Figure 6 ( B ) Frequency of occurrence of differentially regulated genes across significantly deregulated canonical pathways compared to the Log fold change ( Log ( FC ) ) . ( C ) Upstream regulators as identified by the IPA software from differentially regulated genes . ( E ) Microglia were pre - treated with Torin , or medium as indicated and then stimulated with mNSC - CM , mGICPten - / - ; p53 - / - - CM , or GL261 - CM . p - NF - ĸB ( p - P65 ) and p - STAT3 were analysed by immunoblotting of whole cell lysates collected at 4hr . ( F ) Immunofluorescence images of NF - ĸB ( P65 ) in conditioned microglia . Quantification of nuclear translocation of NF - κB from staining . Units represent the % of voxels in the NF - κB channel colocalised with DAPI ( n = 3 ; mean ± SEM ; One - Way ANOVA Tukey test - right ) . Scale bar is 50µm . ( G ) Production of Il - 12 ( Il - 12p40 ) , Tnf , Il6 , and Il10 by conditioned microglia was determined by qPCR . Each treatment was normalised to housekeeping gene and the unconditioned control . ( n = 3 ; mean ± SEM ; Two - Way ANOVA Tukey test ) . ( H , I ) MFI levels of pSTAT3 in TAM - MG ( CD45 + P2RY12 + CD49d - ) ( H ) and p - NF - κB ( p - P65 ) in TAM - BMDM ( CD45 + P2RY12 - CD49d + ) ( I ) from Rheb1fl / fl ( fl / fl , blue , n = 4 ) and Cx3cr1 - Rheb1Δ / Δ ( Δ / Δ , red , n = 5 ) GL261 tumours . Representative FACS plots are displayed for each cell type ( mean ± SEM , Two - Way ANOVA Tukey 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 B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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(B) Frequency of occurrence of differentially regulated genes across significantly deregulated canonical pathways compared to the Log fold change (Log(FC)).(C) Upstream regulators as identified by the IPA software from differentially regulated genes.(E) Microglia were pre-treated with Torin, or medium as indicated and then stimulated with mNSC-CM, mGICPten-/-;p53-/--CM, or GL261-CM. p-NF-ĸB (p-P65) and p-STAT3 were analysed by immunoblotting of whole cell lysates collected at 4hr.(F) Immunofluorescence images of NF-ĸB (P65) in conditioned microglia. Quantification of nuclear translocation of NF-κB from staining. Units represent the % of voxels in the NF-κB channel colocalised with DAPI (n=3; mean ±SEM; One-Way ANOVA Tukey test - right). Scale bar is 50µm.(G) Production of Il-12 (Il-12p40), Tnf, Il6, and Il10 by conditioned microglia was determined by qPCR. Each treatment was normalised to housekeeping gene and the unconditioned control. (n=3; mean ±SEM; Two-Way ANOVA Tukey test).(H,I) MFI levels of pSTAT3 in TAM-MG (CD45+ P2RY12+ CD49d-) (H) and p-NF-κB (p-P65) in TAM-BMDM (CD45+ P2RY12- CD49d+) (I) from Rheb1fl/fl (fl/fl, blue, n=4) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, red, n=5) GL261 tumours. Representative FACS plots are displayed for each cell type (mean ±SEM, Two-Way ANOVA Tukey test).


Figure 7 ( A ) Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM - MG or TAM - BMDM signatures in TCGA GBM transcriptomic data . Comparison carried out on all IDH - wild - type samples , and in a subgroup specific manner according to Wang ' s classifier . Size of circle is indicative of Rsquare value and bold outline represents a p - value ≤ 0 . 05 . ( B ) Separation of IDH - wildtype GBM samples between those displaying high mTOR and microglia enrichment ( + ve correlation , group 1 in orange ) and those without this signature ( group 2 in green ) . ( C ) CIBERSORT cell fractions calculated from the TPM of TCGA IDH - wild type GBM samples from group 1 compared to those from group 2 . ( F ) Staining by flow cytometry of EPSC , YS - EBs and matched - iMGL with indicated markers ( n = 3 ; mean ± SEM ) . ( G , H ) Mature iMGL were immunostained for ( G ) PU1 and CD11b and ( H ) Iba1 and TREM2 . Scale bar 20µm . ( I - K ) ( I ) P - S6 , ( J ) p - STAT3 , and ( K ) p - NF - ĸB ( p - P65 ) were analysed by flow cytometry in iMGL , pre - treated with Torin , as indicated and then stimulated with matched patient EPSC - or hGIC - CM . ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey 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 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 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 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 7(A) Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM-MG or TAM-BMDM signatures in TCGA GBM transcriptomic data. Comparison carried out on all IDH-wild-type samples, and in a subgroup specific manner according to Wang's classifier. Size of circle is indicative of Rsquare value and bold outline represents a p-value ≤0.05.(B) Separation of IDH-wildtype GBM samples between those displaying high mTOR and microglia enrichment (+ve correlation, group 1 in orange) and those without this signature (group 2 in green).(C) CIBERSORT cell fractions calculated from the TPM of TCGA IDH-wild type GBM samples from group 1 compared to those from group 2.(F) Staining by flow cytometry of EPSC, YS-EBs and matched-iMGL with indicated markers (n=3; mean ±SEM).(G,H) Mature iMGL were immunostained for (G) PU1 and CD11b and (H) Iba1 and TREM2. Scale bar 20µm.(I-K) (I) P-S6, (J) p-STAT3, and (K) p-NF-ĸB (p-P65) were analysed by flow cytometry in iMGL, pre-treated with Torin, as indicated and then stimulated with matched patient EPSC- or hGIC-CM. (n=3; mean ±SEM, Two-Way ANOVA Tukey test).


Figure 1 F . A physical 650 - kb window of the genomic distribution of genes surrounding SNP rs4971059 , such as EFNA4 , KRTCAP2 , MUC1 , MIR92B , THBS3 , and DAP3 ( top ) . Total RNAs were extracted from [ GG ] , [ GA ] , or [ AA ] MCF - 10A cells and analyzed for the expression of EFNA4 , KRTCAP2 , TRIM46 , MUC1 , THBS3 , MIR92B , or DAP3 by RT - qPCR . TRIM28 was examined as a negative control ( bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 F. A physical 650-kb window of the genomic distribution of genes surrounding SNP rs4971059, such as EFNA4, KRTCAP2, MUC1, MIR92B, THBS3,and DAP3 (top). Total RNAs were extracted from [GG], [GA], or [AA] MCF-10A cells and analyzed for the expression of EFNA4, KRTCAP2, TRIM46, MUC1, THBS3, MIR92B, or DAP3 by RT-qPCR. TRIM28 was examined as a negative control (bottom).


Figure 2 B . MCF - 7 cells were transfected with a control vector or FLAG - TRIM46 for 40 h and treated with DMSO or MG132 ( 2μM ) for 10h . Cellular lysates were then collected for western blotting with antibodies against the indicated proteins . F . Schematic diagrams of HDAC1 deletion mutants . GST pull - down experiments were performed with bacterially expressed GST - fused proteins as indicated and in vitro transcribed / translated TRIM46 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B. MCF-7 cells were transfected with a control vector or FLAG-TRIM46 for 40 h and treated with DMSO or MG132 (2μM) for 10h. Cellular lysates were then collected for western blotting with antibodies against the indicated proteins.  F. Schematic diagrams of HDAC1 deletion mutants. GST pull-down experiments were performed with bacterially expressed GST-fused proteins as indicated and in vitro transcribed/translated TRIM46.


Figure 3 F . MCF - 7 cells were transfected with empty vector or FLAG - TRIM46 for forty - eight hours and then treated with 50 μg / mL cycloheximide ( CHX ) for the indicated hours . DMSO was added to cells 10 h before cellular proteins were collected for western blotting ( left ) . On the right panel , quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1 . G . MCF - 7 cells were treated as described in F , except that MG132 ( 10 μM ) was added to cells 10 h before cellular proteins were collected for western blotting ( left ) . Quantitation was performed on the right panel as in F . H . MCF - 7 cells were treated with control siRNA or TRIM46 siRNA . Forty - eight hours after transfection , cells were treated with 50 μg / mL CHX for the indicated hours before cellular proteins were extracted for western blotting analysis ( left ) . Quantitation was performed on the right panel as in F .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 F. MCF-7 cells were transfected with empty vector or FLAG-TRIM46 for forty-eight hours and then treated with 50 μg/mL cycloheximide (CHX) for the indicated hours. DMSO was added to cells 10 h before cellular proteins were collected for western blotting (left). On the right panel, quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1. G. MCF-7 cells were treated as described in F, except that MG132 (10 μM) was added to cells 10 h before cellular proteins were collected for western blotting (left). Quantitation was performed on the right panel as in F. H. MCF-7 cells were treated with control siRNA or TRIM46 siRNA. Forty-eight hours after transfection, cells were treated with 50 μg/mL CHX for the indicated hours before cellular proteins were extracted for western blotting analysis (left). Quantitation was performed on the right panel as in F.


Figure 4 In vivo ubiquitination assays performed in MCF - 7 cells . ( B ) Stable shCTR - or shTRIM46 - expressing MCF7 cells were transfected with His - Ub for 40 h . Cells were then treated with MG132 ( 10 μM ) for 12 h before Ni - NTA bead precipitation followed by IB with anti - HDAC1 . ( C ) and ( D ) MCF - 7 cells were co - transfected with the indicated plasmids . Forty - eight hours after transfection , cells were treated with MG132 for 10 h before cellular extracts were harvested for Ni - NTA bead precipitation followed by IB with anti - HDAC1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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  In vivo ubiquitination assays performed in MCF-7 cells. (B) Stable shCTR- or shTRIM46-expressing MCF7 cells were transfected with His-Ub for 40 h. Cells were then treated with MG132 (10 μM) for 12 h before Ni-NTA bead precipitation followed by IB with anti-HDAC1.  (C) and (D) MCF-7 cells were co-transfected with the indicated plasmids. Forty-eight hours after transfection, cells were treated with MG132 for 10 h before cellular extracts were harvested for Ni-NTA bead precipitation followed by IB with anti-HDAC1.


Figure 5 E - G . Verification of representative genes targeted by the TRIM46 - HDAC1 axis . ( E ) MCF - 7 cells were transfected with empty vector , FLAG - TRIM46 , or FLAG - TRIM46 together with FLAG - HDAC1 for 48 h . ( F ) MCF - 7 cells were transfected with control siRNA , TRIM46 siRNA , or TRIM46 siRNA together with HDAC1 siRNA for 72 h . ( G ) MCF - 7 cells were transfected with control siRNA , TRIM46 siRNA , or a siRNA - resistant TRIM46 construct together with TRIM46 siRNA for 72 h . Total RNAs were extracted from cells and RT - qPCR was performed with primers specific for indicted genes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 E-G. Verification of representative genes targeted by the TRIM46-HDAC1 axis. (E) MCF-7 cells were transfected with empty vector, FLAG-TRIM46, or FLAG-TRIM46 together with FLAG-HDAC1 for 48 h. (F) MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or TRIM46 siRNA together with HDAC1 siRNA for 72 h. (G) MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or a siRNA-resistant TRIM46 construct together with TRIM46 siRNA for 72 h. Total RNAs were extracted from cells and RT-qPCR was performed with primers specific for indicted genes.


Figure 6 B . EdU assays were performed in MCF - 7 cells treated with control siRNA , TRIM46 siRNA or TRIM46 siRNA and HDAC1 siRNA for 72 h , or MCF - 10A cells treated with vector , FLAG - TRIM46 or FLAG - TRIM46 and FLAG - HDAC1 for 48 h . Representative images from FACS analysis are shown on the left panel , the percentage numbers of cells in S phase was quantified by FlowJo software on the right panel . G . Accumulation of γH2AX in cells depleted of TRIM46 or HDAC1 after cisplatin stimulation . MCF - 7 cells were treated with 4 μM cisplatin for 8 h before harvesting . Cell lysates were analyzed by western blotting .

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

Figure 6 B. EdU assays were performed in MCF-7 cells treated with control siRNA, TRIM46 siRNA or TRIM46 siRNA and HDAC1 siRNA for 72 h, or MCF-10A cells treated with vector, FLAG-TRIM46 or FLAG-TRIM46 and FLAG-HDAC1 for 48 h. Representative images from FACS analysis are shown on the left panel, the percentage numbers of cells in S phase was quantified by FlowJo software on the right panel.  G. Accumulation of γH2AX in cells depleted of TRIM46 or HDAC1 after cisplatin stimulation. MCF-7 cells were treated with 4 μM cisplatin for 8 h before harvesting. Cell lysates were analyzed by western blotting.


Figure 7 B . MCF - 7 cells stably transfected with indicated shRNAs or expression plasmids or MCF - 10A cells stably transfected with expression plasmids were tested for colony formation assay . D . Control MCF - 7 cells or MCF - 7 cells depleted with TRIM46 were treated with different concentrations of cisplatin ( cis ) , VP16 , or adriamycin ( ADM ) for 72 h , and the proportions of viable cells were examined . E . Control MCF - 10A cells or MCF - 10A cells overexpressed TRIM46 were treated with different concentrations of cisplatin , VP16 , or adriamycin for 72 h , and the proportions of viable cells were examined .

O O O O O O O O O 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 7 B. MCF-7 cells stably transfected with indicated shRNAs or expression plasmids or MCF-10A cells stably transfected with expression plasmids were tested for colony formation assay. D. Control MCF-7 cells or MCF-7 cells depleted with TRIM46 were treated with different concentrations of cisplatin (cis), VP16, or adriamycin (ADM) for 72 h, and the proportions of viable cells were examined. E. Control MCF-10A cells or MCF-10A cells overexpressed TRIM46 were treated with different concentrations of cisplatin, VP16, or adriamycin for 72 h, and the proportions of viable cells were examined.


Figure 8 A . Immunohistochemical staining of tissue arrays containing 142 breast carcinoma samples ( grade I : n = 29 , grade II : n = 69 , grade III : n = 44 ) and 20 normal mammary tissues for TRIM46 and HDAC1 expression . Representative images are shown on the left panel . The positively stained cells were analyzed , and the mean intensity was scored by Image - Pro Plus software on the right panel . In the boxplot , the central lines , the box limits , and the whiskers represented medians , 25th / 75th percentile , and min / max , respectively . ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA ) . B . Total proteins from 20 paired samples of breast cancer " T " versus adjacent normal breast tissues " A " were extracted for western blotting analysis with antibodies against TRIM46 or HDAC1 . E . Kaplan - Meier survival analysis of TCGA BRCA dataset for the correlation between overall survival of breast cancer patients and TRIM46 , BRCA1 , or BLM expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8 A. Immunohistochemical staining of tissue arrays containing 142 breast carcinoma samples (grade I: n=29, grade II: n=69, grade III: n=44) and 20 normal mammary tissues for TRIM46 and HDAC1 expression. Representative images are shown on the left panel. The positively stained cells were analyzed, and the mean intensity was scored by Image-Pro Plus software on the right panel. In the boxplot, the central lines, the box limits, and the whiskers represented medians, 25th/75th percentile, and min/max, respectively. (*P<0.05, **P<0.01, ****P < 0.0001, one-way ANOVA). B. Total proteins from 20 paired samples of breast cancer "T" versus adjacent normal breast tissues "A" were extracted for western blotting analysis with antibodies against TRIM46 or HDAC1. E. Kaplan-Meier survival analysis of TCGA BRCA dataset for the correlation between overall survival of breast cancer patients and TRIM46, BRCA1, or BLM expression.


Figure 1 ( A , B ) Relative change in total abundances between control and ρ0 cells averaged over four independent migration profiles ( Dataset EV1 ) . A , all mitochondrial proteins detected . Grey area indicates proteins that changed less than 2 - fold in both directions . Subunits of selected mitochondrial machineries are highligted as follows : magenta , pyruate dehydrogenase complex ( PDH ) ; blue , mitochondial carriers ( Carrier ) ; brown , translocase of the outer membrane ( TOM ) ; yellow , translocase of the inner membrane ( TIM ) ; green , mitochondrial contact site and cristae organizing system ( MICOS ) ; yellow - green , mitochondrial fusion - fission system ( Dynamics ) ; red , porin or voltage dependent anion channel ( VDAC ) ; B , subunits of OXPHOS complexes ( Complex I - V ) and mitoribosomes ( Ribosome ) . Gene names of the subunit with the highest abundances in ρ0 cells are indicated for each complex . For the full gene name , add NDU ( complex I ) , SDH ( complex II ) , UQC ( R10 ) , COX ( complex IV ) , ATP ( 5L , 5J2 ) , MRP ( Ribosome ) in front of the abbreviated gene names given in the figure .

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

Figure 1(A, B) Relative change in total abundances between control and ρ0 cells averaged over four independent migration profiles (Dataset EV1). A, all mitochondrial proteins detected. Grey area indicates proteins that changed less than 2-fold in both directions. Subunits of selected mitochondrial machineries are highligted as follows: magenta, pyruate dehydrogenase complex (PDH); blue, mitochondial carriers (Carrier); brown, translocase of the outer membrane (TOM); yellow, translocase of the inner membrane (TIM); green, mitochondrial contact site and cristae organizing system (MICOS); yellow-green, mitochondrial fusion-fission system (Dynamics); red, porin or voltage dependent anion channel (VDAC); B, subunits of OXPHOS complexes (Complex I-V) and mitoribosomes (Ribosome). Gene names of the subunit with the highest abundances in ρ0 cells are indicated for each complex. For the full gene name, add NDU (complex I), SDH (complex II), UQC (R10), COX (complex IV), ATP (5L, 5J2), MRP (Ribosome) in front of the abbreviated gene names given in the figure.


Figure 3 ( A , B ) Selected proteins involved in DNA ( A ) and RNA ( B ) maintenance of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . A , for POLG , UNG and TWNK , only abundances > 1 MDa were quantified . Plots and gene names are in corresponding colors . Data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test for individual proteins and two - way ANOVA for changes of multiple proteins in a complex ) .

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

Figure 3(A, B) Selected proteins involved in DNA (A) and RNA (B) maintenance of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, for POLG, UNG and TWNK, only abundances >1 MDa were quantified. Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test for individual proteins and two-way ANOVA for changes of multiple proteins in a complex).


Figure 4 ( A - F ) OXPHOS complexes ( A - C ) and selected enzymes involved in the energy metabolism of control ( 143B , solid lines ) and ρ0 cells ( dashed lines ) . A , Complex ( C ) I , III and IV ; B , F1 , FO domain and inhibitor protein IF1 of complex V ; C , subunits SDHA , SDHB - D and assembly factor SDHAF2 of complex II ; D , oxoglutarate ( OGDH ; average of OGDHL , OGDH , DLST ) and pyruvate dehydrogenase complex ( PDH ; average of PDHA1 , PDHB , PDHX , DLAT ) ; E , subunits of GDP forming ( SUCLG2 ) and ADP forming ( SUCLA2 ) succinyl - CoA ligase ; F , urea cycle enzymes and glutaminase ( GLS ) . Plots and gene names are in corresponding colors .

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

Figure 4(A-F) OXPHOS complexes (A-C) and selected enzymes involved in the energy metabolism of control (143B, solid lines) and ρ0 cells (dashed lines). A, Complex (C) I, III and IV; B, F1, FO domain and inhibitor protein IF1 of complex V; C, subunits SDHA, SDHB-D and assembly factor SDHAF2 of complex II; D, oxoglutarate (OGDH; average of OGDHL, OGDH, DLST) and pyruvate dehydrogenase complex (PDH; average of PDHA1, PDHB, PDHX, DLAT); E, subunits of GDP forming (SUCLG2) and ADP forming (SUCLA2) succinyl-CoA ligase; F, urea cycle enzymes and glutaminase (GLS). Plots and gene names are in corresponding colors.


Figure 5 ( A - D ) Selected metabolite carriers of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . A , isoforms of the nucleotide carrier ( ANT1 - 3 ) and phosphate carrier ( PiC ) ; B , pyruvate carrier ; C , dicarboxylate carriers ; D , mitoferrins ( MFRN1 - 2 ) and glycine transporter ( GlyC ) . Plots and gene names are in corresponding colors . Data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) .

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

Figure 5(A-D) Selected metabolite carriers of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, isoforms of the nucleotide carrier (ANT1-3) and phosphate carrier (PiC); B, pyruvate carrier; C, dicarboxylate carriers; D, mitoferrins (MFRN1-2) and glycine transporter (GlyC). Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01 (Student's t-test).


Figure 6 ( A - D ) Components of the mitochondrial protein import machinery and selected proteins related to this process of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored and dark - grey bars ) . A and B , components of the translocase of the outer membrane ( TOM ) machinery ( TOM avg . : average of TOMM40 , TOMM22 , TOMM5 , TOMM6 , TOMM7 ) ; C , components of the trans locase of the inner membrane ( TIM ) machinery ( TIM22 avg . : TIMM22 , AGK , TIMM10B ; TIM23 avg . : TIMM23 , TIMM17A , TIMM17B ) ; D , components of the TIM23 - associated TIMM21 assembly machinery ( left panel ) and its association with prohibitin ( PHB ) and components of the mitochondrial quality control system ( right panel ) . Plots and gene names are in corresponding colors , except for TIMM44 and TIMM50 ( no migration profiles shown ) . In ( A - C ) data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Components of the mitochondrial protein import machinery and selected proteins related to this process of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored and dark-grey bars). A and B, components of the translocase of the outer membrane (TOM) machinery (TOM avg.: average of TOMM40, TOMM22, TOMM5, TOMM6, TOMM7); C, components of the translocase of the inner membrane (TIM) machinery (TIM22 avg.: TIMM22, AGK, TIMM10B; TIM23 avg.: TIMM23, TIMM17A, TIMM17B); D, components of the TIM23-associated TIMM21 assembly machinery (left panel) and its association with prohibitin (PHB) and components of the mitochondrial quality control system (right panel). Plots and gene names are in corresponding colors, except for TIMM44 and TIMM50 (no migration profiles shown). In (A-C) data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test).


Figure 7Selected proteins and complexes involved in controlling mitochondrial morphology and fusion / fission machinery and related processes of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . A , mitochondrial contact site and cristae organizing system ( MICOS : MIC60 , MIC19 , MIC25 ) and SAM complex ( SAM : SAMM50 , MTX2 , MTX3 ) together forming the MIB complexSelected proteins and complexes involved in controlling mitochondrial morphology and fusion / fission machinery and related processes of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . B , proteins involved in controlling mitochondrial morphology ( orange ) and dynamics ( teal ) ; Selected proteins and complexes involved in controlling mitochondrial morphology and fusion / fission machinery and related processes of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . C , DNM1L / DRP1 and interacting proteins . For DNM1L , MTFR1 and ARL2BP , quantification of the entire migra tion profiles ( Overall ) and the range above 1 , 400 kDa only ( > 1 , 400 kDa ) are shown . Plots and gene names are in corresponding colors , except for ARL2 ( dark - grey ) not shown in the plot . Data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, mitochondrial contact site and cristae organizing system (MICOS: MIC60, MIC19, MIC25) and SAM complex (SAM: SAMM50, MTX2, MTX3) together forming the MIB complexSelected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). B, proteins involved in controlling mitochondrial morphology (orange) and dynamics (teal);Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). C, DNM1L/DRP1 and interacting proteins. For DNM1L, MTFR1 and ARL2BP, quantification of the entire migration profiles (Overall) and the range above 1,400 kDa only (>1,400 kDa) are shown. Plots and gene names are in corresponding colors, except for ARL2 (dark-grey) not shown in the plot. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).


Figure 1 ( a ) Primary siRNA screening analysis . MDA - MB - 231 cells reverse transfected with siRNA pools against MT1 - MMP , TKS5 , or each human RAB GTPase were plated on fluorescein - labeled - gelatin coated optical microplates . Degradation activity was measured by counting the number of cells degrading gelatin identified as dark black foci ( similar results were obtained by measuring the area of degradation / number of cells ) . The inset shows representative images of degraded Oregon green gelatin ( upper panels ) and merged images with TRITC - phalloidin ( red ) and DAPI ( blue ) in scrambled siRNA ( NC ) , or siRNA against RAB28 or RAB42 transfected cells ( lower panel ) . Data are means ± SEM of 3 independent experiments . Bar , 50 µm . ( b ) Secondary siRNA screening analysis was performed on candidate RAB GTPases selected from the primary screen using 3 individual deconvoluted siRNAs . Data are mean ± SD ( n > 5 different fields / experiment ) . ( c - e ) Tertiary siRNA screening . Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5 , used as control , were transfected into MCF10 . DCIS . com cells . Cells plated onto fluorescently conjugated gelatin ( red ) were stimulated with 100 ng / ml HGF overnight in the presence or absence of GM6001 , followed by staining with FITC - phalloidin ( green ) and DAPI ( blue ) ( c ) . Data are the mean ± SEM ( d , error bars ; n > 100 cells / experiment in at least 4 independent ones ) . ( c - e ) Tertiary siRNA screening . Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5 , used as control , were transfected into MCF10 . DCIS . com cells . Efficacy of targeted gene silencing was verified by qPCR ( e ) . Bar , 50 µm . ( f - h ) Inverse invasion assay . An equal number of MDA - MB - 231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes . Invasive activity is shown as the average number of cells / field that migrate into fibronectin - supplemented ( 25 µg / ml ) Matrigel further than 20 µm ( f , g ) . Data are mean ± SD from three different fields from one representative experiments out of 5 that were performed . ( f - h ) Inverse invasion assay . An equal number of MDA - MB - 231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes . Efficacy of targeted gene silencing was verified by qPCR ( h ) . Bar , 250 µm . * P < 0 . 05 . * * * , P < 0 . 001 .

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

Figure 1(a) Primary siRNA screening analysis. MDA-MB-231 cells reverse transfected with siRNA pools against MT1-MMP, TKS5, or each human RAB GTPase were plated on fluorescein-labeled-gelatin coated optical microplates. Degradation activity was measured by counting the number of cells degrading gelatin identified as dark black foci (similar results were obtained by measuring the area of degradation/number of cells). The inset shows representative images of degraded Oregon green gelatin (upper panels) and merged images with TRITC-phalloidin (red) and DAPI (blue) in scrambled siRNA (NC), or siRNA against RAB28 or RAB42 transfected cells (lower panel). Data are means ± SEM of 3 independent experiments. Bar, 50 µm.(b) Secondary siRNA screening analysis was performed on candidate RAB GTPases selected from the primary screen using 3 individual deconvoluted siRNAs. Data are mean ± SD (n> 5 different fields/experiment).(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Cells plated onto fluorescently conjugated gelatin (red) were stimulated with 100 ng/ml HGF overnight in the presence or absence of GM6001, followed by staining with FITC-phalloidin (green) and DAPI (blue) (c). Data are the mean ± SEM (d, error bars; n > 100 cells/experiment in at least 4 independent ones).(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Efficacy of targeted gene silencing was verified by qPCR (e). Bar, 50 µm.(f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Invasive activity is shown as the average number of cells/field that migrate into fibronectin-supplemented (25 µg/ml) Matrigel further than 20 µm (f, g). Data are mean ± SD from three different fields from one representative experiments out of 5 that were performed.(f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Efficacy of targeted gene silencing was verified by qPCR (h). Bar, 250 µm. *P < 0.05. ***, P < 0.001.


Figure 2 ( a ) Doxycycline - inducible empty vector ( EV ) or RAB2A - shRNA MDA - MB - 231 cells were cultured in the absence ( - ) or presence ( + ) of doxycycline for three days , and immunoblotted with anti - RAB2A and anti - actin antibodies . The intensity of RAB2A relative to actin is shown on the bottom . Relative density in uninduced EV cells was set to 1 . ( b ) Doxycycline - inducible RAB2A - shRNA MDA - MB - 231 cells were cultured in the absence ( CTR ) or presence ( shRAB2A ) of doxycycline for three days . Invasion of MDA - MB - 231 cells through Matrigel - coated transwells toward HGF was assessed after 24 hours . Representative images are shown . Data are the mean ± SD ( n = 10 field / condition in at least 3 experiments ) . Bar , 200 µm . ( c - e ) EV and shRAB2AMDA - MB - 231 cells were embedded in Spheroid Formation ECMR in the absence ( - ) or presence ( + ) of doxycycline and / or GM6001 . After 3 days , the cells were transferred into Invasive MatrixR . Phase contrast light microscopy images of representative spheroids are shown after 9 days ( c ) . Dashed circle indicates the size of spheroids at day 0 . Bars , 500 µm . Invasive activity into ECM was expressed as total spheroid area ( relative to control cells at day 0 ) quantified by ImageJ ( d ) . The number of cells at the indicated days was quantified by CyQUANTR Cell Proliferation Assay Kit ( e ) . Data are the mean ± SD ( n = 3 independent experiments ) . ( f - h ) Doxycycline - inducible murine RAB2A ( mRAB2A ) - expression MCF10 . DCIS . com cells were transfected with scrambled siRNA or siRNA against human RAB2A ( siRAB2A ) in the absence ( - ) or presence ( + ) of doxycycline . Serum - starved cells were plated onto fluorescently conjugated gelatin ( red ) , stimulated with HGF ( 100 ng / ml ) overnight , and stained with phalloidin ( green ) and DAPI ( blue ) ( f ) . Bar , 50 μm . Data are the mean ± SEM ( error bars ; n > 100 cells / experiment in 4 independent ones ) ( g ) . Silencing of endogenous RAB2A protein ( lower band ) and ectopic RAB2A expression ( upper band ) was verified by immunoblotting ( h ) . * P < 0 . 05 , * * , P < 0 . 01 , * * * , P < 0 . 001 , NS , not significant .

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

Figure 2(a) Doxycycline-inducible empty vector (EV) or RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (-) or presence (+) of doxycycline for three days, and immunoblotted with anti-RAB2A and anti-actin antibodies. The intensity of RAB2A relative to actin is shown on the bottom. Relative density in uninduced EV cells was set to 1.(b) Doxycycline-inducible RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days. Invasion of MDA-MB-231 cells through Matrigel-coated transwells toward HGF was assessed after 24 hours. Representative images are shown. Data are the mean ± SD (n=10 field/condition in at least 3 experiments). Bar, 200 µm.(c-e) EV and shRAB2AMDA-MB-231 cells were embedded in Spheroid Formation ECMR in the absence (-) or presence (+) of doxycycline and/or GM6001. After 3 days, the cells were transferred into Invasive MatrixR. Phase contrast light microscopy images of representative spheroids are shown after 9 days (c). Dashed circle indicates the size of spheroids at day 0. Bars, 500 µm. Invasive activity into ECM was expressed as total spheroid area (relative to control cells at day 0) quantified by ImageJ (d). The number of cells at the indicated days was quantified by CyQUANTR Cell Proliferation Assay Kit (e). Data are the mean ± SD (n=3 independent experiments).(f-h) Doxycycline-inducible murine RAB2A (mRAB2A)-expression MCF10.DCIS.com cells were transfected with scrambled siRNA or siRNA against human RAB2A (siRAB2A) in the absence (-) or presence (+) of doxycycline. Serum-starved cells were plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue) (f). Bar, 50 μm. Data are the mean ± SEM (error bars; n > 100 cells/experiment in 4 independent ones) (g). Silencing of endogenous RAB2A protein (lower band) and ectopic RAB2A expression (upper band) was verified by immunoblotting (h). * P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.


Figure 3Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort ( n = 622 ) operated in the European Institute of Oncology , Milano , Italy , in the year 2000 . Expression was measured by IHC on TMA . ( a ) Images are representative of RAB2A expression scoring according to intensity staining in TMA . In tumor tissues the IHC signals were associated with the tumor cell component and not with the adjacent or infiltrating stroma . Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort ( n = 622 ) operated in the European Institute of Oncology , Milano , Italy , in the year 2000 . Expression was measured by IHC on TMA . ( b ) Correlation of RAB2A expression with clinicopathological parameters . Only for 520 of 622 samples expression data for RAB2A was available . Note that the number of scored cases is lower than the total number of cases since : i ) in some cases , individual cores detached from the slides during the manipulations ; ii ) clinical information was not available for all patients . In tumor tissues the IHC signal was associated with the tumor cell component and not with the adjacent or infiltrating stroma . Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort ( n = 622 ) operated in the European Institute of Oncology , Milano , Italy , in the year 2000 . Expression was measured by IHC on TMA . ( c ) Kaplan - Meier Distant Recurrence Free Interval survival ( DRFI [ 70 ] ) and ( d ) Univariate and Multivariate Cox - regression analysis , shows that in BC patients ' high levels of RAB2A protein predict a poor prognosis , and that RAB2A expression is an independent predictor of higher risk of developing a distant metastatic recurrence .

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 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

Figure 3Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(a) Images are representative of RAB2A expression scoring according to intensity staining in TMA. In tumor tissues the IHC signals were associated with the tumor cell component and not with the adjacent or infiltrating stroma.Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(b) Correlation of RAB2A expression with clinicopathological parameters. Only for 520 of 622 samples expression data for RAB2A was available. Note that the number of scored cases is lower than the total number of cases since: i) in some cases, individual cores detached from the slides during the manipulations; ii) clinical information was not available for all patients. In tumor tissues the IHC signal was associated with the tumor cell component and not with the adjacent or infiltrating stroma.Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(c) Kaplan-Meier Distant Recurrence Free Interval survival (DRFI [70]) and (d) Univariate and Multivariate Cox-regression analysis, shows that in BC patients' high levels of RAB2A protein predict a poor prognosis, and that RAB2A expression is an independent predictor of higher risk of developing a distant metastatic recurrence.


Figure 4 ( a , b ) MCF10 . DCIS . com cells were transfected with FLAG - empty vector ( EV ) , constitutively active RAB2A - Q65L , or dominant negative RAB2A - S20N mutant . Cells were immunostained with anti - FLAG ( red ) and RAB7 ( a , green ) or anti - EEA1 ( b ) antibodies . Merged images with DAPI ( blue ) are shown on the right . Enlarged endosomes in the boxed region are highly magnified in the insets . Diameters of RAB7 positive vesicles are also shown . n , number of cells scored for each cell population . The RGB profiler plugin from ImageJ was used to plot colocalization in the lines drawn in the inset images . Bars , 10 μm , ( inset 2 μm ) . ( c ) MDA - MB - 231 cells were transfected with EGFP - fused EV , RAB2A - Q65L , or RAB2A - S20N . Cells are immunostained with anti - RAB7 ( blue ) and anti - MT1 - MMP ( red ) antibodies , and their merged images are shown . Merged images are shown on the right . Enlarged endosomes containing MT1 - MMP in the boxed region are highly magnified in the insets . Note that RAB2A - Q65L and RAB7 - positive vesicles contain MT1 - MMP ( arrows ) . Bars , 10 μm , ( inset 2 μm ) . ( d - f ) MDA - MB - 231 cells expressing MT1 - MMP - pHluorin transfected with scrambled siRNA ( siNC ) or siRNA against RAB2A were seeded on type I collagen , and images were acquired for 20 min period by TIRF microscopy . Frequency of MT1 - MMP - pHluorin exocytic events was quantified ( e ) . The corresponding time - lapse series are shown in Movie 1 , while an entire cell shown in Movie 2 . Efficacy of RAB2A gene silencing was verified by qPCR ( f ) . Bars , 2 μm . * * , P < 0 . 01 .

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 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

Figure 4(a, b) MCF10.DCIS.com cells were transfected with FLAG-empty vector (EV), constitutively active RAB2A-Q65L, or dominant negative RAB2A-S20N mutant. Cells were immunostained with anti-FLAG (red) and RAB7 (a, green) or anti-EEA1 (b) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. The RGB profiler plugin from ImageJ was used to plot colocalization in the lines drawn in the inset images. Bars, 10 μm, (inset 2 μm).(c) MDA-MB-231 cells were transfected with EGFP-fused EV, RAB2A-Q65L, or RAB2A-S20N. Cells are immunostained with anti-RAB7 (blue) and anti-MT1-MMP (red) antibodies, and their merged images are shown. Merged images are shown on the right. Enlarged endosomes containing MT1-MMP in the boxed region are highly magnified in the insets. Note that RAB2A-Q65L and RAB7-positive vesicles contain MT1-MMP (arrows). Bars, 10 μm, (inset 2 μm).(d-f) MDA-MB-231 cells expressing MT1-MMP-pHluorin transfected with scrambled siRNA (siNC) or siRNA against RAB2A were seeded on type I collagen, and images were acquired for 20 min period by TIRF microscopy. Frequency of MT1-MMP-pHluorin exocytic events was quantified (e). The corresponding time-lapse series are shown in Movie 1, while an entire cell shown in Movie 2. Efficacy of RAB2A gene silencing was verified by qPCR (f). Bars, 2 μm. **, P < 0.01.


Figure 5 ( a ) Total cellular lysates from HEK293T cells transfected with myc - VPS39 or VPS41 were incubated with GST - empty vector ( EV ) , RAB2A - S20N ( SN ) , or RAB2A - Q65L ( QL ) proteins bound to glutathione sepharose beads . Immunoblotting was performed with anti - myc antibody . Input lysates are also shown . GST proteins loaded in gels were stained with CBB . ( b ) FLAG - EV or RAB2A were transiently co - transfected with EGFP - VPS39 or VPS41 into HEK293T cells . Immunoprecipitated ( IP ) fraction from cell lysates with anti - FLAG antibody and total lysates were immunoblotted with the antibodies indicated on the right . ( c , d ) MDA - MB - 231 cells were transfected with EGFP - fused empty EV , VPS39 , or VPS41 . Cells were immunostained with RAB7 ( c , red ) or anti - MT1 - MMP ( d ) antibodies . Merged images with DAPI ( blue ) are shown on the right . Enlarged endosomes in the boxed region are highly magnified in the insets . Diameters of RAB7 positive vesicles are also shown . n , number of cells scored for each cell population . Bars , 10 μm , ( inset 5 μm ) . ( e - i ) Scrambled siRNA ( siNC ) or siRNA against RAB2A ( siRAB2A ) , VPS39 ( siVPS39 ) , or VPS41 ( siVPS41 ) were transfected into MCF10 . DCIS . com cells . Serum - starved cells were then plated onto fluorescent - conjugated gelatin ( red ) , stimulated with HGF ( 100 ng / ml ) overnight , and stained with phalloidin ( green ) and DAPI ( blue ) . Quantification of gelatin degradation ( f ) and efficacy of targeted gene silencing verified by qPCR ( g - i ) . Data are the mean ± SEM ( error bars ; n > 100 cells / experiment in at least 4 independent ones ) . Bars , 50 µm . ( j - l ) Inverse invasion assay of MDA - MB - 231 cells transfected with siRNA oligonucleotides targeting interest genes . Invasive activity is the numbers of cells / field that migrate through Matrigel further than 50 µm ( k ) . Efficacy of targeted gene silencing was verified by qPCR ( l ) . Data are mean ± SD from 3 different fields . Bar , 250 µm . ( m - q ) Doxycycline - inducible murine RAB2A - expression MCF10 . DCIS . com cells were transfected with siNC , siRAB2A , or siVPS39 in the absence ( CTR ) or presence ( RAB2A ) of doxycycline . Their ECM degradation activities were quantified ( n ) . Data are the mean ± SEM ( error bars ; n > 100 cells / experiment in 3 independent ones ) . Total cell lysates were immunoblotted by anti - RAB2A and anti - tubulin antibodies ( o ) . Efficacy of targeted gene silencing was verified by qPCR ( p - q ) . Bar , 50 μm . * * , P < 0 . 01 , * * * , P < 0 . 001 .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Total cellular lysates from HEK293T cells transfected with myc-VPS39 or VPS41 were incubated with GST-empty vector (EV), RAB2A-S20N (SN), or RAB2A-Q65L (QL) proteins bound to glutathione sepharose beads. Immunoblotting was performed with anti-myc antibody. Input lysates are also shown. GST proteins loaded in gels were stained with CBB.(b) FLAG-EV or RAB2A were transiently co-transfected with EGFP-VPS39 or VPS41 into HEK293T cells. Immunoprecipitated (IP) fraction from cell lysates with anti-FLAG antibody and total lysates were immunoblotted with the antibodies indicated on the right.(c, d) MDA-MB-231 cells were transfected with EGFP-fused empty EV, VPS39, or VPS41. Cells were immunostained with RAB7 (c, red) or anti-MT1-MMP (d) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. Bars, 10 μm, (inset 5 μm).(e-i) Scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A), VPS39 (siVPS39), or VPS41 (siVPS41) were transfected into MCF10.DCIS.com cells. Serum-starved cells were then plated onto fluorescent-conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue). Quantification of gelatin degradation (f) and efficacy of targeted gene silencing verified by qPCR (g-i). Data are the mean ± SEM (error bars; n > 100 cells/experiment in at least 4 independent ones). Bars, 50 µm.(j-l) Inverse invasion assay of MDA-MB-231 cells transfected with siRNA oligonucleotides targeting interest genes. Invasive activity is the numbers of cells/field that migrate through Matrigel further than 50 µm (k). Efficacy of targeted gene silencing was verified by qPCR (l). Data are mean ± SD from 3 different fields. Bar, 250 µm.(m-q) Doxycycline-inducible murine RAB2A-expression MCF10.DCIS.com cells were transfected with siNC, siRAB2A, or siVPS39 in the absence (CTR) or presence (RAB2A) of doxycycline. Their ECM degradation activities were quantified (n). Data are the mean ± SEM (error bars; n > 100 cells/experiment in 3 independent ones). Total cell lysates were immunoblotted by anti-RAB2A and anti-tubulin antibodies (o). Efficacy of targeted gene silencing was verified by qPCR (p-q). Bar, 50 μm. **, P < 0.01, ***, P < 0.001.


Figure 6 ( a ) Doxycycline - inducible RAB2A - shRNA MCF10 . DCIS . com cells were incubated on a thick layer of Matrigel for 4 days to allow spheroid formation in the absence ( CTR ) or presence ( shRAB2A ) of doxycycline . Cells were stimulated with 5 ng / ml EGF and 20 ng / ml HGF ( EGF + HGF ) or left starved ( STV ) for 9 days . Representative phase contrast light microscopy images are shown . Invasive activity of the cells was expressed by circularity ( with a value of 1 representing perfect circularity = no invasion ) of each acini . Bar , 100 µm . ( b - d ) MCF10 . DCIS . com cells ( 104 cells ) transfected with scrambled siRNA ( siNC ) or siRNA against RAB2A ( siRAB2A ) were plated onto 6 - well dish . Representative phase contrast light microscopy images ( b ) and the distribution of cell numbers per cluster of the indicated cell number ( c ) are shown . Data are means ± SD of 10 fields ( n > 60 cells / field ) of independent 4 independent experiments . Bars , 100 μm . Efficacy of RAB2A gene silencing was verified by qPCR ( d ) . Bar , 200 µm . ( e ) Doxycycline - inducible RAB2A - shRNA MCF10 . DCIS . com cells were cultured in the absence ( CTR ) or presence ( shRAB2A ) of doxycycline for three days , and immunostained with antibodies indicated on the top ( green ) and DAPI ( blue ) . ( f , g ) MCF - 10AneoT ( f ) cells were transfected with scrambled siRNA ( siNC ) or siRNA against RAB2A , and immunostained with antibodies indicated on the top ( green ) and DAPI ( blue ) . Bars , 10 μm . ( f , g ) MCF10 - CA1a ( g ) cells were transfected with scrambled siRNA ( siNC ) or siRNA against RAB2A , and immunostained with antibodies indicated on the top ( green ) and DAPI ( blue ) . Bars , 10 μm . ( h ) Total cell lysates from ( e ) , ( f ) , and ( g ) were immunoblotted by anti - E - cadherin , RAB2A , and anti - actin antibodies . * * * , P < 0 . 001 , NS , not significant .

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

Figure 6(a) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were incubated on a thick layer of Matrigel for 4 days to allow spheroid formation in the absence (CTR) or presence (shRAB2A) of doxycycline. Cells were stimulated with 5 ng/ml EGF and 20 ng/ml HGF (EGF+HGF) or left starved (STV) for 9 days. Representative phase contrast light microscopy images are shown. Invasive activity of the cells was expressed by circularity (with a value of 1 representing perfect circularity = no invasion) of each acini. Bar, 100 µm.(b-d) MCF10.DCIS.com cells (104 cells) transfected with scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A) were plated onto 6-well dish. Representative phase contrast light microscopy images (b) and the distribution of cell numbers per cluster of the indicated cell number (c) are shown. Data are means ± SD of 10 fields (n > 60 cells/field) of independent 4 independent experiments. Bars, 100 μm. Efficacy of RAB2A gene silencing was verified by qPCR (d). Bar, 200 µm.(e) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days, and immunostained with antibodies indicated on the top (green) and DAPI (blue).(f, g) MCF-10AneoT (f) cells were transfected with scrambled siRNA (siNC) or siRNA against RAB2A, and immunostained with antibodies indicated on the top (green) and DAPI (blue). Bars, 10 μm.(f, g) MCF10-CA1a (g) cells were transfected with scrambled siRNA (siNC) or siRNA against RAB2A, and immunostained with antibodies indicated on the top (green) and DAPI (blue). Bars, 10 μm.(h) Total cell lysates from (e), (f), and (g) were immunoblotted by anti-E-cadherin, RAB2A, and anti-actin antibodies. ***, P < 0.001, NS, not significant.


Figure 7 ( a , b ) Doxycycline - inducible RAB2A - expression MCF10 . DCIS . com ( a ) and MCF10A ( b ) cells were cultured in the absence ( CTR ) or presence ( RAB2A ) of doxycycline , and immunostained with anti - RAB2A ( blue ) , anti - E - cadherin ( green ) , and anti - TGN46 ( red ) antibodies . Merged images with DAPI ( magenta ) are shown on the right . Bars , 10 μm . ( c ) CTR and RAB2AMCF10A cells ( 104 cells ) were plated onto 6 - well dish . Representative phase contrast light microscopy images ( left ) and the distribution of cell numbers per cluster composed of the indicated number of cells are shown . Data are means ± SD of 10 fields ( n > 20 cells / field ) from 3 independent experiments . Bar , 100 μm . ( d ) The circularity of CTR and RAB2A MCF10A cells and the percentage of cells with protrusive structure were quantified . Data are means ± SD of ten independent fields ( n > 100 cells / field ) . n , number of cells scored for each cell population . ( e ) Total cell lysate from ( c ) were immunoblotted with antibodies anti - E - cadherin , which recognize either an epitope in the cytoplasmic domain ( IC ) , an epitope in the extracellular domain of mature E - cadherin ( SHE78 - 7 ) , respectively , or anti - RAB2A , and anti - actin . An arrow indicates the unprocessed form of E - cadherin . ( f - h ) MCF10A cells were transfected with scrambled siRNA ( siNC ) or siRNA against Furin , and immunostained with anti - E - cadherin ( green ) and anti - TGN46 ( red ) antibodies . Merged images with DAPI ( blue ) are shown on the right ( f ) . Bar , 20 μm . Total cell lysate were immunoblotted with antibodies indicated on the right ( g ) . Efficacy of Furin silencing was verified by qPCR ( h ) . ( i ) Representative electron microscopy of Golgi stacks ( arrows ) of CTR and RAB2A MCF10 . DCIS . com cells . Bars , 1 μm . ( j ) CTR and RAB2A MCF10A cells were transfected with Str - KDEL _ SBP - EGFP - E - cadherin ( green ) . At the indicated time points after the addition of biotin , cells were fixed , and stained without permeabilization with anti - GFP antibody , followed by Alexa568 - conjugated secondary antibody ( red ) . The cell surface and total levels of SBP - EGFP - E - cadherin at 60 min after the addition of biotin were quantified using ImageJ software on non - saturated images , and surface / total ratio was expressed as a surface GFP index ( relative to control cells ) . Bar , 20 µm . * * * , P < 0 . 001 .

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

Figure 7(a, b) Doxycycline-inducible RAB2A-expression MCF10.DCIS.com (a) and MCF10A (b) cells were cultured in the absence (CTR) or presence (RAB2A) of doxycycline, and immunostained with anti-RAB2A (blue), anti-E-cadherin (green), and anti-TGN46 (red) antibodies. Merged images with DAPI (magenta) are shown on the right. Bars, 10 μm.(c) CTR and RAB2AMCF10A cells (104 cells) were plated onto 6-well dish. Representative phase contrast light microscopy images (left) and the distribution of cell numbers per cluster composed of the indicated number of cells are shown. Data are means ± SD of 10 fields (n > 20 cells/field) from 3 independent experiments. Bar, 100 μm.(d) The circularity of CTR and RAB2A MCF10A cells and the percentage of cells with protrusive structure were quantified. Data are means ± SD of ten independent fields (n > 100 cells/field). n, number of cells scored for each cell population.(e) Total cell lysate from (c) were immunoblotted with antibodies anti-E-cadherin, which recognize either an epitope in the cytoplasmic domain (IC), an epitope in the extracellular domain of mature E-cadherin (SHE78-7), respectively, or anti-RAB2A, and anti-actin. An arrow indicates the unprocessed form of E-cadherin.(f-h) MCF10A cells were transfected with scrambled siRNA (siNC) or siRNA against Furin, and immunostained with anti-E-cadherin (green) and anti-TGN46 (red) antibodies. Merged images with DAPI (blue) are shown on the right (f). Bar, 20 μm. Total cell lysate were immunoblotted with antibodies indicated on the right (g). Efficacy of Furin silencing was verified by qPCR (h).(i) Representative electron microscopy of Golgi stacks (arrows) of CTR and RAB2A MCF10.DCIS.com cells. Bars, 1 μm.(j) CTR and RAB2A MCF10A cells were transfected with Str-KDEL_SBP-EGFP-E-cadherin (green). At the indicated time points after the addition of biotin, cells were fixed, and stained without permeabilization with anti-GFP antibody, followed by Alexa568-conjugated secondary antibody (red). The cell surface and total levels of SBP-EGFP-E-cadherin at 60 min after the addition of biotin were quantified using ImageJ software on non-saturated images, and surface/total ratio was expressed as a surface GFP index (relative to control cells). Bar, 20 µm. ***, P < 0.001.


Figure 1 ( b ) Estimation of gene editing at the Trim28 loci using NGS - sequencing of amplicons . Black bars indicate % of frameshift indels . Columns show an average of two biological replicates per guide RNA and error bars show mean ± SD . ( c ) Western blot confirmed the loss of Trim28 expression upon Trim28 - KO in mouse NPCs . ( d ) RNA - seq analysis of the expression of TE - families using TEtranscripts ( e ) The significantly upregulated TE families with a fold change larger than 0 . 5 upon Trim28 - KO in mouse NPCs . The dashed line indicates significance . ( f ) CUT & RUN analysis of H3K9me3 in mouse NPCs of all full length MMERVK10Cs ( right panel ) RNA - seq analysis of the Trim28 - KO and control samples , visualising full length MMERVK10C elements . The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram . ( g ) Example of transcriptional readthrough outside the full length MMERVK10Cs into a nearby gene .

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

Figure 1(b) Estimation of gene editing at the Trim28 loci using NGS-sequencing of amplicons. Black bars indicate % of frameshift indels. Columns show an average of two biological replicates per guide RNA and error bars show mean ± SD. (c) Western blot confirmed the loss of Trim28 expression upon Trim28-KO in mouse NPCs.  (d) RNA-seq analysis of the expression of TE-families using TEtranscripts (e) The significantly upregulated TE families with a fold change larger than 0.5 upon Trim28-KO in mouse NPCs. The dashed line indicates significance.  (f) CUT&RUN analysis of H3K9me3 in mouse NPCs of all full length MMERVK10Cs (right panel) RNA-seq analysis of the Trim28-KO and control samples, visualising full length MMERVK10C elements. The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram.  (g) Example of transcriptional readthrough outside the full length MMERVK10Cs into a nearby gene.


Figure 2 ( b ) Estimation of gene editing at the Trim28 loci using NGS - sequencing of amplicons from DNA isolated from 50 . 000 RFP + nuclei per animal . One animal per group was analyzed . Black bars indicate % of the detected indels that disrupted the frameshift . Gene editing of the Trim28 - loci resulted in a robust loss of Trim28 protein , as evaluated by IHC where the expression of Trim28 in RFP + cells were quantified and are displayed as mean ± SEM . Approximately 600 RFP + cells per animal and group was evaluated . Scale bar 30 μm . Gene editing of the Trim28 - loci resulted in a robust loss of Trim28 protein , as evaluated by IHC where the expression of Trim28 in RFP + cells were quantified and are displayed as mean ± SEM . Approximately 600 RFP + cells per animal and group was evaluated . Scale bar 30 μm . ( e ) RNA - seq analysis of the expression of TE - families using TEtranscripts . ( f ) RNA - seq analysis of the Trim28 - KO and control samples , visualising full length MMERVK10C elements ( left panel ) and ChIP - seq analysis of H3K9me3 in adult forebrain neurons ( right panel ) . The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (b) Estimation of gene editing at the Trim28 loci using NGS-sequencing of amplicons from DNA isolated from 50.000 RFP+ nuclei per animal. One animal per group was analyzed. Black bars indicate % of the detected indels that disrupted the frameshift.  Gene editing of the Trim28-loci resulted in a robust loss of Trim28 protein, as evaluated by IHC where the expression of Trim28 in RFP+ cells were quantified and are displayed as mean ± SEM. Approximately 600 RFP+ cells per animal and group was evaluated. Scale bar 30 μm.  Gene editing of the Trim28-loci resulted in a robust loss of Trim28 protein, as evaluated by IHC where the expression of Trim28 in RFP+ cells were quantified and are displayed as mean ± SEM. Approximately 600 RFP+ cells per animal and group was evaluated. Scale bar 30 μm.  (e) RNA-seq analysis of the expression of TE-families using TEtranscripts.  (f) RNA-seq analysis of the Trim28-KO and control samples, visualising full length MMERVK10C elements (left panel) and ChIP-seq analysis of H3K9me3 in adult forebrain neurons (right panel). The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram.


Figure 3 ( b ) IHC for Trim28 in the adult cortex revealed that the protein was lost in cells exposed to Cre - activity during brain development ( GFP + cells ) . Scale bars : low magnification 75 μm , high magnification 20μm . ( c ) RNA - seq analysis of the expression of TE - families using TEtranscripts ( d ) Significantly upregulated TE families upon the Trim28 - KO , in which the families with the highest fold change are listed . ( e ) RNA - seq analysis of full length MMERVK10Cs in the adult tissue . The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram .

Figure 3(b) IHC for Trim28 in the adult cortex revealed that the protein was lost in cells exposed to Cre-activity during brain development (GFP+ cells). Scale bars: low magnification 75 μm, high magnification 20μm. (c) RNA-seq analysis of the expression of TE-families using TEtranscripts (d) Significantly upregulated TE families upon the Trim28-KO, in which the families with the highest fold change are listed.  (e) RNA-seq analysis of full length MMERVK10Cs in the adult tissue. The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram.


Figure 4 ( e - h ) A selection of significant cell type specific changes in gene expression between Emx1 - Cre / Trim28 - KO animals and controls as revealed by single - nuclei RNA - seq . The black dots represent the mean value value ( Wilcoxon rank sum test , ( p - adj value < 0 . 01 ) , n = 2 .

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Figure 4 (e-h) A selection of significant cell type specific changes in gene expression between Emx1-Cre/Trim28-KO animals and controls as revealed by single-nuclei RNA-seq. The black dots represent the mean value value (Wilcoxon rank sum test, (p-adj value < 0.01), n=2.


Figure 5 ( b ) Mean plots show the changes of TE subfamily expression in each cell cluster upon the Emx1 - Cre / Trim28 - KO . A differential expression analysis performed with DESeq2 ( described in material and methods ) showed upregulated TEs in cell types in which Trim28 was deleted ( excitatory neurons , astrocytes , oligodendrocytes and oligodendrocyte precursors ) , ( indicated with red dots : p - adj < 0 . 01 , log2FC > 3 ) . The specific upregulated elements and their fold changes are listed in bar graphs under each mean plot . In cell types in which Trim28 was not deleted , TE expression remained unaffected ( inhibitory neurons and microglia ) .

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

Figure 5 (b) Mean plots show the changes of TE subfamily expression in each cell cluster upon the Emx1-Cre/Trim28-KO. A differential expression analysis performed with DESeq2 (described in material and methods) showed upregulated TEs in cell types in which Trim28 was deleted (excitatory neurons, astrocytes, oligodendrocytes and oligodendrocyte precursors), (indicated with red dots: p-adj < 0.01, log2FC > 3). The specific upregulated elements and their fold changes are listed in bar graphs under each mean plot. In cell types in which Trim28 was not deleted, TE expression remained unaffected (inhibitory neurons and microglia).


Figure 6 ( a ) IHC analysis for the microglia marker Iba1 in Emx1 - Cre / Trim28 - KO animals and controls . Scale bars : Low magnification 75 μm , high magnification 10 μm . ( b - c ) The morphology of microglia in cortex and striatum ( control region ) of Emx1 - Cre / Trim28 - KO animals and controls were quantified by high content screening of Iba1 immunoreactivity , revealing differences in process length , area and number of branch points specifically in cortex , error bars mean ± SEM ( unpaired t - test . p - values : 1 . 8x10 - 4 , 9 . 6x10 - 4 and 5 . 2x10 - 6 , respectively ) . A large number of photos from three control and two KO animals were analysed ( d ) Western blot revealed an increased expression of CD68 in the Emx1 - Cre / Trim28 - KO animals ( n = 5 per group ) , unpaired t - test , * * p = 0 . 0036 , error bars mean ± SEM . The star indicates an unspecific band . ( e ) An increased expression of the ERV derived protein IAP - Gag was detected in the cortex of Emx1 - Cre / Trim28 - KO animals ( n = 5 per group ) , unpaired t - test , * * * p = 0 . 0008 , error bars mean ± SEM . ( f ) Immunohistochemistry for IAP - gag visualized the presence of ERV proteins in the cortex of Emx1 - Cre / Trim28 - KO animals . The distribution of IAP - gag was heterogenous among Trim28 - KO neurons , where it was either accumulated in a low ( # ) or high ( ° ) number of aggregate - like structures in the cytoplasm , or as weak homogenous staining throughout the cytoplasm ( # ) or not present at all ( * ) . Scale bars : low magnification 75μm , high magnification 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IHC analysis for the microglia marker Iba1 in Emx1-Cre/Trim28-KO animals and controls. Scale bars: Low magnification 75 μm, high magnification 10 μm. (b-c) The morphology of microglia in cortex and striatum (control region) of Emx1-Cre/Trim28-KO animals and controls were quantified by high content screening of Iba1 immunoreactivity, revealing differences in process length, area and number of branch points specifically in cortex, error bars mean ± SEM (unpaired t-test. p-values: 1.8x10-4, 9.6x10-4 and 5.2x10-6, respectively). A large number of photos from three control and two KO animals were analysed  (d) Western blot revealed an increased expression of CD68 in the Emx1-Cre/Trim28-KO animals (n=5 per group), unpaired t-test, **p=0.0036, error bars mean ± SEM. The star indicates an unspecific band.  (e) An increased expression of the ERV derived protein IAP-Gag was detected in the cortex of Emx1-Cre/Trim28-KO animals (n=5 per group), unpaired t-test, ***p=0.0008, error bars mean ± SEM.  (f) Immunohistochemistry for IAP-gag visualized the presence of ERV proteins in the cortex of Emx1-Cre/Trim28-KO animals. The distribution of IAP-gag was heterogenous among Trim28-KO neurons, where it was either accumulated in a low (#) or high (°) number of aggregate-like structures in the cytoplasm, or as weak homogenous staining throughout the cytoplasm (#) or not present at all (*). Scale bars: low magnification 75μm, high magnification 5 μm.


Figure 1U2OS cells were transfected , synchronized at the G1 / S - border by thymidine for 24 hours and then either released into nocodazole for 16 hours ( unperturbed mitotic entry ) or released for 7 hours , treated with 0 . 5 μM Adriamycin for one hour and after 16 hour G2 arrest were induced to recover by addition of caffeine ( checkpoint recovery ) . Mitotic index was scored based on the percentage of Histone H3 - pS10 positive DAPI - nuclei and normalized to the untransfected controls . Black diamonds indicate individual siRNA - targeted genes from the library , light grey diamonds indicate positive ( Wip1 - depleted ) and negative ( untransfected and GAPDH - depleted ) controls and dark grey diamonds indicate the hits based on the two screens . Dotted lines indicate selection criteria for recovery - specific genes .

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

Figure 1U2OS cells were transfected, synchronized at the G1/S-border by thymidine for 24 hours and then either released into nocodazole for 16 hours (unperturbed mitotic entry) or released for 7 hours, treated with 0.5 μM Adriamycin for one hour and after 16 hour G2 arrest were induced to recover by addition of caffeine (checkpoint recovery). Mitotic index was scored based on the percentage of Histone H3-pS10 positive DAPI-nuclei and normalized to the untransfected controls. Black diamonds indicate individual siRNA-targeted genes from the library, light grey diamonds indicate positive (Wip1-depleted) and negative (untransfected and GAPDH-depleted) controls and dark grey diamonds indicate the hits based on the two screens. Dotted lines indicate selection criteria for recovery-specific genes.


Figure 2A ) U2OS cells were transfected with four independent siRNA ' s from the pools used in the screen , treated as in figure 1 and analyzed for mitotic index . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 ) B ) U2OS cells were synchronized with a single thymidine block , released into G2 and damaged with 0 . 5 μM Adriamycin for one hour . After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels . C ) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells . Cumulative percentage of cells entering mitosis were scored and plotted . D ) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA # 3 , synchronized and damaged in G2 . Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS . Percentages of cells in each quadrant are indicated . E ) Tlk2Δ cells were generated using CRISPR / Cas9 genome editing . Cells were synchronized in G2 by thymidine release , damaged with 0 . 5 μM Adriamycin for one hour . After a 16 - hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS . Error bars represent SD , n = 4 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * * for P ≤ 0 . 001 ) . F ) U2TR cells stably expressing Tlk2 siRNA # 3 - insensitive tetracycline - inducible FLAG - Tlk2 - wt or FLAG - Tlk2 - D613A were thymidine synchronized and damaged in G2 . Tetracycline was present form the start of the experiment where indicated . G ) Caffeine induced recovery assay of cell lines shown in 2F . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 , * * * for P ≤ 0 . 001 ) .

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

Figure 2A) U2OS cells were transfected with four independent siRNA's from the pools used in the screen, treated as in figure 1 and analyzed for mitotic index. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01)B) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels.C) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells. Cumulative percentage of cells entering mitosis were scored and plotted.D) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA #3, synchronized and damaged in G2. Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS. Percentages of cells in each quadrant are indicated.E) Tlk2Δ cells were generated using CRISPR/Cas9 genome editing. Cells were synchronized in G2 by thymidine release, damaged with 0.5 μM Adriamycin for one hour. After a 16-hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS. Error bars represent SD, n=4. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, *** for P≤0.001).F) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt or FLAG-Tlk2-D613A were thymidine synchronized and damaged in G2. Tetracycline was present form the start of the experiment where indicated.G) Caffeine induced recovery assay of cell lines shown in 2F. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01, *** for P≤0.001).


Figure 3A ) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release . Cells were fixed at the indicated times after adriamycin - induced DNA damage and stained for DAPI and γH2AX . Scale bar indicates 10 μm . B ) Quantification of cells in 3A . Each dot represents the total signal of γH2AX in the nucleus of a single cell . Bars indicate the mean and error bars indicate the SD of the data points in each condition . Whiskers represent 5 - 95 % of data points . Statistical significance was tested using an unpaired two - tailed t - test ( NS for P > 0 . 05 , * * * * for P ≤ 0 . 0001 ) . C ) U2OS cells were synchronized in G2 and harvested at the indicated times after induction of adriamycin - induced DNA damage with or without Tlk2 - depletion . Samples were analyzed by western blotting for the indicated proteins . D ) U2OS cells were synchronized in G2 and damaged with 6 Gy of IR . Samples were taken at the indicated times after induction of damage with or without Tlk2 - depletion . Samples were analyzed by western blot for the indicated proteins . Band signal intensity for Asf1A and Asf1B was measured and corrected for Ponceau S staining . E ) U2OS cells were transfected with the indicated siRNA ' s and treated as in 2A . Mitotic index was analyzed by FACS . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 ) . F ) U2OS cells were synchronized with a single thymidine block , released into G2 and damaged with 0 . 5 μM Adriamycin for one hour . After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for the indicated protein levels . Lanes were all present on the same blot but pasted together for comparison as indicated by the lines . G ) Cells were synchronized in S - or G2 - phase by a single thymidine block . Damage was induced by 0 . 5 μM Adriamycin for one hour and cells were allowed to incubate for 16 hours afterwards . Samples were fixed at the indicated cell cycle stages and stained for Asf1A - pS166 . Nuclear fluorescence intensity was measured relative to DAPI . Whiskers represent 5 - 95 % of data points . H ) Cells were treated and analyzed as in 2E . Induction of FLAG - Asf1A - 4D was induced using doxycycline . Error bars represent SD , n = 5 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 ) . I ) U2OS cells were synchronized in G2 by a single thymidine block and where indicated treated with 0 . 5 μM Adriamycin for one hour and after the indicated time points were pre - extracted and fixed . Cells were stained for total levels of Histone H3 and quantified relative to DAPI . Scale bar indicates 10 μm . Whiskers represent 5 - 95 % of data points . J ) FLAG - Asf1A - 4D inducible cells were treated as in 3I and expression was induced using doxycycline where indicated . Whiskers represent 5 - 95 % of data points . Statistical significance was tested using an unpaired two - tailed t - test ( * * * * for P ≤ 0 . 0001 ) .

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

Figure 3A) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release. Cells were fixed at the indicated times after adriamycin-induced DNA damage and stained for DAPI and γH2AX. Scale bar indicates 10 μm. B) Quantification of cells in 3A. Each dot represents the total signal of γH2AX in the nucleus of a single cell. Bars indicate the mean and error bars indicate the SD of the data points in each condition. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (NS for P>0.05, **** for P≤0.0001).C) U2OS cells were synchronized in G2 and harvested at the indicated times after induction of adriamycin-induced DNA damage with or without Tlk2-depletion. Samples were analyzed by western blotting for the indicated proteins.D) U2OS cells were synchronized in G2 and damaged with 6 Gy of IR. Samples were taken at the indicated times after induction of damage with or without Tlk2-depletion. Samples were analyzed by western blot for the indicated proteins. Band signal intensity for Asf1A and Asf1B was measured and corrected for Ponceau S staining.E) U2OS cells were transfected with the indicated siRNA's and treated as in 2A. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).F) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for the indicated protein levels. Lanes were all present on the same blot but pasted together for comparison as indicated by the lines.G) Cells were synchronized in S- or G2-phase by a single thymidine block. Damage was induced by 0.5 μM Adriamycin for one hour and cells were allowed to incubate for 16 hours afterwards. Samples were fixed at the indicated cell cycle stages and stained for Asf1A-pS166. Nuclear fluorescence intensity was measured relative to DAPI. Whiskers represent 5-95% of data points.H) Cells were treated and analyzed as in 2E. Induction of FLAG-Asf1A-4D was induced using doxycycline. Error bars represent SD, n=5. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).I) U2OS cells were synchronized in G2 by a single thymidine block and where indicated treated with 0.5 μM Adriamycin for one hour and after the indicated time points were pre-extracted and fixed. Cells were stained for total levels of Histone H3 and quantified relative to DAPI. Scale bar indicates 10 μm. Whiskers represent 5-95% of data points.J) FLAG-Asf1A-4D inducible cells were treated as in 3I and expression was induced using doxycycline where indicated. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (**** for P≤0.0001).


Figure 4A ) U2OS cells were treated as in 2B after which mRNA was isolated and analyzed by qPCR . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 ) . B ) Tlk2Δ 9 . 3 . 1 cells were treated and analyzed as in 3A . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 ) . C ) U2OS cells were synchronized in G2 and harvested or synchronized in G2 and harvested 16 hours after adriamycin - induced DNA damage . Samples were analyzed by western blot for the indicated proteins . D ) U2OS cells were transfected with the indicated siRNA ' s , synchronized in G2 and damaged with 6 Gy of IR . Cells were harvested 24 hours later and the mitotic index was analyzed by FACS . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * * for P ≤ 0 . 01 ) . E ) Cells were treated as in 4C . F ) U2OS cells were transfected with the indicated siRNA ' s , arrested in G2 with Adriamycin and induced to recover by addition of caffeine . Mitotic index was analyzed by FACS . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * * for P ≤ 0 . 01 ) . G ) U2OS cells were synchronized in G2 and harvested 16 hours after adriamycin - induced DNA damage . Samples were analyzed by western blot for the indicated proteins . H ) U2TR cells stably expressing Tlk2 siRNA # 3 - insensitive tetracycline - inducible FLAG - Tlk2 - wt were thymidine synchronized , damaged in G2 and harvested 24 hours afterwards . Tetracycline was either absent ( off ) , added from the start of the experiment ( early ) or for 16 hours after induction of DNA damage ( late ) . I ) Cells were treated as in 5G and recovery was induced by addition of caffeine for 8 hours . Mitotic index was analyzed by FACS and normalized to the control . Error bars represent SD of three independent experiments . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * * for P ≤ 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START 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 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 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 4A) U2OS cells were treated as in 2B after which mRNA was isolated and analyzed by qPCR. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).B) Tlk2Δ 9.3.1 cells were treated and analyzed as in 3A. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01).C) U2OS cells were synchronized in G2 and harvested or synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins.D) U2OS cells were transfected with the indicated siRNA's, synchronized in G2 and damaged with 6 Gy of IR. Cells were harvested 24 hours later and the mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).E) Cells were treated as in 4C.F) U2OS cells were transfected with the indicated siRNA's, arrested in G2 with Adriamycin and induced to recover by addition of caffeine. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).G) U2OS cells were synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins.H) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt were thymidine synchronized, damaged in G2 and harvested 24 hours afterwards. Tetracycline was either absent (off), added from the start of the experiment (early) or for 16 hours after induction of DNA damage (late).I) Cells were treated as in 5G and recovery was induced by addition of caffeine for 8 hours. Mitotic index was analyzed by FACS and normalized to the control. Error bars represent SD of three independent experiments. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).