of n = 3 biological replicates . Cluster analysis of clinical samples .

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Figure 5(C-F) Relative expressions of chemokines CCL5, CXCL10, ICAM1 (C), and Caspase 3, Caspase 7, Caspase 8 (F) in Mtb-stimulated Socs6+/+ or Socs6-/- BMDMs were detected by qRT-PCR. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. Cluster analysis of clinical samples.


Figure 6 ( C ) Socs6 - / - BMDMs were transfected with plasmids expressing Myc - RIPK3 or Myc - RIPK3 K51A mutant for 24 hours . Afterwards , transfected cells were stimulated with Mtb for 12 hours , cell lysates were collected and immunoprecipitated using an anti - RIPK1 antibody . The recruitment of Caspase 8 , RIPK3 , FADD and MLKL was analyzed by immunoblot . The lower panel represents the immunoblot analysis of whole cell lysates . Representative blots from n = 3 biological replicates are shown . ( D - F ) Cell viabilities ( D ) , cell death mechanisms ( E ) , or Mtb growth rates ( F ) of Mtb - infected Socs6 - / - BMDMs that were transfected with plasmids expressing Myc - RIPK3 or Myc - RIPK3 K51A mutant as indicated . Z - VAD treatment concentration was 20 μM and the treatment time was 24 hours . Data are shown as the mean ± s . e . m . 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 O O O O O O O O O O O O O O O O O O O O O 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

Figure 6(C) Socs6-/- BMDMs were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant for 24 hours. Afterwards, transfected cells were stimulated with Mtb for 12 hours, cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of Caspase 8, RIPK3, FADD and MLKL was analyzed by immunoblot. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown.(D-F) Cell viabilities (D), cell death mechanisms (E), or Mtb growth rates (F) of Mtb-infected Socs6-/- BMDMs that were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant as indicated. Z-VAD treatment concentration was 20 μM and the treatment time was 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


Figure 7 ( E - G ) RAW264 . 7 cells were transfected with plasmids expressing A20 and RIPK3 ( E ) , or A20 and RIPK3 mutants ( G ) as indicated , cell lysates were collected for immunoprecipitation and immunoblot . Myc - A20 or Flag - RIPK3 purified from transfected HEK - 293T cells was incubated with ATP , E1 , E2 and ubiquitin as indicated . The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti - Ub antibody ( F ) . Representative blots from n = 3 biological replicates are shown .

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

Figure 7(E-G) RAW264.7 cells were transfected with plasmids expressing A20 and RIPK3 (E), or A20 and RIPK3 mutants (G) as indicated, cell lysates were collected for immunoprecipitation and immunoblot. Myc-A20 or Flag-RIPK3 purified from transfected HEK-293T cells was incubated with ATP, E1, E2 and ubiquitin as indicated. The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti-Ub antibody (F). Representative blots from n = 3 biological replicates are shown.


Figure 8 ( A , B ) Production of cytokines TNF - α , IL - 1 , IL - 6 , CXCL15 ( A ) , or chemokines Ccl5 , Cxcl10 , Icam1 ( B ) in the A20 - / - Socs6 + / + , A20 + / + Socs6 + / + , A20 - / - Socs6 - / - and A20 + / + Socs6 - / - BMDMs after Mtb stimulation . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

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Figure 8(A, B) Production of cytokines TNF-α, IL-1, IL-6, CXCL15 (A), or chemokines Ccl5, Cxcl10, Icam1 (B) in the A20-/-Socs6+/+, A20+/+Socs6+/+, A20-/-Socs6-/- and A20+/+Socs6-/- BMDMs after Mtb stimulation. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


Figure 1 ( B ) Genome browser representation of the SOX2 locus . The promoter is defined as the unmethylated region spanning the SOX2 transcriptional start site in MCF7 - control . Genomic distribution of ( C ) identified ZF - D3A binding sites , and ( D ) differentially methyl - ated regions ( DMRs ) between MCF7 - control and ZF - D3A + dox . ( E ) Shown for all DMRs identified between ZF - D3A + dox and MCF7 - control is : ∆ mCG ( difference in the methylation level [ ratio of C base calls to total base calls for all CG dinucleotides in region , mCG / CG ] between ZF - D3A + dox and MCF7 - control ) , ZF - D3A ChIP - seq signal ( normalized ChIP - seq read density , ± 1kb flanking DMR center ) , CpG dinucleotide density ( CpG / 100bp , ± 1kb flanking DMR center ) , and classification of each DMR as promoter or non - promoter located . ( F ) DNA methylation level in MCF7 - control and ZF - D3A + dox cells flanking ( ± 1 kb , 100 bp bins ) the center of ZF - D3A ChIP - seq peaks identified in ZF - D3A + dox . ( G ) Genomic distribution of unmethylated regions ( UMRs ) in MCF7 - control . ( H ) DMRs and UMRs with an intersection of ≥ 1 bp .

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 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 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

Figure 1(B) Genome browser representation of the SOX2 locus. The promoter is defined as the unmethylated region spanning the SOX2 transcriptional start site in MCF7-control.Genomic distribution of (C) identified ZF-D3A binding sites, and (D) differentially methyl- ated regions (DMRs) between MCF7-control and ZF-D3A +dox.(E) Shown for all DMRs identified between ZF-D3A +dox and MCF7-control is: ∆mCG (difference in the methylation level [ratio of C base calls to total base calls for all CG dinucleotides in region, mCG/CG] between ZF-D3A +dox and MCF7-control), ZF-D3A ChIP-seq signal (normalized ChIP-seq read density, ±1kb flanking DMR center), CpG dinucleotide density (CpG/100bp, ±1kb flanking DMR center), and classification of each DMR as promoter or non-promoter located.(F) DNA methylation level in MCF7-control and ZF-D3A +dox cells flanking (±1 kb, 100 bp bins) the center of ZF-D3A ChIP-seq peaks identified in ZF-D3A +dox.(G) Genomic distribution of unmethylated regions (UMRs) in MCF7-control. (H) DMRs and UMRs with an intersection of ≥1 bp.


Figure 2 ( A ) Line histogram of DMR methylation levels in each sample . Kernel density estimate smoothed scatterplot comparison of DMR methylation levels in MCF7 - control and ( B ) ZF - D3A + dox , or ( C ) ZF - D3A dox - wd . ( D ) Line histogram of UMR methylation levels in each sample . Scatterplot compari - son of UMR methylation levels in MCF7 - control and ( E ) ZF - D3A + dox , or ( F ) ZF - D3A dox - wd . ( G ) Heat - maps of DMRs ( ordered by decreasing H3K4me3 read density ) showing : DNA methylation levels ( ± 2 . 5 kb ) , differences ( ΔmCG ) , classification as promoter or non - promoter located , CpG dinucleotide density ( ± 2 . 5 kb ) , and histone modification ChIP - seq read density ( ± 2 . 5 kb ) ; ( H ) As in ( G ) but for the 6 , 000 DMRs with the lowest H3K4me3 levels , ordered by decreasing H3K4me1 read density , and DMR classification as enhancer or non - enhancer in MCF7 ( ENCODE ) .

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 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

Figure 2(A) Line histogram of DMR methylation levels in each sample. Kernel density estimate smoothed scatterplot comparison of DMR methylation levels in MCF7-control and (B) ZF-D3A +dox, or (C) ZF-D3A dox-wd.(D) Line histogram of UMR methylation levels in each sample. Scatterplot compari- son of UMR methylation levels in MCF7-control and (E) ZF-D3A +dox, or (F) ZF-D3A dox-wd.(G) Heat- maps of DMRs (ordered by decreasing H3K4me3 read density) showing: DNA methylation levels (±2.5 kb), differences (ΔmCG), classification as promoter or non-promoter located, CpG dinucleotide density (±2.5 kb), and histone modification ChIP-seq read density (±2.5 kb); (H) As in (G) but for the 6,000 DMRs with the lowest H3K4me3 levels, ordered by decreasing H3K4me1 read density, and DMR classification as enhancer or non-enhancer in MCF7 (ENCODE).


Figure 3 ( A ) H3K4me3 ChIP - seq read density flanking ( ± 1 kb , 50 bp bins ) the centre of DMRs with ∆ mCG > 0 . 2 ( top ) , and H3K4me3 ChIP - seq peaks with ∆ mCG < 0 . 2 ( bottom ) . ( B ) H3K4me3 ChIP - seq normalized read counts in DMRs with ∆ mCG > 0 . 2 , from H3K4me3 ChIP - seq in ZF - D3A no - dox and ZF - D3A + dox cells . Black line represents equal normalized read counts in ZF - D3A no - dox and ZF - D3A + dox cells ; blue line represents a linear regression of the data . ( C ) Comparison of UMR ∆ mCG and difference in H3K4me3 ChIP - seq normalized read counts between ZF - D3A no - dox and ZF - D3A + dox . Blue line represents a linear regression of the data . ( D ) DNA methylation levels of UMRs that intersect with H3K4me3 ChIP - seq peaks in ZF - D3A no - dox and ZF - D3A + dox cells , for genomic DNA or DNA immunoprecipitated with an anti - H3K4me3 antibody . ( E ) DNA methylation levels of UMRs that intersect with phos - pho - ser5 RNA polymerase II ChIP - seq peaks in ZF - D3A no - dox and ZF - D3A + dox cells , for genomic DNA or DNA immunoprecipitated with an anti - phospho - ser5 RNA polymerase II antibody .

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 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 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 3(A) H3K4me3 ChIP-seq read density flanking (±1 kb, 50 bp bins) the centre of DMRs with ∆mCG >0.2 (top), and H3K4me3 ChIP-seq peaks with ∆mCG <0.2 (bottom).(B) H3K4me3 ChIP-seq normalized read counts in DMRs with ∆mCG >0.2, from H3K4me3 ChIP-seq in ZF-D3A no-dox and ZF-D3A +dox cells. Black line represents equal normalized read counts in ZF-D3A no-dox and ZF-D3A +dox cells; blue line represents a linear regression of the data.(C) Comparison of UMR ∆mCG and difference in H3K4me3 ChIP-seq normalized read counts between ZF-D3A no-dox and ZF-D3A +dox. Blue line represents a linear regression of the data.(D) DNA methylation levels of UMRs that intersect with H3K4me3 ChIP-seq peaks in ZF-D3A no-dox and ZF-D3A +dox cells, for genomic DNA or DNA immunoprecipitated with an anti-H3K4me3 antibody.(E) DNA methylation levels of UMRs that intersect with phos- pho-ser5 RNA polymerase II ChIP-seq peaks in ZF-D3A no-dox and ZF-D3A +dox cells, for genomic DNA or DNA immunoprecipitated with an anti-phospho-ser5 RNA polymerase II antibody.


Figure 4 ( A ) Western blot measurement of ZF - D3A protein abundance upon doxycycline induction and withdrawal . ( B ) Box and whisker plots of the DNA methylation levels of the 121 CpG sites in the DACH1 promoter region through - out doxycycline induction and withdrawal . Whiskers indicate 1 . 5 times the interquartile range or the most extreme data point , whichever is lower . ( C ) Average DNA methylation level at the 121 CpG sites in the DACH1 promoter region throughout doxycycline induction followed by doxycycline withdrawal and cell cycle inhibition by growth in doxycycline - free media containing different cell cycle inhibitors ( Lovastatin , RO - 3306 , thymidine ) . ( D ) DMR mCG and hmCG levels in MCF7 - control and ZF - D3A + dox cells , ordered by the difference in DMR hmCG level ( hmCG / CG ) between MCF7 - control and ZF - D3A + dox cells , and heatmap representation of H3K4me3 levels in ZF - D3A + dox cells flanking ( ± 2 . 5 kb , 50 bp bins ) the centre of the DMRs . ( E ) Heatmap of hmCG levels flanking ( ± 2 . 5 kb , 50 bp bins ) the centre of the DMRs , ordered by DMR hmCG level in MCF7 - control cells .

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 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 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 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 4(A) Western blot measurement of ZF-D3A protein abundance upon doxycycline induction and withdrawal.(B) Box and whisker plots of the DNA methylation levels of the 121 CpG sites in the DACH1 promoter region through- out doxycycline induction and withdrawal. Whiskers indicate 1.5 times the interquartile range or the most extreme data point, whichever is lower.(C) Average DNA methylation level at the 121 CpG sites in the DACH1 promoter region throughout doxycycline induction followed by doxycycline withdrawal and cell cycle inhibition by growth in doxycycline-free media containing different cell cycle inhibitors (Lovastatin, RO-3306, thymidine).(D) DMR mCG and hmCG levels in MCF7-control and ZF-D3A +dox cells, ordered by the difference in DMR hmCG level (hmCG/CG) between MCF7-control and ZF-D3A +dox cells, and heatmap representation of H3K4me3 levels in ZF-D3A +dox cells flanking (±2.5 kb, 50 bp bins) the centre of the DMRs.(E) Heatmap of hmCG levels flanking (±2.5 kb, 50 bp bins) the centre of the DMRs, ordered by DMR hmCG level in MCF7-control cells.


Figure 5 ( A ) Scatter plot of the difference in UMR DNA methylation levels , versus the fold change in mRNA abun - dance of UMR - associated expressed genes , between MCF7 - control and ZF - D3A + dox . Point color indicates the gene differential expression significance : red indicates FDR < 0 . 01 , black indicates FDR > 0 . 01 . Trend line ( green ) was fitted using a generalized additive model , and contour lines represent the relative density of UMRs . ( B ) Distribution of the ratios of mRNA levels between ZF - D3A + dox and MCF7 - control , for expressed genes that have an associated UMR with a ∆ mCG > 0 . 3 . Column color indicates the class of mRNA abundance ratios between ZF - D3A + dox and MCF7 - control : blue = no decrease ; green = small decrease ; red = decrease . ( C ) Representative genes with high induction of promoter DNA methylation in ZF - D3A + dox and no reduction in mRNA abundance .

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

Figure 5(A) Scatter plot of the difference in UMR DNA methylation levels, versus the fold change in mRNA abun- dance of UMR-associated expressed genes, between MCF7-control and ZF-D3A +dox. Point color indicates the gene differential expression significance: red indicates FDR <0.01, black indicates FDR >0.01. Trend line (green) was fitted using a generalized additive model, and contour lines represent the relative density of UMRs.(B) Distribution of the ratios of mRNA levels between ZF-D3A +dox and MCF7-control, for expressed genes that have an associated UMR with a ∆mCG >0.3. Column color indicates the class of mRNA abundance ratios between ZF-D3A +dox and MCF7-control: blue = no decrease; green = small decrease; red = decrease.(C) Representative genes with high induction of promoter DNA methylation in ZF-D3A +dox and no reduction in mRNA abundance.


Figure 6Fluorescence distribution of cells stained with RNA - FISH probes targeting ( A ) GAPDH or ( B ) PSME1 mRNA before and after FACS . mRNA quantitation after FACS sorting of ( C ) GAPDH by RT - qPCR , and ( D ) PSME1 by RNA - seq . DNA methylation levels of single CpGs at the promoter region of ( E ) GAPDH , and ( F ) PSME1 , for the low ( x axis ) and high ( y axis ) expressing sorted cell populations . ( G ) Genome browser representation of the GAPDH ( top ) and PSME1 ( bottom ) promoter regions showing DNA methylation in MCF7 - control and low and high expressing sorted cell populations . ( H ) Distribution of per - read methylation levels ( blue line ) for selected ZF - D3A + dox WGBS reads ( ≥ 10 cytosines in the CpG context ) that align within robustly methylated DMRs ( DMR mean methylation level [ mCG / CG ] of 0 . 4 - 0 . 6 in ZF - D3A + dox and < 0 . 1 in MCF7 - control ) , and average CG methylation levels from ZF - D3A + dox WGBS data of the DMR genomic intervals covered by the selected reads ( red line ) .

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 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 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

Figure 6Fluorescence distribution of cells stained with RNA-FISH probes targeting (A) GAPDH or (B) PSME1 mRNA before and after FACS.mRNA quantitation after FACS sorting of (C) GAPDH by RT-qPCR, and (D) PSME1 by RNA-seq.DNA methylation levels of single CpGs at the promoter region of (E) GAPDH, and (F) PSME1, for the low (x axis) and high (y axis) expressing sorted cell populations.(G) Genome browser representation of the GAPDH (top) and PSME1 (bottom) promoter regions showing DNA methylation in MCF7-control and low and high expressing sorted cell populations.(H) Distribution of per-read methylation levels (blue line) for selected ZF-D3A +dox WGBS reads (≥10 cytosines in the CpG context) that align within robustly methylated DMRs (DMR mean methylation level [mCG/CG] of 0.4-0.6 in ZF-D3A +dox and <0.1 in MCF7-control), and average CG methylation levels from ZF-D3A +dox WGBS data of the DMR genomic intervals covered by the selected reads (red line).


Figure 1a ) Auto - ubiquitination assays in which the E3 acts as both an E3 and as proxy substrate were performed with GST - HHARIRBR , T7 - Triad1ΔAri , GST - ParkinRBR , and Flag - BRCA1 / BARD1 and either UbcH5WT or UbcH5L104Q as the E2 . Products were visualized by western blotting against indicated tags on E3s . Times given are post - ATP addition . b ) Overlay of ( 1H , 15N ) - HSQC - TROSY spectra of 15N - UbcH5 - O - 15N - Ub in the absence ( black ) and presence ( red ) of 0 . 5mol equiv . HHARI RING1 . A subset of UbcH5 peaks , but not Ub peaks , shift and broaden upon HHARI RING1 binding .

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 O O O O O O O O O O O O O O O O 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 1a) Auto-ubiquitination assays in which the E3 acts as both an E3 and as proxy substrate were performed with GST-HHARIRBR, T7-Triad1ΔAri, GST-ParkinRBR, and Flag-BRCA1/BARD1 and either UbcH5WT or UbcH5L104Q as the E2. Products were visualized by western blotting against indicated tags on E3s. Times given are post-ATP addition.b) Overlay of (1H,15N)-HSQC-TROSY spectra of 15N-UbcH5-O-15N-Ub in the absence (black) and presence (red) of 0.5mol equiv. HHARI RING1. A subset of UbcH5 peaks, but not Ub peaks, shift and broaden upon HHARI RING1 binding.


Figure 2a ) Histogram of CSPs between free 15N - UbcH7 and 15N - UbcH7 - O - Ub identifies UbcH7 residues affected by conjugation to Ub . Active site ( Ser86 ) is indicated with a star and cross - over helix residues ( 101 - 113 ) are marked with a gray cylinder . INSET . CSPs greater than 1 stdv ( > 0 . 115 ppm ) are highlighted in yellow on a surface representation of UbcH7 ( PDB 1fbv ) . The view on the left shows the surface that contains the UbcH7 cross - over helix . b ) Region of 1H - 15N - HSQC TROSY spectra containing resonances of the cross - over helix residues S107 and Q106 are overlaid : unconjugated 15N - UbcH7 ( blue ) , 15N - UbcH7 - O - Ub ( black ) , and 15N - UbcH7 - O - UbI44A ( red ) . Peaks representing S107 and Q106 in the context of UbcH7 ~ Ub - I44A ( red ) are closer to peaks observed in free UbcH7 ( blue ) than UbcH7 ~ Ub - WT ( black ) indicating that I44A of Ub disrupts interactions with the cross - over helix of UbcH7 .

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 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

Figure 2a) Histogram of CSPs between free 15N-UbcH7 and 15N-UbcH7-O-Ub identifies UbcH7 residues affected by conjugation to Ub. Active site (Ser86) is indicated with a star and cross-over helix residues (101-113) are marked with a gray cylinder. INSET. CSPs greater than 1 stdv (> 0.115 ppm) are highlighted in yellow on a surface representation of UbcH7 (PDB 1fbv). The view on the left shows the surface that contains the UbcH7 cross-over helix.b) Region of 1H-15N-HSQC TROSY spectra containing resonances of the cross-over helix residues S107 and Q106 are overlaid: unconjugated 15N-UbcH7 (blue), 15N-UbcH7-O-Ub (black), and 15N-UbcH7-O-UbI44A (red). Peaks representing S107 and Q106 in the context of UbcH7~Ub-I44A (red) are closer to peaks observed in free UbcH7 (blue) than UbcH7~Ub-WT (black) indicating that I44A of Ub disrupts interactions with the cross-over helix of UbcH7.


Figure 3a ) Region of 1H - 15N - HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid : free 15N - Ub ( blue ) , UbcH7 - O - 15N - Ub ( black ) , and HHARI RING1 - bound UbcH7 - O - 15N - Ub ( red ) . Black arrow highlights perturbation upon conjugation to UbcH7 ( black arrow ) and red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7 . b ) Region of 1H - 13C - HSQC spectra of 13C - UbcH7 ( blue ) , 13C - UbcH7 - O - Ub ( black ) , and HHARI RING1 - bound 13C - UbcH7 - O - Ub ( red ) that includes the methyl ( 13CH3 ) resonance of the surface - exposed UbcH7 cross - over helix residue , Ala 110 ( black arrow ) is shown . The 13CH3 peak of Ala110 either broadens dramatically or shifts to an unknown position in the spectrum of 13C - UbcH7 - O - Ub ( black ) and reappears at its position in free UbcH7 in the presence of HHARI RING1 , consistent with disruption of closed UbcH7 ~ Ub conformations . Pairwise overlays and larger spectra are provided in Appendix Fig S3 . c ) Regions of 1H - 15N - HSQC - TROSY spectra of UbcH7 - O - 15N - Ub in the absence ( black ) and presence ( red ) of a RING1 domain from the RBR E3s HHARI ( left ) or RNF144 ( middle ) or a canonical RING domain of BRCA1 / BARD1 ( right ) . The perturbations on Ub due to binding of HHARI RING1 and RNF144 RING1 are remarkably similar , while binding of the canonical RING domain of BRCA1 / BARD1 has no observable effect on the Ub spectrum . Gray boxes mark area expanded in a )

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 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 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

Figure 3a) Region of 1H-15N-HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid: free 15N-Ub (blue), UbcH7-O-15N-Ub (black), and HHARI RING1-bound UbcH7-O-15N-Ub (red). Black arrow highlights perturbation upon conjugation to UbcH7 (black arrow) and red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7.b) Region of 1H-13C-HSQC spectra of 13C-UbcH7 (blue), 13C-UbcH7-O-Ub (black), and HHARI RING1-bound 13C-UbcH7-O-Ub (red) that includes the methyl (13CH3) resonance of the surface-exposed UbcH7 cross-over helix residue, Ala 110 (black arrow) is shown. The 13CH3 peak of Ala110 either broadens dramatically or shifts to an unknown position in the spectrum of 13C-UbcH7-O-Ub (black) and reappears at its position in free UbcH7 in the presence of HHARI RING1, consistent with disruption of closed UbcH7~Ub conformations. Pairwise overlays and larger spectra are provided in Appendix Fig S3.c) Regions of 1H-15N-HSQC-TROSY spectra of UbcH7-O-15N-Ub in the absence (black) and presence (red) of a RING1 domain from the RBR E3s HHARI (left) or RNF144 (middle) or a canonical RING domain of BRCA1/BARD1 (right). The perturbations on Ub due to binding of HHARI RING1 and RNF144 RING1 are remarkably similar, while binding of the canonical RING domain of BRCA1/BARD1 has no observable effect on the Ub spectrum. Gray boxes mark area expanded in a)


Figure 4b ) LEFT . Auto - ubiquitination assays were performed with wild - type HHARIRBR or an active site - dead mutant ( C357A - HHARIRBR ) . Products were visualized by western blotting against GST . The zero time point was taken immediately prior to ATP addition , all other times are post - ATP addition . RIGHT . Identical assays as shown on left were performed with the UBR - hybrid . The active site - dead mutant ( C357A ) retains substantial auto - ubiquitination activity signifying that UbcH5 ~ Ub is able to transfer Ub directly onto the GST - UBR - hybrid construct , bypassing the RING2 active site Cys .

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

Figure 4b) LEFT. Auto-ubiquitination assays were performed with wild-type HHARIRBR or an active site-dead mutant (C357A- HHARIRBR). Products were visualized by western blotting against GST. The zero time point was taken immediately prior to ATP addition, all other times are post-ATP addition. RIGHT. Identical assays as shown on left were performed with the UBR-hybrid. The active site-dead mutant (C357A) retains substantial auto-ubiquitination activity signifying that UbcH5~Ub is able to transfer Ub directly onto the GST-UBR-hybrid construct, bypassing the RING2 active site Cys.


Figure 5a ) LEFT . E3 auto - ubiquitination assays were performed using various Ub mutants ( I44A , R42A , Q49A , Q49E , and V70A ) and the RBR E3s HHARIRBR ( left ) and ParkinRBR ( right ) . Products were visualized by western blotting against HA - Ub . Samples were analyzed 30 minutes after ATP addition . c ) UbcH7 ~ Ub conjugates were preformed with either with WT - Ub or I44A - Ub . After the addition of apyrase to quench the charging reaction , UbcH7 ~ UbWT ( left ) or UbcH7 ~ UbI44A ( right ) was incubated with ParkinRBR . The disappearance of each UbcH7 ~ Ub species and appearance of auto - ubiquitinated E3 was visualized under non - reducing conditions by western blotting for HA - Ub . Time was recorded post addition of ParkinRBR . E2 ~ Ub conjugated with I44A - Ub does not disappear over the time course of the reaction . d ) UbcH7 conjugated with WT - Ub , I44A - Ub , or V70A Ub as indicated was incubated with either H887A - HOIPRBR - LDD ( left ) or H359A - HHARIRBR ( right ) mutants that allow trapping of the E3 ~ Ub thioester with WT - Ub ( Stieglitz et al , 2013 ; [ 29 ] ) . While a HOIP ~ Ub thioester species is observed when UbcH7 was charged with WT - Ub , no detectable transfer occurs with the I44A - Ub conjugate ( left ) . Similarly , V70A - Ub shows reduced formation of the HHARI ~ Ub thioester ( right ) . In addition to blotting for HA - Ub , the blot on the right was also blotted for GST - HHARI .

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 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 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

Figure 5a) LEFT. E3 auto-ubiquitination assays were performed using various Ub mutants (I44A, R42A, Q49A, Q49E, and V70A) and the RBR E3s HHARIRBR (left) and ParkinRBR (right). Products were visualized by western blotting against HA-Ub. Samples were analyzed 30 minutes after ATP addition.c) UbcH7~Ub conjugates were preformed with either with WT-Ub or I44A-Ub. After the addition of apyrase to quench the charging reaction, UbcH7~UbWT (left) or UbcH7~UbI44A (right) was incubated with ParkinRBR. The disappearance of each UbcH7~Ub species and appearance of auto-ubiquitinated E3 was visualized under non-reducing conditions by western blotting for HA-Ub. Time was recorded post addition of ParkinRBR. E2~Ub conjugated with I44A-Ub does not disappear over the time course of the reaction.d) UbcH7 conjugated with WT-Ub, I44A-Ub, or V70A Ub as indicated was incubated with either H887A-HOIPRBR-LDD (left) or H359A-HHARIRBR (right) mutants that allow trapping of the E3~Ub thioester with WT-Ub (Stieglitz et al, 2013;[29]). While a HOIP~Ub thioester species is observed when UbcH7 was charged with WT-Ub, no detectable transfer occurs with the I44A-Ub conjugate (left). Similarly, V70A-Ub shows reduced formation of the HHARI~Ub thioester (right). In addition to blotting for HA-Ub, the blot on the right was also blotted for GST-HHARI.


Figure 6a ) Top panel . Regions of 1H - 15N - HSQC TROSY spectra of 15N - HHARI RING2 in the absence ( black ) and presence of excess Ub ( red ) . Middle panel . Identical spectral region , but in the presence of Ub - V70A ( red ) . Bottom panel . Identical spectral region , but spectra are from a truncated HHARI RING2 construct that lacks N - terminal residues 325 - 335 ( HHARI RING2 - ΔL ) . d ) Mutations in the Ub binding surface of RING2 decrease activity in E3 auto - ubiquitination assays . Time points ( 10min ) are visualized by western blotting for the GST - tag on HHARI . Relative activity of HHARI WT and mutant forms is clearest when the intensity of the unmodified HHARI band is compared . e ) 1H - 15N - HSQC TROSY spectra of 15N - Ub demonstrate binding by WT HHARI RING2 , but not by mutant HHARI RING2 constructs that exhibit decreased auto - ubiquitination activity . Overlay of 15N - Ub spectra in the absence ( black ) and presence of WT - HHARI RING2 ( red ) , W336A - HHARI RING2 ( blue ) , T341N - HHARI RING2 ( green ) , or E352A - HHARI RING2 ( purple ) . HHARI mutations that exhibited reduced binding to Ub also show decreased ubiquitination activity in panel d ) .

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

Figure 6a) Top panel. Regions of 1H-15N-HSQC TROSY spectra of 15N-HHARI RING2 in the absence (black) and presence of excess Ub (red). Middle panel. Identical spectral region, but in the presence of Ub-V70A (red). Bottom panel. Identical spectral region, but spectra are from a truncated HHARI RING2 construct that lacks N-terminal residues 325-335 (HHARI RING2-ΔL).d) Mutations in the Ub binding surface of RING2 decrease activity in E3 auto-ubiquitination assays. Time points (10min) are visualized by western blotting for the GST-tag on HHARI. Relative activity of HHARI WT and mutant forms is clearest when the intensity of the unmodified HHARI band is compared.e) 1H-15N-HSQC TROSY spectra of 15N-Ub demonstrate binding by WT HHARI RING2, but not by mutant HHARI RING2 constructs that exhibit decreased auto-ubiquitination activity. Overlay of 15N-Ub spectra in the absence (black) and presence of WT-HHARI RING2 (red), W336A-HHARI RING2 (blue), T341N-HHARI RING2 (green), or E352A-HHARI RING2 (purple). HHARI mutations that exhibited reduced binding to Ub also show decreased ubiquitination activity in panel d).


Figure 2G , Fluorescent in situ hybridization for cd74a , tnfa , and ctsc at 7 dpi , respectively . mpeg1 was used to label all MC cells . The blue boxed region is highlighted in the zoom - in image to the right . Scale bar = 50 μm . A stands for arium , V stand for ventricle and OFT stands for outflow tract . White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane .

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Figure 2G, Fluorescent in situ hybridization for cd74a, tnfa, and ctsc at 7 dpi, respectively. mpeg1 was used to label all MC cells. The blue boxed region is highlighted in the zoom-in image to the right. Scale bar = 50 μm. A stands for arium, V stand for ventricle and OFT stands for outflow tract. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane.


Figure 3F , In situ hybridization showing temporal spatial expression patterns of postnb . The white boxed region is shown in zoom - in images at the bottom . Scale bar = 25 μm . White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane . A stands for arium , V stand for ventricle and OFT stands for outflow tract . H , Fluorescent in situ hybridization for postnb , tnfα , and tnfrsf1a in the injury area at 7 dpf , respectively . The white boxed regions are shown in their respective zoom - in images to the right . White dashed lines outline cardiac apex and the yellow dashed lines indicate approximate resection plane . Scale bar = 25 μm . J , Violin plots showing expression of cxcl18b , sele , atf3 , and fosl1a in each eEC subpopulation . K , Fluorescent in situ hybridization for fosl1a at 7 dpi . The white boxed region is shown in a higher magnification image to the right for the injury area . cdh5 was used to label all EC cells and the red boxed region is shown in a higher magnification image to highlight a pan - EC cdh5 expression in green . White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane . Scale bar = 25 μm . A stands for arium , V stand for ventricle and OFT stands for outflow tract .

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 B-PANEL_START 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

Figure 3F, In situ hybridization showing temporal spatial expression patterns of postnb. The white boxed region is shown in zoom-in images at the bottom. Scale bar = 25 μm. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane. A stands for arium, V stand for ventricle and OFT stands for outflow tract.H, Fluorescent in situ hybridization for postnb, tnfα, and tnfrsf1a in the injury area at 7 dpf, respectively. The white boxed regions are shown in their respective zoom-in images to the right. White dashed lines outline cardiac apex and the yellow dashed lines indicate approximate resection plane. Scale bar = 25 μm.J, Violin plots showing expression of cxcl18b, sele, atf3, and fosl1a in each eEC subpopulation.K, Fluorescent in situ hybridization for fosl1a at 7 dpi. The white boxed region is shown in a higher magnification image to the right for the injury area. cdh5 was used to label all EC cells and the red boxed region is shown in a higher magnification image to highlight a pan-EC cdh5 expression in green. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane. Scale bar = 25 μm. A stands for arium, V stand for ventricle and OFT stands for outflow tract.


Figure 4A , Wildtype or kit mutant stained with AFOG at 14 days and 30 dpi . Sham - operated zebrafish hearts serve as control . Scale bar = 100 μm . C , PCNA / Mef2 double staining showing the proliferating CMs in wildtype and mutant hearts at 7 dpi . The white boxed regions are shown in zoom - in images to their right . Blue dashed lines indicate approximate resection plane . White arrows point to the PCNA / Mef2 double positive nuclei . Scale bar = 50 μm . I , Immunostaining for MC marker IB4 in WT ( left ) and mutant hearts ( middle ) at 7 dpi . Quantification of IB4 + cell number per unit area ( right ) . The white boxed regions are shown in their respective zoom - in images to the right . White dashed lines indicate approximate resection plane .

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 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

Figure 4A, Wildtype or kit mutant stained with AFOG at 14 days and 30 dpi. Sham-operated zebrafish hearts serve as control. Scale bar = 100 μm.C, PCNA/Mef2 double staining showing the proliferating CMs in wildtype and mutant hearts at 7 dpi. The white boxed regions are shown in zoom-in images to their right. Blue dashed lines indicate approximate resection plane. White arrows point to the PCNA/Mef2 double positive nuclei. Scale bar = 50 μm.I, Immunostaining for MC marker IB4 in WT (left) and mutant hearts (middle) at 7 dpi. Quantification of IB4+ cell number per unit area (right). The white boxed regions are shown in their respective zoom-in images to the right. White dashed lines indicate approximate resection plane.


Figure 5D , F , Violin plots showing the expression of il1b in MC1 ( D ) , and ctsd expression in MC ( F ) . E , G , Expression of il1b ( E ) and ctsd ( G ) in WT and kit mutant hearts determined by qRT - PCR . O , Violin plots showing expression of junba in eEC . P , Expression of junba in WT and kit mutant hearts determined by qRT - PCR . Q , Representative image of hearts from 7 dpi tcf21 : nucGFP transgenic fish treated by PBS liposome , Clodronate liposome and Dex , respectively . The red boxes mark the peripheral area , and the white boxes mark the injury area and are shown in their respective zoom - in images to the right . . White dashed lines indicate approximate resection plane .

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 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

Figure 5D, F, Violin plots showing the expression of il1b in MC1 (D), and ctsd expression in MC (F). E, G, Expression of il1b (E) and ctsd (G) in WT and kit mutant hearts determined by qRT-PCR.O, Violin plots showing expression of junba in eEC. P, Expression of junba in WT and kit mutant hearts determined by qRT-PCR.Q, Representative image of hearts from 7 dpi tcf21:nucGFP transgenic fish treated by PBS liposome, Clodronate liposome and Dex, respectively. The red boxes mark the peripheral area, and the white boxes mark the injury area and are shown in their respective zoom-in images to the right.. White dashed lines indicate approximate resection plane.


Figure 1 ( A ) Serum starvation induces CP110 degradation . RPE - 1 cells were either mock - treated or serum starved for 0 . 5 , 1 , 2 , 4 , 6 and 24 h and then lysed and subjected to immunoblotting with anti - CP110 antibodies ( top panel ) and anti - actin antibodies ( loading control ; bottom panel ) . ( B ) Graph shows CP110 levels normalized to actin . Normal distribution was determined by the Shapiro - Wilk normality test . ( Inhibition of proteasomal degradation leads to CP110 stabilization on the m - centriole and impaired ciliogenesis . RPE - 1 cells on cover - slips were either grown in media with serum , serum starved for 4 h in the absence of MG132 , or serum starved for 4 h in the presence of MG132 prior to fixation and immunostaining with antibodies for acetylated tubulin ( to detect cilia ) and anti - CP110 antibodies . ( E ) Representative micrographs showing MG132 treatment impedes removal of CP110 from the m - centriole and impairs ciliogenesis . Bar , 10 μm . ( F ) Graph quantifying percent of cells displaying primary cilia . Normal distribution was determined by the D ' Agostino and Pearson normality test . ( H - I ) EHD1 depletion prevents CP110 degradation upon serum starvation . ( H ) Mock - transfected RPE - 1 cells or EHD1 siRNA - transfected RPE - 1 cells were grown in media containing serum , or serum starved for 4 h in the absence or presence of MG132 , and then lysed , separated by SDS - PAGE and immunoblotted with anti - CP110 antibodies ( upper panel ) or anti - actin antibodies ( loading control ; lower panel ) . Validation of EHD1 depletion is shown in the right panel . ( I ) Graph displaying densitometric analysis of the intensity of the CP110 bands shown in H normalized to actin ( means and p - values are representative of 3 independent experiments ) . Normal distribution was determined by the Shapiro - Wilk normality 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 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 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

Figure 1(A) Serum starvation induces CP110 degradation. RPE-1 cells were either mock-treated or serum starved for 0.5, 1, 2, 4, 6 and 24 h and then lysed and subjected to immunoblotting with anti-CP110 antibodies (top panel) and anti-actin antibodies (loading control; bottom panel). (B) Graph shows CP110 levels normalized to actin. Normal distribution was determined by the Shapiro-Wilk normality test. (Inhibition of proteasomal degradation leads to CP110 stabilization on the m-centriole and impaired ciliogenesis. RPE-1 cells on cover-slips were either grown in media with serum, serum starved for 4 h in the absence of MG132, or serum starved for 4 h in the presence of MG132 prior to fixation and immunostaining with antibodies for acetylated tubulin (to detect cilia) and anti-CP110 antibodies. (E) Representative micrographs showing MG132 treatment impedes removal of CP110 from the m-centriole and impairs ciliogenesis. Bar, 10 μm. (F) Graph quantifying percent of cells displaying primary cilia. Normal distribution was determined by the D'Agostino and Pearson normality test.(H-I) EHD1 depletion prevents CP110 degradation upon serum starvation. (H) Mock-transfected RPE-1 cells or EHD1 siRNA-transfected RPE-1 cells were grown in media containing serum, or serum starved for 4 h in the absence or presence of MG132, and then lysed, separated by SDS-PAGE and immunoblotted with anti-CP110 antibodies (upper panel) or anti-actin antibodies (loading control; lower panel). Validation of EHD1 depletion is shown in the right panel. (I) Graph displaying densitometric analysis of the intensity of the CP110 bands shown in H normalized to actin (means and p-values are representative of 3 independent experiments). Normal distribution was determined by the Shapiro-Wilk normality test.


Figure 2 ( A - B ) PCM1 - depletion impedes CP110 ubiquitination upon serum starvation . ( A ) Mock - transfected RPE - 1 cells were maintained in media with serum , and both mock - transfected and PCM1 siRNA - transfected cells were serum starved for 4 h in the presence of MG132 prior to lysis and immunoblotting with anti - CP110 antibodies ( second panel from top ) , or first immunoprecipitated with anti - CP110 antibodies and then immunoblotted with antibodies to detect ubiquitinated CP110 ( top panel ) . Bottom panels depict the efficacy of PCM1 depletion upon PCM1 siRNA - transfection , with actin as a loading control . ( B ) Graph displaying densitometric analysis of ubiquitinated CP110 levels ( from the upper panel of A ) normalized to total CP110 immunoprecipitated . Normal distribution was determined by the Shapiro - Wilk normality test . ( D - G ) Serum starved mock - transfected RPE - 1 cells on cover - slips were fixed and immunostained with antibodies directed against acetylated tubulin and CP110 and display primary cilia generation ( marked by acetylated tubulin ; D , F and inset in G ) and removal of CP110 from the m - centriole ( E - G ; see arrow marking CP110 only on the daughter centriole ) . ( H - K ) Serum starved PCM siRNA - transfected RPE - 1 cells on cover - slips were fixed and immunostained with antibodies directed against acetylated tubulin and CP110 and display limited ciliogenesis ( H , J and inset in K ) and failure to remove CP110 from the m - centriole ( I - K ; see arrows identifying CP110 on both centrioles ) . EHD1 regulates the distribution of centriolar satellites to the centrosomal region . Mock - transfected RPE - 1 cells on cover - slips were serum starved and immunostained with antibodies against acetylated tubulin and PCM1 and EHD1 siRNA - transfected cells on cover - slips were serum starved and immunostained with antibodies against acetylated tubulin and PCM1 The p - values are calculated for all non - ciliated and ciliated mock - transfected cells compared to all EHD1 siRNA non - ciliated and ciliated cells . Note that as expected , among the EHD1 - depleted cells , there are minimal cells that display ciliation .

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 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 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

Figure 2(A-B) PCM1-depletion impedes CP110 ubiquitination upon serum starvation. (A) Mock-transfected RPE-1 cells were maintained in media with serum, and both mock-transfected and PCM1 siRNA-transfected cells were serum starved for 4 h in the presence of MG132 prior to lysis and immunoblotting with anti-CP110 antibodies (second panel from top), or first immunoprecipitated with anti-CP110 antibodies and then immunoblotted with antibodies to detect ubiquitinated CP110 (top panel). Bottom panels depict the efficacy of PCM1 depletion upon PCM1 siRNA-transfection, with actin as a loading control. (B) Graph displaying densitometric analysis of ubiquitinated CP110 levels (from the upper panel of A) normalized to total CP110 immunoprecipitated. Normal distribution was determined by the Shapiro-Wilk normality test.(D-G) Serum starved mock-transfected RPE-1 cells on cover-slips were fixed and immunostained with antibodies directed against acetylated tubulin and CP110 and display primary cilia generation (marked by acetylated tubulin; D, F and inset in G) and removal of CP110 from the m-centriole (E-G; see arrow marking CP110 only on the daughter centriole). (H-K) Serum starved PCM siRNA-transfected RPE-1 cells on cover-slips were fixed and immunostained with antibodies directed against acetylated tubulin and CP110 and display limited ciliogenesis (H, J and inset in K) and failure to remove CP110 from the m-centriole (I-K; see arrows identifying CP110 on both centrioles).EHD1 regulates the distribution of centriolar satellites to the centrosomal region. Mock-transfected RPE-1 cells on cover-slips were serum starved and immunostained with antibodies against acetylated tubulin and PCM1 and EHD1 siRNA-transfected cells on cover-slips were serum starved and immunostained with antibodies against acetylated tubulin and PCM1 The p-values are calculated for all non-ciliated and ciliated mock-transfected cells compared to all EHD1 siRNA non-ciliated and ciliated cells. Note that as expected, among the EHD1-depleted cells, there are minimal cells that display ciliation.


Figure 3 ( A ) Validation of siRNA depletion . RPE - 1 cells were mock - transfected or transfected with siRNA oligonucleotides to impair translation of Cullin3 , HERC2 , or MIB1 . 72 h later , the cells were lysed , subjected to SDS - PAGE and immunoblotted . Immunoblotting with anti - actin antibodies was done as a loading control . ( B ) HERC2 or MIB1 depletion impairs CP110 ubiquitination . RPE - 1 cells were grown in serum ( first lane from left ) , or with MG132 in the absence of serum for 4 h ( second lane from left ) , transfected with Cullin3 siRNA - oligonucleotides and treated with MG132 in the absence of serum for 4 h ( third lane from left ) , transfected with HERC2 siRNA - oligonucleotides and treated with MG132 in the absence of serum for 4 h ( fourth lane from left ) , or transfected with MIB1 siRNA - oligonucleotides and treated with MG132 in the absence of serum for 4 h ( fifth lane from left ) . Cells were lysed and immunoprecipitated with anti - CP110 antibodies , and the resultant immunoprecipitates were immunoblotted with anti - ubiquitin to identify ubiquitinated CP110 ( upper panel ) . The blot was then stripped and re - immunoblotted with anti - CP110 to reveal the amount of CP110 pulled - down from the lysate ( lower panel ) . ( D ) CP110 interacts with HERC2 and MIB1 , but not Cullin3 . RPE - 1 cells were lysed and immunoprecipitated with beads only or anti - CP110 , and then subjected to SDS - PAGE and immunoblotting with CP110 to validate the immunoprecipitation ( lower panel of each gel ) and either Cullin3 ( left gel , upper panel ) , HERC2 ( middle gel , upper panel ) or MIB1 ( right gel , upper panel ) . 7 % inputs are shown on the left of each gel .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3(A) Validation of siRNA depletion. RPE-1 cells were mock-transfected or transfected with siRNA oligonucleotides to impair translation of Cullin3, HERC2, or MIB1. 72 h later, the cells were lysed, subjected to SDS-PAGE and immunoblotted. Immunoblotting with anti-actin antibodies was done as a loading control.(B) HERC2 or MIB1 depletion impairs CP110 ubiquitination. RPE-1 cells were grown in serum (first lane from left), or with MG132 in the absence of serum for 4 h (second lane from left), transfected with Cullin3 siRNA-oligonucleotides and treated with MG132 in the absence of serum for 4 h (third lane from left), transfected with HERC2 siRNA-oligonucleotides and treated with MG132 in the absence of serum for 4 h (fourth lane from left), or transfected with MIB1 siRNA-oligonucleotides and treated with MG132 in the absence of serum for 4 h (fifth lane from left). Cells were lysed and immunoprecipitated with anti-CP110 antibodies, and the resultant immunoprecipitates were immunoblotted with anti-ubiquitin to identify ubiquitinated CP110 (upper panel). The blot was then stripped and re-immunoblotted with anti-CP110 to reveal the amount of CP110 pulled-down from the lysate (lower panel).(D) CP110 interacts with HERC2 and MIB1, but not Cullin3. RPE-1 cells were lysed and immunoprecipitated with beads only or anti-CP110, and then subjected to SDS-PAGE and immunoblotting with CP110 to validate the immunoprecipitation (lower panel of each gel) and either Cullin3 (left gel, upper panel), HERC2 (middle gel, upper panel) or MIB1 (right gel, upper panel). 7% inputs are shown on the left of each gel.


Figure 4 ( B - M ) ( B - D ) Mock - transfected cells , ( E - G ) MIB1 siRNA - transfected cells , ( H - J ) HERC2 siRNA - transfected cells , or ( K - M ) MIB1 and HERC2 siRNA - transfected cells on cover - slips were fixed and immunostained with antibodies against acetylated tubulin ( insets ; C , F , I , L : merged field ; B , E , H , K ) and CP110 ( insets ; D , G , J , M : merged field ; B , E , H , K ) . Bars ( B , E , H , K ) , 10 mm . Bars ( C , D , F , G , I , J , L , M ) , 2 μm .

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Figure 4(B-M) (B-D) Mock-transfected cells, (E-G) MIB1 siRNA-transfected cells, (H-J) HERC2 siRNA-transfected cells, or (K-M) MIB1 and HERC2 siRNA-transfected cells on cover-slips were fixed and immunostained with antibodies against acetylated tubulin (insets; C, F, I, L: merged field; B, E, H, K) and CP110 (insets; D, G, J, M: merged field; B, E, H, K). Bars (B, E, H, K), 10 mm. Bars (C, D, F, G, I, J, L, M), 2 μm.


Figure 5 ( A ) EHD1 is required for the interaction between HERC2 and CP110 . RPE - 1 cells were mock - transfected or transfected with EHD1 siRNA oligonucleotides for 72 h . Cells were lysed and immunoprecipitated with anti - CP110 or beads only ( control ) and separated by SDS - PAGE along with 7 % of the lysate ( input ) . After transfer to nitrocellulose , antibodies were used to detect CP110 ( lower panel ) and HERC2 ( upper panel ) . ( C - D ) HERC2 partially localizes to PCM1 - containing centriolar satellites . RPE - 1 cells were serum starved for 4 h , fixed and immunostained with antibodies against PCM1 ( red ) and HERC2 ( green ) . The micrographs show a single representative cell ( C ) and an inset ( D ) . Bar ( C ) , 10 μm . Bar ( D ) , 1 μm . ( G - H ) Representative micrographs of RPE - 1 cells on cover - slips that were mock - transfected ( G ) or EHD1 siRNA - transfected ( H ) and immunostained with antibodies to acetylated tubulin ( red ) or HERC2 ( green ) . Dashed yellow regions of interest highlight the centrosomal area . Arrows denote the HERC2 signal observed in each micrograph . Bars , 1 μm .

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 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 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

Figure 5(A) EHD1 is required for the interaction between HERC2 and CP110. RPE-1 cells were mock-transfected or transfected with EHD1 siRNA oligonucleotides for 72 h. Cells were lysed and immunoprecipitated with anti-CP110 or beads only (control) and separated by SDS-PAGE along with 7% of the lysate (input). After transfer to nitrocellulose, antibodies were used to detect CP110 (lower panel) and HERC2 (upper panel).(C-D) HERC2 partially localizes to PCM1-containing centriolar satellites. RPE-1 cells were serum starved for 4 h, fixed and immunostained with antibodies against PCM1 (red) and HERC2 (green). The micrographs show a single representative cell (C) and an inset (D). Bar (C), 10 μm. Bar (D), 1 μm.(G-H) Representative micrographs of RPE-1 cells on cover-slips that were mock-transfected (G) or EHD1 siRNA-transfected (H) and immunostained with antibodies to acetylated tubulin (red) or HERC2 (green). Dashed yellow regions of interest highlight the centrosomal area. Arrows denote the HERC2 signal observed in each micrograph. Bars, 1 μm.


Figure 6 ( A - D ) RPE - 1 cells on coverslips were mock - transfected ( A ) , transfected with CP110 siRNA - oligonucleotides ( B ) transfected with HERC2 siRNA - oligonucleotides ( C ) , or transfected with both CP110 and HERC2 siRNA - oligonucleotides ( D ) . Cells were fixed and immunostained with acetylated tubulin to detect primary cilia ( yellow arrows ) . Bars , 10 μm . : Graphs show standard deviation and p - values from 3 independent experiments . The micrographs ( A - D ) are representative ones from the 3 individual biological experiments . More than 50 cells were quantified in each experiment , although fewer ciliated cells were observed and measured under the HERC2 knock - down condition . ( E ) Immunoblot demonstrating the efficacy of depletion for CP110 ( middle panel ) , HERC2 ( top panel ) , and simultaneous CP110 and HERC2 depletion ( middle and top panels ) . Actin was used as a loading control ( bottom panel ) . : Graphs show standard deviation and p - values from 3 independent experiments . immunoblots ( E ) are representative ones from the 3 individual biological experiments .

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Figure 6(A-D) RPE-1 cells on coverslips were mock-transfected (A), transfected with CP110 siRNA-oligonucleotides (B) transfected with HERC2 siRNA-oligonucleotides (C), or transfected with both CP110 and HERC2 siRNA-oligonucleotides (D). Cells were fixed and immunostained with acetylated tubulin to detect primary cilia (yellow arrows). Bars, 10 μm. : Graphs show standard deviation and p-values from 3 independent experiments. The micrographs (A-D) are representative ones from the 3 individual biological experiments. More than 50 cells were quantified in each experiment, although fewer ciliated cells were observed and measured under the HERC2 knock-down condition.(E) Immunoblot demonstrating the efficacy of depletion for CP110 (middle panel), HERC2 (top panel), and simultaneous CP110 and HERC2 depletion (middle and top panels). Actin was used as a loading control (bottom panel). : Graphs show standard deviation and p-values from 3 independent experiments. immunoblots (E) are representative ones from the 3 individual biological experiments.


Figure 1 ( A ) THP - 1 cells were subjected to knockdowns as indicated and treated with 100 ng / mL LPS overnight , followed by 0 . 25 mM LLOMe for 3 h , and the levels of IL - 1β were determined in supernatants . ( B ) THP - 1 cells were subjected to GABARAP knockdown and processed as in A . ( C ) IL - 1β levels in supernatants of THP - 1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe . ( D ) Confocal microscopy of THP - 1 cells treated sequentially with LPS and LLOMe , and stained for LC3B and TRIM16 . Line tracings correspond to arrows . Arrowheads indicate colocalization . Scale bars , 5 μm . ( E ) IL - 1β levels in supernatants of THP - 1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe . ( F ) Levels of IL - 1β were determine in supernatants from THP - 1 cells subjected to knockdown as indicated , treated with 100 ng / mL LPS for overnight and then with 0 . 25mM of Silica , Alum , or monosodium urate ( MSU ) . ( G ) IL - 1β in supernatants of primary human macrophages ( MDM ) subjected to knockdowns and sequentially treated with LPS and LLOMe . Data , means ± SEM ; n ≥ 5 ( except for B where a representative data set from 3 repeats ) . * P < 0 . 05 ( t test or ANOVA ) .

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Figure 1(A) THP-1 cells were subjected to knockdowns as indicated and treated with 100 ng/mL LPS overnight, followed by 0.25 mM LLOMe for 3 h, and the levels of IL-1β were determined in supernatants.(B) THP-1 cells were subjected to GABARAP knockdown and processed as in A.(C) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.(D) Confocal microscopy of THP-1 cells treated sequentially with LPS and LLOMe, and stained for LC3B and TRIM16. Line tracings correspond to arrows. Arrowheads indicate colocalization. Scale bars, 5 μm.(E) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.(F) Levels of IL-1β were determine in supernatants from THP-1 cells subjected to knockdown as indicated, treated with 100 ng/mL LPS for overnight and then with 0.25mM of Silica, Alum, or monosodium urate (MSU).(G) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. Data, means ± SEM; n≥5 (except for B where a representative data set from 3 repeats). *P < 0.05 (t test or ANOVA).


Figure 2 ( A and B ) Levels of IL - 1β in supernatants from THP - 1 cells that were subjected to knockdown as indicated and were treated with LPS , and then ( A ) treated with LLOMe or ( B ) starved in EBSS . ( C ) IL - 1β in supernatants of primary human macrophages ( MDM ) subjected to knockdowns and sequentially treated with LPS and LLOMe . ( D ) Co - immunoprecipitation ( co - IP ) of flag - galectin - 8 ( Gal - 8 ) and GFP - TRIM16 ( T16 ) in HEK293T cells . ( E ) Co - IP analysis of endogenous galectin - 8 and TRIM16 in protein complexes from LLOMe - treated THP - 1 cells . ( F ) Confocal microscopy of THP - 1 cells treated with LPS and LLOMe , and stained for TRIM16 and galectin - 8 . Line tracings correspond to arrowed dashed lines . Pearson correlation coefficient was calculated using SlideBook as described previously . Scale bars , 5 μm . ( G ) In vitro translated and radiolabeled [ 35S ] myc - HA - TRIM16 was incubated with GST - galectin - 8 in the presence ( + ) or absence ( - ) of flag - ULK1 and cold ATP , GST pulldowns were performed , and [ 35S ] radiolabeled Myc - HA - TRIM16 in pulled - down material detected by PAGE and autoradiography . Amounts of GST fusion proteins are shown in coommassie brilliant blue ( CBB ) - stained gels . Data , means  SEM ; n ≥ 5 , except for immunoblot quantifications where n ≥ 3 . * P < 0 . 05 , † P ≥ 0 . 05 ( t test or ANOVA ) .

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Figure 2(A and B) Levels of IL-1β in supernatants from THP-1 cells that were subjected to knockdown as indicated and were treated with LPS, and then (A) treated with LLOMe or (B) starved in EBSS.(C) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.(D) Co-immunoprecipitation (co-IP) of flag-galectin-8 (Gal-8) and GFP-TRIM16 (T16) in HEK293T cells.(E) Co-IP analysis of endogenous galectin-8 and TRIM16 in protein complexes from LLOMe-treated THP-1 cells.(F) Confocal microscopy of THP-1 cells treated with LPS and LLOMe, and stained for TRIM16 and galectin-8. Line tracings correspond to arrowed dashed lines. Pearson correlation coefficient was calculated using SlideBook as described previously. Scale bars, 5 μm.(G) In vitro translated and radiolabeled [35S] myc-HA-TRIM16 was incubated with GST-galectin-8 in the presence (+) or absence (-) of flag-ULK1 and cold ATP, GST pulldowns were performed, and [35S] radiolabeled Myc-HA-TRIM16 in pulled-down material detected by PAGE and autoradiography. Amounts of GST fusion proteins are shown in coommassie brilliant blue (CBB)-stained gels. Data, means  SEM; n≥5, except for immunoblot quantifications where n≥3. *P < 0.05, †P ≥ 0.05 (t test or ANOVA).


Figure 3 ( A and B ) Immunoblot analyses of indicated membrane fractions prepared by differential centrifugation . HeLa cells reconstituted with flag - pro - IL - 1β and myc - pro - Caspase - 1 were treated with 1mM LLOMe for 1h , and lysates were subjected to differential centrifugations at 3 , 000xg ( 3k ) , 25 , 000xg ( 25k ) and 100 , 000xg ( 100k ) as depicted in the cartoon . Arrows , two ways the membranes were analyzed - pellets from velocity sedimentation were analyzed in panels B - D , or by further sucrose and OptiPrep density separation of 25k pellets in panels F and G . ( C ) IL - 1β levels in 25k membrane fractions ( pellets ) from HeLa cells and their CRISPR knockout derivatives ( TRIM16KO ) that were reconstituted for IL - 1β secretion and treated with 1mM LLOMe for 1 h . ( D ) Quantification of analyses in ( B ) . RI , relative intensity . ( E ) Whole cell lysates ( immunoblots ) analyzed in F and G . ( F , G ) Sucrose density gradient analysis of 25k membranes . Note that mature IL - 1β ( mIL - 1β ) is recruited to and co - fractionates with TRIM16 + , Sec22b + , and LC3 - II + membranes in TRIM16wt HeLa cells reconstituted with pro - IL - 1β and pro - caspase 1 , but not in TRIM16KO mutant cells . ( H ) Confocal images of LC3 in HeLa cells expressing GFP - Sec22b treated with LLOMe or untreated ( control ) . Arrowheads , colocalization . Scale bars , 5 μm . ( I ) GFP - Sec22b and LC3 overlapping area obtained by high content microscopy . ( J ) Co - IP analysis between flag - TRIM16 with GFP - Sec22b or GFP - syntaxin - 17 co - expressed in HEK293T cells . ( K ) Co - IP analysis of endogenous proteins , TRIM16 and Sec22b , in lysates from THP - 1 cells treated with LLOMe . ( L ) Co - IP analysis of interactions between deletion variants of flag - TRIM16 with GFP - Sec22b in HEK293T cells . ( M and N ) IL - 1β secretion complementation experiments of TRIM16KO cells with TRIM16 Sec22b - interacting and its Sec22b - nonbinding mutant shown . ( O ) Two color super - resolution image showing co - clustering of flag - TRIM16 ( red ) and GFP - Sec22b ( green ) ; Scale bar , 200 nm . ( P ) Cross - correlation analysis of flag - TRIM16 and GFP - Sec22b super - resolution data showing a characteristic separation of ~ 70 nm .

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Figure 3(A and B) Immunoblot analyses of indicated membrane fractions prepared by differential centrifugation. HeLa cells reconstituted with flag-pro-IL-1β and myc-pro-Caspase-1 were treated with 1mM LLOMe for 1h, and lysates were subjected to differential centrifugations at 3,000xg (3k), 25,000xg (25k) and 100,000xg (100k) as depicted in the cartoon. Arrows, two ways the membranes were analyzed - pellets from velocity sedimentation were analyzed in panels B-D, or by further sucrose and OptiPrep density separation of 25k pellets in panels F and G.(C) IL-1β levels in 25k membrane fractions (pellets) from HeLa cells and their CRISPR knockout derivatives (TRIM16KO) that were reconstituted for IL-1β secretion and treated with 1mM LLOMe for 1 h.(D) Quantification of analyses in (B). RI, relative intensity.(E) Whole cell lysates (immunoblots) analyzed in F and G.(F,G) Sucrose density gradient analysis of 25k membranes. Note that mature IL-1β (mIL-1β) is recruited to and co-fractionates with TRIM16+, Sec22b+, and LC3-II+ membranes in TRIM16wt HeLa cells reconstituted with pro-IL-1β and pro-caspase 1, but not in TRIM16KO mutant cells.(H) Confocal images of LC3 in HeLa cells expressing GFP-Sec22b treated with LLOMe or untreated (control). Arrowheads, colocalization. Scale bars, 5 μm. (I) GFP-Sec22b and LC3 overlapping area obtained by high content microscopy.(J) Co-IP analysis between flag-TRIM16 with GFP-Sec22b or GFP-syntaxin-17 co-expressed in HEK293T cells.(K) Co-IP analysis of endogenous proteins, TRIM16 and Sec22b, in lysates from THP-1 cells treated with LLOMe.(L) Co-IP analysis of interactions between deletion variants of flag-TRIM16 with GFP-Sec22b in HEK293T cells.(M and N) IL-1β secretion complementation experiments of TRIM16KO cells with TRIM16 Sec22b-interacting and its Sec22b-nonbinding mutant shown.(O) Two color super-resolution image showing co-clustering of flag-TRIM16 (red) and GFP-Sec22b (green); Scale bar, 200 nm. (P) Cross-correlation analysis of flag-TRIM16 and GFP-Sec22b super-resolution data showing a characteristic separation of ~ 70 nm.


Figure 4 ( A and B ) IL - 1β levels in supernatants of THP - 1 cells subjected to knockdowns , treated with LPS , and then ( A ) with LLOMe or ( B ) starved in EBSS . ( C ) IL - 1β levels in supernatants of primary human macrophages ( MDM ) subjected to knockdowns and sequentially treated with LPS and LLOMe . ( D ) Immunoblot analyses of p62 levels in THP - 1 cells subjected to knockdowns as indicated and starved for 3 h . ( E ) mRFP - GFP overlapping areas of tandem HeLa cells reconstituted with flag - pro - IL - 1β and myc - Caspase 1 and starved for 3 h . Mask overlay , iDev software - defined objects ( primary objects , cell outlines ; internal secondary objects , GFP ( top ) , mRFP ( middle ) , or their overlapping area ( bottom ) ) . Bars , 10 μm . ( F ) IL - 1β levels in supernatants of starved tandem HeLa cells reconstituted with flag - pro - IL - 1β and myc - Caspase 1 as in E .

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Figure 4(A and B) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then (A) with LLOMe or (B) starved in EBSS.(C) IL-1β levels in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.(D) Immunoblot analyses of p62 levels in THP-1 cells subjected to knockdowns as indicated and starved for 3 h.(E) mRFP-GFP overlapping areas of tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 and starved for 3 h. Mask overlay, iDev software-defined objects (primary objects, cell outlines; internal secondary objects, GFP (top), mRFP (middle), or their overlapping area (bottom)). Bars, 10 μm.(F) IL-1β levels in supernatants of starved tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 as in E.


Figure 5 ( A - C ) IL - 1β levels in supernatants of THP - 1 cells subjected to knockdowns , treated with LPS , and then with LLOMe ( A and C ) or starved in EBSS ( B ) . ( A - C ) IL - 1β levels in supernatants of THP - 1 cells subjected to knockdowns , treated with LPS , and then with LLOMe ( A and C ) .

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Figure 5(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C) or starved in EBSS (B).(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C).


Figure 6 ( C ) Ferritin levels in supernatants of THP - 1 cells subjected to knockdowns as indicated and sequentially treated with LPS and LLOMe . ( D ) Ferritin levels in supernatants of THP - 1 cells subjected to knockdowns as indicated and infected with M . tuberculosis as indicated . ( E ) Levels of ferritin were determine in supernatants from THP - 1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe . ( F ) Confocal microscopy of HeLa cells expressing GFP - Sec22b were treated with LLOMe , and stained for FTH1 . Line tracings correspond to arrows . Scale bars , 5 μm ;

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Figure 6(C) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and sequentially treated with LPS and LLOMe.(D) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and infected with M. tuberculosis as indicated.(E) Levels of ferritin were determine in supernatants from THP-1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe.(F) Confocal microscopy of HeLa cells expressing GFP-Sec22b were treated with LLOMe, and stained for FTH1. Line tracings correspond to arrows. Scale bars, 5 μm;