The oxygen consumption rate ( OCR ) of 1 × 105 differentiated myeloid cells were monitored after the addition of oligomycin ( OA ) ( 1 µM ) , the uncoupler carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 1 µM ) , and the electron transport inhibitor rotenone and antimycin A ( R / AA , 5 µM ) at indicated time points . The basal OCR , basal extracellular acidification rate ( ECAR ) , spare respiratory capacity , ATP production and maximal respiration based on the OCR value were quantified .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O 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-MEASURED_VAR O O O O O O O O O O O O

The oxygen consumption rate (OCR) of 1×105 differentiated myeloid cells were monitored after the addition of oligomycin (OA) (1 µM), the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA, 5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, ATP production and maximal respiration based on the OCR value were quantified.


Mean fluorescence intensity ( MFI ) of intracellular phosphor - mTOR ( pS2448 ) in polarized myeloid cells as determined by flow

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

Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells as determined by flow cytometry


Mean fluorescence intensity ( MFI ) of intracellular phosphor - mTOR ( pS2448 ) in polarized myeloid cells lysed for western blot .

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

Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells lysed for western blot.


Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206 + ( black ) and CD206 - ( grey ) cells .

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

Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206+ (black) and CD206- (grey) cells.


Balb / c mice were inoculated with 5 × 105 CT26 tumor cells subcutaneously . Since day 6 , vehicle or DGAT inhibitors ( iDGAT ) were administrated via intra - tumoral injection once daily .

Balb/c mice were inoculated with 5×105 CT26 tumor cells subcutaneously. Since day 6, vehicle or DGAT inhibitors (iDGAT) were administrated via intra-tumoral injection once daily.


At day 13 , tumor infiltrated immune cells were isolated and stained for CD206 and CD11b .

At day 13, tumor infiltrated immune cells were isolated and stained for CD206 and CD11b.


5 × 105 MCA205 were injected subcutaneously into C57BL / 6 mice . PBS , liposome control or iDGAT - liposome was injected into the peritoneum every other day starting at day 7 . Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis . Immune cells from spleen , tumor - draining lymph nodes ( TDLN ) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells .

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

5×105 MCA205 were injected subcutaneously into C57BL/6 mice. PBS, liposome control or iDGAT-liposome was injected into the peritoneum every other day starting at day 7. Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis. Immune cells from spleen, tumor-draining lymph nodes (TDLN) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells.


Tumor infiltrating CD8 + T cells ( red ) were stained together with nuclei ( DAPI / blue ) after sacrificing the tumor bearing mice .

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

Tumor infiltrating CD8+ T cells (red) were stained together with nuclei (DAPI/blue) after sacrificing the tumor bearing mice.


1 × 106 CD14 + myeloid cells from healthy donors were polarized by 1 ng / ml GM - CSF in the presence of BSA - control ( C ) , 0 . 2 mM oleate ( O ) or 0 . 2 mM stearate ( S ) over a 6 - day period . The expression of CD206 , CD204 and CD38 was analyzed via flow cytometry .

O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

1×106 CD14+ myeloid cells from healthy donors were polarized by 1 ng/ml GM-CSF in the presence of BSA-control (C), 0.2 mM oleate (O) or 0.2 mM stearate (S) over a 6-day period. The expression of CD206, CD204 and CD38 was analyzed via flow cytometry.


The level of lipid droplets was determined via BODIPY staining .

The level of lipid droplets was determined via BODIPY staining.


The oxygen consumption rate ( OCR ) of differentiated myeloid cells was monitored as described in Materials and Methods .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

The oxygen consumption rate (OCR) of differentiated myeloid cells was monitored as described in Materials and Methods.


( D ) The purified CFSE - labeled autologous CD4 + T cells were co - cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay .

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

(D) The purified CFSE-labeled autologous CD4+ T cells were co-cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay.


Tumor tissue ( tumor ) as well as corresponding non - tumorous adjacent tissue ( control ) were collected from colorectal cancer patients and prepared for histopathology . The expression of CD68 ( red ) , CD206 ( green ) and ADRP ( blue ) indicate the lipid droplets in tumor infiltrating myeloid cells . Nuclei ( white ) were stained using DAPI . The absolute number of positive cells was quantified in 5 high - power fields ( hpf ) ( scale bar = 20 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

Tumor tissue (tumor) as well as corresponding non-tumorous adjacent tissue (control) were collected from colorectal cancer patients and prepared for histopathology. The expression of CD68 (red), CD206 (green) and ADRP (blue) indicate the lipid droplets in tumor infiltrating myeloid cells. Nuclei (white) were stained using DAPI. The absolute number of positive cells was quantified in 5 high-power fields (hpf) (scale bar = 20 µm).


A ) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1 . Coimmunoprecipitation ( CoIP ) experiments using TAP - tagged Nrd1 ( either wt or ∆ CID ) as the bait . Representative gel of one out of two independent experiments .

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

A) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1. Coimmunoprecipitation (CoIP) experiments using TAP-tagged Nrd1 (either wt or ∆CID) as the bait. Representative gel of one out of two independent experiments.


C ) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1 . CoIP experiments using Nrd1 - TAP as the bait in a SEN1 or sen1 ∆ NIM background . Representative gel of one out of two independent experiments .

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

C) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1. CoIP experiments using Nrd1-TAP as the bait in a SEN1 or sen1∆NIM background. Representative gel of one out of two independent experiments.


D ) CoIP experiments using HA - tagged Sen1 , either wt or ∆ NIM , as the bait . Representative gel of one out of two independent experiments . Protein extracts were treated with RNaseA prior to immunoprecipitation . In these experiment , Sen1 could not be detected in the input extracts .

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

D) CoIP experiments using HA-tagged Sen1, either wt or ∆NIM, as the bait. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA prior to immunoprecipitation. In these experiment, Sen1 could not be detected in the input extracts.


A ) Deletion of the N - terminal of Sen1 is lethal . A ∆ sen1 strain ( YDL2767 ) covered by an URA3 - containing plasmid ( pFL38 ) expressing wt Sen1 was transformed with a TRP1 - plasmid ( pFL39 ) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left . After over - night growth in non - selective medium , cells were plated on minimal medium ( CSM ) containing 5 - fluorootic acid ( 5 - FOA ) to select those that have lost the URA3 plasmid . The absence of growth in 5 - FOA implies that the SEN1 version expressed in the TRP1 - plasmid does not support viability .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

A) Deletion of the N-terminal of Sen1 is lethal. A ∆sen1 strain (YDL2767) covered by an URA3-containing plasmid (pFL38) expressing wt Sen1 was transformed with a TRP1-plasmid (pFL39) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left. After over-night growth in non-selective medium, cells were plated on minimal medium (CSM) containing 5-fluorootic acid (5-FOA) to select those that have lost the URA3 plasmid. The absence of growth in 5-FOA implies that the SEN1 version expressed in the TRP1-plasmid does not support viability.


B ) Deletion of the N - terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo . Northern blot analyses of three well - characterized NNS - targets , the CUT NEL025c and the snoRNAs SNR13 and SNR33 , in a Sen1 - AID ( auxin - induced degron ) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1 . Sen1 - AID was depleted for 2h by the addition of 500 μM indole - 3 - acetic acid ( IAA ) to monitor the capacity of the plasmid - borne versions of SEN1 to induce transcription termination . The expected RNA species resulting from inefficient termination ( RT for readthrough species ) are indicated . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Deletion of the N-terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo. Northern blot analyses of three well-characterized NNS-targets, the CUT NEL025c and the snoRNAs SNR13 and SNR33, in a Sen1-AID (auxin-induced degron) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1. Sen1-AID was depleted for 2h by the addition of 500 μM indole-3-acetic acid (IAA) to monitor the capacity of the plasmid-borne versions of SEN1 to induce transcription termination. The expected RNA species resulting from inefficient termination (RT for readthrough species) are indicated. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.


C ) Overexpression of sen1 ∆ Nter restores viability . Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter ( pGAL ) in the presence of galactose .

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

C) Overexpression of sen1∆Nter restores viability. Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter (pGAL) in the presence of galactose.


D ) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N - terminal domain . Northern blot analysis of a typical NNS - target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control .

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

D) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N-terminal domain. Northern blot analysis of a typical NNS-target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . A ) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site ( PAS ) of mRNAs . Values on the y axis correspond to the median coverage . B ) Box - plot representation of the readthrough ( RT ) index as a readout of the termination efficiency at each mRNA . The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS , divided by the average signal from the TSS to the PAS of each mRNA . Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median . The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1 . 5 * IQR ( where IQR is the interquartile distance or distance between the first and the third quartile ) . The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1 . 5 * IQR . For each mRNA ( n = 5752 ) , the values correspond to the average of two independent biological replicates . Data populations were compared using a t - test ( bilateral distributions , unpaired data ) and p - values ≥ 0 . 05 are indicated as ns ( not significant ) .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. A) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site (PAS) of mRNAs. Values on the y axis correspond to the median coverage. B) Box-plot representation of the readthrough (RT) index as a readout of the termination efficiency at each mRNA. The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS, divided by the average signal from the TSS to the PAS of each mRNA. Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR (where IQR is the interquartile distance or distance between the first and the third quartile). The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. For each mRNA (n=5752), the values correspond to the average of two independent biological replicates. Data populations were compared using a t-test (bilateral distributions, unpaired data) and p-values≥ 0.05 are indicated as ns (not significant).


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . C - D ) IGV screenshots of two well - characterized protein - coding genes displaying substantial defects in premature termination in some mutants . The values correspond to the counts per million of counts ( CPM ) multiplied by 10 .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. C-D) IGV screenshots of two well-characterized protein-coding genes displaying substantial defects in premature termination in some mutants. The values correspond to the counts per million of counts (CPM) multiplied by 10.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . E ) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site ( TTS ) as the reference point . F ) Analysis of termination defects in the different mutants at CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS , divided by the average signal over a window of 100 nt downstream from the TSS ( CUT100 ) . For each CUT ( n = 329 ) , the values correspond to the average of two independent biological replicates . P - values < 0 . 001 are indicated by three asterisks .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. E) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site (TTS) as the reference point. F) Analysis of termination defects in the different mutants at CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS, divided by the average signal over a window of 100 nt downstream from the TSS (CUT100). For each CUT (n=329), the values correspond to the average of two independent biological replicates. P-values < 0.001 are indicated by three asterisks.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . G - H ) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. G-H) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS , divided by the average signal over the mature snoRNA .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS, divided by the average signal over the mature snoRNA.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs.


A ) Deletion of Sen1 N - terminal domain does not prevent the interaction of Sen1 with RNAPII . CoIP experiments using Rbp3 - FLAG as the bait . Assays were performed in a Sen1 - AID strain harboring a plasmid expressing either SEN1 or sen1 ∆ Nter upon depletion of Sen1 - AID in the presence of IAA for 2h . An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1 ∆ Nter . Nrd1 is detected as a positive control . Representative gel of one out of two independent experiments . Protein extracts were treated with RNaseA before immunoprecipitation .

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

A) Deletion of Sen1 N-terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3-FLAG as the bait. Assays were performed in a Sen1-AID strain harboring a plasmid expressing either SEN1 or sen1∆Nter upon depletion of Sen1-AID in the presence of IAA for 2h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1∆Nter. Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.


B ) Deletion of the Sen1 N - terminal domain reduces the interaction of Sen1 with the S5P - CTD . CoIP experiments using TAP - Sen1 as the bait . Sen1 proteins were expressed from pGAL in the presence of galactose . Nab3 is detected as a positive control . Representative gel of one out of three independent experiments . Protein extracts were treated with RNaseA before immunoprecipitation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Deletion of the Sen1 N-terminal domain reduces the interaction of Sen1 with the S5P-CTD. CoIP experiments using TAP-Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.


C ) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability . Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1 - plasmid carrying the SEN1 versions indicated in the scheme on the left . The growth of the strain expressing the Nrd1 CID - sen1 ∆ Nter chimera in 5 - FOA implies that this gene can support viability .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

C) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1-plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID-sen1∆Nter chimera in 5-FOA implies that this gene can support viability.


D ) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1 ∆ Nter mutant . Northern blot assays performed in a Sen1 - AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B . Experiments performed in a ∆ rrp6 background . Representative gel of one out of two independent experiments . The U4 RNA is used as a loading control .

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

D) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1∆Nter mutant. Northern blot assays performed in a Sen1-AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B. Experiments performed in a ∆rrp6 background. Representative gel of one out of two independent experiments.The U4 RNA is used as a loading control.


E ) Sen1 Nter interacts with the C - terminal domain ( Cter ) of Sen1 both in the presence and in the absence of the NIM in vitro . Pull - down experiments using either a wt or a ∆ NIM version of recombinant Sen1 Cter immobilized on glutathione - sepharose beads and a TAP - tagged version of Sen1 Nter expressed in yeast . Representative gel of one out of three independent experiments .

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

E) Sen1 Nter interacts with the C-terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro. Pull-down experiments using either a wt or a ∆NIM version of recombinant Sen1 Cter immobilized on glutathione-sepharose beads and a TAP-tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments.


A - C ) Comparison of the distribution of Sen1 and RNAPII ( Rpb1 ) at mRNAs and CUTs . Values on the y axis correspond to the median coverage . Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot .

A-C) Comparison of the distribution of Sen1 and RNAPII (Rpb1) at mRNAs and CUTs. Values on the y axis correspond to the median coverage. Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot.


D - F ) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots .

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D-F) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots.


G ) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point . Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background .

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G) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point. Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background.


H - I ) Screenshots of two well - characterized protein - coding genes that are regulated by NNS - dependent premature termination . Values correspond to unnormalized Sen1 occupancy .

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H-I) Screenshots of two well-characterized protein-coding genes that are regulated by NNS-dependent premature termination. Values correspond to unnormalized Sen1 occupancy.


J ) Profile of Sen1 binding at CUTs calculated as for mRNAs .

J) Profile of Sen1 binding at CUTs calculated as for mRNAs.


K - L ) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter . Plots correspond to unnormalized data .

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K-L) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter. Plots correspond to unnormalized data.


M - O ) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs .

M-O) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs.


P ) Comparison of the total levels of the different Sen1 variants at mRNAs , CUTs and the termination region of snoRNAs ( i . e . a window of 200 nt downstream of the 3 ' end of the mature snoRNA , snoRNA - ter ) . Boxes in box - plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median . The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1 . 5 * IQR . The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1 . 5 * IQR . Datasets for the different Sen1 versions were compared using a t - test ( bilateral distributions , unpaired data ) . p - values ≥ 0 . 05 are indicated as ns ( not significant ) , one asterisk is used when 0 . 05 > p - value ≥ 0 . 01 , two asterisks when 0 . 01 > p - value ≥ 0 . 001 and three asterisks for p - value < 0 . 001 .

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P) Comparison of the total levels of the different Sen1 variants at mRNAs, CUTs and the termination region of snoRNAs (i.e. a window of 200 nt downstream of the 3' end of the mature snoRNA, snoRNA-ter). Boxes in box-plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR. The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. Datasets for the different Sen1 versions were compared using a t-test (bilateral distributions, unpaired data). p-values≥ 0.05 are indicated as ns (not significant), one asterisk is used when 0.05>p-value≥0.01, two asterisks when 0.01>p-value≥0.001 and three asterisks for p-value <0.001.


A ) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter . Northern blot assays performed in a Sen1 - AID , ∆ rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments . Where indicated , cells were treated with 50 mg / L of 6 - azauracile ( 6AU ) for 2h . Representative gel of one out of two independent biological replicates . The U4 RNA is used as a loading control .

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A) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter. Northern blot assays performed in a Sen1-AID, ∆rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments. Where indicated, cells were treated with 50 mg/L of 6-azauracile (6AU) for 2h. Representative gel of one out of two independent biological replicates.The U4 RNA is used as a loading control.


Flow cytometry analysis of PD - L1 expression in ( A ) KPC and ( B ) Pan02 cells following treatment with gemcitabine and RT . Gemcitabine was diluted in DMSO ( vehicle ) and DMSO was used for the ' untreated ' control group ( mean ± SD , n = 3 , One - way ANOVA , Bonferroni test ) .

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Flow cytometry analysis of PD-L1 expression in (A) KPC and (B) Pan02 cells following treatment with gemcitabine and RT. Gemcitabine was diluted in DMSO (vehicle) and DMSO was used for the 'untreated' control group (mean ± SD, n=3, One-way ANOVA, Bonferroni test).


Western blot analysis of indicated proteins in both KPC ( C ) and Pan02 ( D ) cells following radiation and chemotherapy ± AG490 , a JAK / Stat kinase inhibitor . Actin represents loading control .

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Western blot analysis of indicated proteins in both KPC (C) and Pan02 (D) cells following radiation and chemotherapy ± AG490, a JAK/Stat kinase inhibitor. Actin represents loading control.


Flow cytometry analysis of PD - L1 after gemcitabine and RT ( as described above ) in KPC ( E ) and Pan02 ( F ) following Stat1 downregulation by siRNA ( mean ± SD , n = 3 , Student ' s t - test ) . MFI , mean fluorescence index ;

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Flow cytometry analysis of PD-L1 after gemcitabine and RT (as described above) in KPC (E) and Pan02 (F) following Stat1 downregulation by siRNA (mean ± SD, n=3, Student's t-test). MFI, mean fluorescence index;


( A ) Tumor growth delay was measured in the different groups , as indicated ( n = 6 mice per group ) . Anti - PD - L1 was given at days 0 3 , 6 and 9 ( black arrows ) . The average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 is shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . No weight loss was observed in the in vivo

(A) Tumor growth delay was measured in the different groups, as indicated (n=6 mice per group). Anti-PD-L1 was given at days 0 3, 6 and 9 (black arrows). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment


( B ) Quantitative data on the percentage of gated cells are shown ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

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(B) Quantitative data on the percentage of gated cells are shown (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


( A ) Tumor growth delay was measured by treating tumor with RT ( 12 Gy or 5x3 Gy daily ; initiated at day 0 ) and anti - PD - L1 alone or combination of RT ( n = 6 mice per group ) . Anti - PD - L1 was given at days 0 , 3 , 6 and 9 ( black arrows ) . The radiosensitizing effect of anti - PD - L1 after 12 Gy and 5x3 Gy was assessed as shown by the capped lines ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . No weight loss was observed in the in vivo experiment .

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(A) Tumor growth delay was measured by treating tumor with RT (12 Gy or 5x3 Gy daily; initiated at day 0) and anti-PD-L1 alone or combination of RT (n=6 mice per group). Anti-PD-L1 was given at days 0, 3, 6 and 9 (black arrows). The radiosensitizing effect of anti-PD-L1 after 12 Gy and 5x3 Gy was assessed as shown by the capped lines (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment.


( A ) KPC tumor growth delay after treatment with either 12 Gy ( day 0 ) , anti - PD - L1 ( days 0 , 3 , 6 and 9 ; black arrows ) , anti - CD8 alone ( days 0 3 , 6 and 9 ) or their combinations , as indicated .

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(A) KPC tumor growth delay after treatment with either 12 Gy (day 0), anti-PD-L1 (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0 3, 6 and 9) or their combinations, as indicated.


( B ) KPC tumor growth delay after treatment with either 5x3 Gy ( day 0 - 4 ) , anti - PD - L1 alone ( days 0 , 3 , 6 and 9 ; black arrows ) , anti - CD8 alone ( days 0 , 3 , 6 and 9 ) or their combinations , as indicated .

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(B) KPC tumor growth delay after treatment with either 5x3 Gy (day 0-4), anti-PD-L1 alone (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0, 3, 6 and 9) or their combinations, as indicated.


( C ) KPC tumor growth delay after either simultaneous ( day 0 , 3 , 6 and 9 ; black arrows - upper row ) or sequential ( day 6 , 9 , 13 and 16 ; black arrows - lower row ) addition of anti - PD - L1 to RT ( day 0 ) . The average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A - C is also shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . No weight loss was observed in the in vivo experiments .

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(C) KPC tumor growth delay after either simultaneous (day 0, 3, 6 and 9; black arrows-upper row) or sequential (day 6, 9, 13 and 16; black arrows-lower row) addition of anti-PD-L1 to RT (day 0). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A-C is also shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiments.


( A ) Mice were treated with either gemcitabine ( day 0 , 3 ) , anti - PD - L1 ( day 4 , 7 , 10 and 13 ; black arrows ) , 12 Gy ( day 4 ) alone or in combinations , as indicated ( n = 8 mice per group ) . The average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 is shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) No weight loss was observed in the in vivo experiment .

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(A) Mice were treated with either gemcitabine (day 0, 3), anti-PD-L1 (day 4, 7, 10 and 13; black arrows), 12 Gy (day 4) alone or in combinations, as indicated (n=8 mice per group). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test) No weight loss was observed in the in vivo experiment.


RT plus anti - PD - L1 ( B ) reverses T - cell exhaustion . Bar graphs show quantitation of T cell activation markers in tumor samples based on flow cytometry analysis , as indicated ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

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RT plus anti-PD-L1 (B) reverses T-cell exhaustion . Bar graphs show quantitation of T cell activation markers in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


RT plus anti - PD - L1 ( C ) increases CD8 : Treg ratio . Bar graphs show ratio of CD8 : Treg ratio in tumor samples based on flow cytometry analysis , as indicated ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

RT plus anti-PD-L1 (C) increases CD8:Treg ratio. Bar graphs show ratio of CD8:Treg ratio in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


( A ) Representative images of livers from the different treatment groups , as indicated . The corresponding liver weights are shown below ( n = 6 mice per group ) .

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(A) Representative images of livers from the different treatment groups, as indicated. The corresponding liver weights are shown below (n=6 mice per group).


( B ) Flow cytometry analysis of CD45 + CD8 + , CD11b + Gr1 + and CD4 + CD25 + FOXP3 + cell populations in the livers of the mice from the different treatment groups , as indicated ( n = 5 per group ; ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

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(B) Flow cytometry analysis of CD45+CD8+, CD11b+Gr1+ and CD4+CD25+FOXP3+ cell populations in the livers of the mice from the different treatment groups, as indicated (n=5 per group; (means ± SD, n=1, One-way ANOVA, Bonferroni test).


A . The survival of age - and sex - matched HDAC6 + / + mice and HDAC6 - / - mice was monitored for 9 days after intravenous VSV - Indiana infection ( 2 × 108 pfu / mouse ; n = 12 per group ; log - rank test ) .

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A. The survival of age- and sex-matched HDAC6+/+ mice and HDAC6-/- mice was monitored for 9 days after intravenous VSV-Indiana infection (2 × 108 pfu/mouse; n = 12 per group; log-rank test).


B . Determination of the viral load in organs by standard plaque assay . HDAC6 + / + and HDAC6 - / - mice were intravenously infected with VSV - Indiana , and the brain and spleen were collected at 5 dpi ( 2 × 108 pfu / mouse ; n = 6 per group ; Mann - Whitney test ) .

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B. Determination of the viral load in organs by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-Indiana, and the brain and spleen were collected at 5 dpi (2 × 108 pfu/mouse; n = 6 per group; Mann-Whitney test).


C . Determination of viral loads in organs by Q - PCR of VSV viral transcripts . The brain and spleen were collected at 5 dpi after HDAC6 + / + and HDAC6 - / - mice were intravenously infected with VSV - Indiana ( 2 × 108 pfu / mouse ; n = 9 per group ; Mann - Whitney test ) .

C. Determination of viral loads in organs by Q-PCR of VSV viral transcripts. The brain and spleen were collected at 5 dpi after HDAC6+/+and HDAC6-/- mice were intravenously infected with VSV-Indiana (2 × 108 pfu/mouse; n = 9 per group; Mann-Whitney test).


D . The viral load in the serum was assessed by standard plaque assay . HDAC6 + / + and HDAC6 - / - mice were intravenously infected with VSV - GFP . Serum was collected at the indicated time points and analyzed ( 4 × 108 pfu / mouse ; n = 3 per group ; Student ' s t - test ) .

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D. The viral load in the serum was assessed by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-GFP. Serum was collected at the indicated time points and analyzed (4 × 108 pfu/mouse; n = 3 per group; Student's t-test).


E . ELISA of IFN - β ( left ) and IL - 6 ( right ) in the serum of mice described in ( d ) ( Student ' s t - test ) .

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E. ELISA of IFN-β (left) and IL-6 (right) in the serum of mice described in (d) (Student's t-test).


F . ELISA of serum IFN - β ( left ) and IL - 6 ( right ) after poly ( I : C ) injection . HDAC6 + / + and HDAC6 - / - mice were intravenously injected with poly ( I : C ) . Serum was collected at the indicated time points and analyzed ( 200 μg / mouse ; n = 3 per group ; Student ' s t - test ) .

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F. ELISA of serum IFN-β (left) and IL-6 (right) after poly(I:C) injection. HDAC6+/+ and HDAC6-/- mice were intravenously injected with poly(I:C). Serum was collected at the indicated time points and analyzed (200 μg/mouse; n = 3 per group; Student's t-test).


A , B . virus replication at 12 and 24 hpi , in HDAC6 + / + and HDAC6 - / - BMDMs in response to VSV - GFP ( MOI = 10 ) infection ( A ) and PR8 - GFP ( MOI = 5 ) infection ( B ) .

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A, B. virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- BMDMs in response to VSV-GFP (MOI = 10) infection (A) and PR8-GFP (MOI = 5) infection (B).


C . Virus replication in HDAC6 + / + and HDAC6 - / - BMDMs in response to HSV - GFP ( MOI = 2 ) infection .

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C. Virus replication in HDAC6+/+ and HDAC6-/- BMDMs in response to HSV-GFP (MOI = 2) infection.


D . Virus replication in HDAC6 + / + and HDAC6 - / - PBMCs in response to VSV - GFP ( MOI = 10 ) infection .

D. Virus replication in HDAC6+/+ and HDAC6-/- PBMCs in response to VSV-GFP (MOI = 10) infection.


E . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) levels in the supernatant of ( A ) and ( B ) , and in HDAC6 + / + and HDAC6 - / - BMDMs treated with poly ( I : C ) ( 20 µg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) .

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E. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in HDAC6+/+ and HDAC6-/- BMDMs treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).


F . Immunoblot analysis of the phosphorylated and inactive forms of IRF3 , IKBα , TBK1 , RIG - I , MAVS , HDAC6 , and β - actin at the indicated times ( 0 , 2 , 4 , 8 , and 16 h ) in HDAC6 + / + and HDAC6 - / - BMDMs . BMDMs were stimulated with PR8 - GFP ( MOI = 3 ) .

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F. Immunoblot analysis of the phosphorylated and inactive forms of IRF3, IKBα, TBK1, RIG-I, MAVS, HDAC6, and β-actin at the indicated times (0, 2, 4, 8, and 16 h) in HDAC6+/+ and HDAC6-/- BMDMs. BMDMs were stimulated with PR8-GFP (MOI = 3).


G . Induction of mRNA for type I IFN , IL - 6 , and other IFN - related antiviral genes in HDAC6 + / + and HDAC6 - / - BMDMs in response to a RIG - I agonist stimulation at 6 h . HDAC6 + / + and HDAC6 - / - BMDMs were stimulated with 5 ' ppp - dsRNA ( 0 . 5 µg / ml ) for 6 h .

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G. Induction of mRNA for type I IFN, IL-6, and other IFN-related antiviral genes in HDAC6+/+ and HDAC6-/- BMDMs in response to a RIG-I agonist stimulation at 6 h. HDAC6+/+ and HDAC6-/- BMDMs were stimulated with 5'ppp-dsRNA (0.5 µg/ml) for 6 h.


A , B . Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi , and virus replication at 12 and 24 hpi , in control and HDAC6 knockdown RAW264 . 7 cells in response to VSV - GFP ( MOI = 1 ) infection ( A ) and PR8 - GFP ( MOI = 1 ) infection ( B ) . bar ,

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A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in control and HDAC6 knockdown RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µm


C . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) levels in the supernatant of ( A ) and ( B ) , and in control and HDAC6 knockdown RAW264 . 7 cells treated with poly ( I : C ) ( 20 µg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) .

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C. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in control and HDAC6 knockdown RAW264.7 cells treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).


D , E . Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi , and virus replication at 12 and 24 hpi , in HDAC6 + / + and HDAC6 - / - MEFs in response to VSV - GFP ( MOI = 1 ) infection ( D ) and PR8 - GFP ( MOI = 1 ) infection ( E ) . bar ,

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D, E. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- MEFs in response to VSV-GFP (MOI = 1) infection (D) and PR8-GFP (MOI = 1) infection (E). bar, 50µm


F . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) levels in the supernatant of ( D ) and ( E ) , and in HDAC6 + / + and HDAC6 - / - MEFs transfected with poly ( I : C ) ( 1 µg / ml ) .

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F. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (D) and (E), and in HDAC6+/+ and HDAC6-/- MEFs transfected with poly(I:C) (1 µg/ml).


A , B . Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi , and virus replication at 12 and 24 hpi , in vector , HDAC6 , or HDAC6 - CDM - overexpressing stable RAW264 . 7 cells in response to VSV - GFP ( MOI = 1 ) infection ( A ) and PR8 - GFP ( MOI = 1 ) infection ( B ) . bar , 100µm .

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

A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µm.


C . ELISA of IFN - b ( upper ) and IL - 6 ( lower ) levels in the supernatant of ( A ) , ( B ) , and in vector , HDAC6 , or HDAC6 - CDM - overexpressing stable RAW264 . 7 cells treated with poly ( I : C ) ( 20 μg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O

C. ELISA of IFN-b (upper) and IL-6 (lower) levels in the supernatant of (A), (B), and in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells treated with poly(I:C) (20 μg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).


D , E . Luciferase assay in 293T cells transfected with an IFN - β luciferase promoter and TK - Renilla together with mutant HDAC6 or HDAC6 - CDM ( 100 , 200 , 400 , or 800 ng ) , followed by PR8 - GFP ( MOI = 2 ) infection ( D ) or by poly ( I : C ) ( E ) ( 1 μg / ml ) transfection for another 12 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

D, E. Luciferase assay in 293T cells transfected with an IFN-β luciferase promoter and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng), followed by PR8-GFP (MOI = 2) infection (D) or by poly(I:C) (E) (1 μg/ml) transfection for another 12 h.


F , G . Luciferase assay in 293T cells transfected with RIG - I ( F ) , MDA - 5 ( G ) , an IFN - β luciferase promoter , and TK - Renilla together with mutant HDAC6 or HDAC6 - CDM ( 100 , 200 , 400 , or 800 ng ) . 24 h later , IFN - β activity was measured by luciferase reporter assay .

F, G. Luciferase assay in 293T cells transfected with RIG-I (F), MDA-5 (G), an IFN-β luciferase promoter, and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng). 24 h later, IFN-β activity was measured by luciferase reporter assay.


A . 293T cells were transfected with poly ( I : C ) and cell lysates were prepared at the indicated times for co - immunoprecipitation analysis of endogenous HDAC6 and RIG - I . Actin was used as the loading control .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 293T cells were transfected with poly(I:C) and cell lysates were prepared at the indicated times for co-immunoprecipitation analysis of endogenous HDAC6 and RIG-I. Actin was used as the loading control.


B . BMDMs were isolated from HDAC6 + / + and HDAC6 - / - mice and were transfected with 5ˊppp - dsRNA . Whole cell lysates were prepared after 8 h of transfection and co - immunoprecipitation analysis of RIG - I and HDAC6 was performed .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O 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. BMDMs were isolated from HDAC6+/+ and HDAC6-/- mice and were transfected with 5ˊppp-dsRNA. Whole cell lysates were prepared after 8 h of transfection and co-immunoprecipitation analysis of RIG-I and HDAC6 was performed.


C . HeLa cells were transfected with 5ˊppp - dsRNA . Cells were stained with anti - RIG - I ( red ) and anti - HDAC6 H300 ( green ) antibodies after 8 h of transfection . Nuclei were stained with DAPI ( blue ) ( bar , 20µm ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HeLa cells were transfected with 5ˊppp-dsRNA. Cells were stained with anti-RIG-I (red) and anti-HDAC6 H300 (green) antibodies after 8 h of transfection. Nuclei were stained with DAPI (blue) (bar, 20µm).


D . Fluorescence microscopy , green fluorescence absorbance and virus replication at 24hpi in RIG - I + / + and RIG - I - / - MEFs . Cells were transfected with si - HDAC6 and si - control for 36 h , followed infection with VSV - GFP ( MOI = 1 ) . bar , 100µm .

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

D. Fluorescence microscopy, green fluorescence absorbance and virus replication at 24hpi in RIG-I+/+ and RIG-I-/- MEFs. Cells were transfected with si-HDAC6 and si-control for 36 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm.


E . HDAC6 + / + , HDAC6 - / - MEFs , and HDAC6 - / - MEFs reconstituted with HDAC6 wild - type were subjected to immunoprecipitation using an anti - RIG - I antibody and immunoblotted with anti - acetyl - lysine and anti - RIG - I antibodies . Acetyl - tubulin was measured in total lysates to determine HDAC6 deacetylase activity .

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

E. HDAC6+/+, HDAC6-/- MEFs, and HDAC6-/- MEFs reconstituted with HDAC6 wild-type were subjected to immunoprecipitation using an anti-RIG-I antibody and immunoblotted with anti-acetyl-lysine and anti-RIG-I antibodies. Acetyl-tubulin was measured in total lysates to determine HDAC6 deacetylase activity.


F . Whole cell lysates from HDAC6 + / + and HDAC6 - / - MEFs were prepared and used in dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with anti - RIG - I , anti - HDAC6 , and anti - β - actin antibodies . Intensity of pull - downed RIG - I was quantified .

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

F. Whole cell lysates from HDAC6+/+ and HDAC6-/- MEFs were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I, anti-HDAC6, and anti-β-actin antibodies. Intensity of pull-downed RIG-I was quantified.


G . Whole cell lysates from HDAC6 - / - MEFs and HDAC6 - / - MEFs reconstituted with HDAC6 wild - type and HDAC6 - CDM were prepared and used in dsRNA pull - down assays . Samples were analyzed by western blotting with anti - RIG - I and anti - HDAC6 antibodies . Intensity of pull - downed RIG - I was quantified .

G. Whole cell lysates from HDAC6-/- MEFs and HDAC6-/- MEFs reconstituted with HDAC6 wild-type and HDAC6-CDM were prepared and used in dsRNA pull-down assays. Samples were analyzed by western blotting with anti-RIG-I and anti-HDAC6 antibodies. Intensity of pull-downed RIG-I was quantified.


A , B . 293T cells were transfected with the indicated expression plasmids . At 1 day post - transfection , whole cell lysates were subjected to dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with an anti - FLAG antibody .

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

A, B. 293T cells were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were subjected to dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with an anti-FLAG antibody.


C . A luciferase assay was performed using 293T cells transfected with the IFN - βluciferase promoter and TK - Renilla together with 2 ng of RIG - I wild - type , RIG - I 858Q , RIG - I 858R , RIG - I K909Q , or RIG - I K909R mutant , followed by transfection with poly ( I : C ) ( 1 μg / ml ) for another 12 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O

C. A luciferase assay was performed using 293T cells transfected with the IFN-βluciferase promoter and TK-Renilla together with 2 ng of RIG-I wild-type, RIG-I 858Q, RIG-I 858R, RIG-I K909Q, or RIG-I K909R mutant, followed by transfection with poly(I:C) (1 μg/ml) for another 12 h.


D . Fluorescence microscopy , green fluorescence absorbance , and virus replication at 24 hpi in RIG - I - / - MEFs . Cells were transiently transfected with empty vector , RIG - I wild - type , RIG - I K909Q , or RIG - I K909R mutant for 24 h , followed infection with VSV - GFP ( MOI = 1 ) . bar ,

D. Fluorescence microscopy, green fluorescence absorbance, and virus replication at 24 hpi in RIG-I-/- MEFs. Cells were transiently transfected with empty vector, RIG-I wild-type, RIG-I K909Q, or RIG-I K909R mutant for 24 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm


E . HDAC6 - / - MEFs were transfected with the indicated expression plasmids . At 1 day post - transfection , whole cell lysates were prepared and used in dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with anti - RIG - I and anti - β - actin antibodies .

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

E. HDAC6-/- MEFs were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I and anti-β-actin antibodies.


F . 293T cells were transfected with 2 μg of FLAG - tagged RIG - I and 1 , 5 , or 10 μg of V5 - tagged HDAC6 . At 36 h post - transfection , whole cell lysates were prepared for co - immunoprecipitation analysis of V5 - tagged HDAC6 and acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody .

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

F. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I and 1, 5, or 10 μg of V5-tagged HDAC6. At 36 h post-transfection, whole cell lysates were prepared for co-immunoprecipitation analysis of V5-tagged HDAC6 and acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody.


G . 293T cells were transfected with 2 μg of FLAG - tagged RIG - I . At 24 h post - transfection , cells were infected with VSV - GFP ( MOI = 0 . 1 ) . At 2 and 4 h post - infection , whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody . Intensity of ac - 909K normalized to Flag - RIG - I was quantified .

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

G. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I. At 24 h post-transfection, cells were infected with VSV-GFP (MOI = 0.1). At 2 and 4 h post-infection, whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to Flag-RIG-I was quantified.


H . whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody . Intensity of ac - 909K normalized to RIG - I was quantified .

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

H. whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to RIG-I was quantified.


A . The structural modeling result of RIG - I / dsRNA complex and positions of the lysine residue were presented . RIG - I helicase A and B are shown as green and pale blue ribbons . The dsRNA strands were presented as purple and yellow rods , respectively . Two lysine residues , K907 and K909 , on RIG - I that interact with dsRNA are indicated as violet sticks . K907 forms hydrogen bonding with neither 5 ´ - ppp dsRNA nor K907 . B , C . Acetylated K909 and K907 on RIG - I are indicated as green sticks . There were hydrogen bonding between NH2 of K907 and oxygen of acetyl - K909 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. The structural modeling result of RIG-I/dsRNA complex and positions of the lysine residue were presented. RIG-I helicase A and B are shown as green and pale blue ribbons. The dsRNA strands were presented as purple and yellow rods, respectively. Two lysine residues, K907 and K909, on RIG-I that interact with dsRNA are indicated as violet sticks. K907 forms hydrogen bonding with neither 5´-ppp dsRNA nor K907.B, C. Acetylated K909 and K907 on RIG-I are indicated as green sticks. There were hydrogen bonding between NH2 of K907 and oxygen of acetyl-K909.


B - J : Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs . Antibodies against Tuj1 , Gfap and Olig2 were used to recognize neurons , astrocytes and oligodendrocytes . In D , G and J the percentage of cells positive ( mean ± SEM ) for each marker was counted over the total of DAPI positive cells ( 100 % ) . No difference between genotypes was highlighted ( Two - way ANOVA ) ; the variation through time resulted significant for the levels of Tuj1 and Gfap ( Two - way ANOVA : F ( 1 , 16 ) = 9 . 542 , p - value < 0 . 05 and F ( 1 , 10 ) = 68 . 95 , p - value < 0 . 0001 respectively ) . Sample size : n ≥ 3 wells obtained from two independent preparations ; for each well 10 random fields were acquired . Scale bars : 30 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

B-J: Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs. Antibodies against Tuj1, Gfap and Olig2 were used to recognize neurons, astrocytes and oligodendrocytes. In D, G and J the percentage of cells positive (mean ± SEM) for each marker was counted over the total of DAPI positive cells (100%). No difference between genotypes was highlighted (Two-way ANOVA); the variation through time resulted significant for the levels of Tuj1 and Gfap (Two-way ANOVA: F(1,16)=9.542, p-value<0.05 and F(1,10)=68.95, p-value<0.0001 respectively). Sample size: n ≥3 wells obtained from two independent preparations; for each well 10 random fields were acquired. Scale bars: 30 µm.


P : Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14 . For each gene Two - way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) . Values are represented as mean ± SEM . Sample size : ≥ 5 samples obtained from two independent preparations .

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

P: Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14. For each gene Two-way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Values are represented as mean ± SEM. Sample size: ≥5 samples obtained from two independent preparations.


H - K : Raster plots ( H and I ) and their quantitation under basal conditions and upon 100µM 4 - AP exposure . The magnitude of the response after stimulation is expressed either as fold change ( J ) or as the averaged maximal values reached after 4 - AP exposure ( K ) . Dotted line in J represents untreated controls ( set to 1 ) . Values are represented as mean ± SEM . Student ' s t - test , * : p - value < 0 . 05 . Sample size n = 8 wells deriving from three independent preparations .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H-K: Raster plots (H and I) and their quantitation under basal conditions and upon 100µM 4-AP exposure. The magnitude of the response after stimulation is expressed either as fold change (J) or as the averaged maximal values reached after 4-AP exposure (K). Dotted line in J represents untreated controls (set to 1). Values are represented as mean ± SEM. Student's t-test, *: p-value<0.05. Sample size n=8 wells deriving from three independent preparations.


L : Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points . The binary mask is representative of a neuron used to study morphology . Scale bars : 30 µm . M - Q : Each graph represents a different morphological parameter analysed at selected time point . Two - way ANOVA indicated both a significant time effect ( ( F 4 , 20 ) = 282 , 5 and p < 0 , 0001 for the number of intersections ; ( F 4 , 26 ) = 104 , 5 and p < 0 , 0001 for the total dendritic length ; ( F 4 , 20 ) = 58 , 51 and p < 0 , 0001 for the maximal dendritic length ; ( F 3 , 22 ) = 38 , 32 and p < 0 , 0001 for polarization ) and genotype effect ( ( F 1 , 20 ) = 25 , 1 and p < 0 , 0001 for the number of intersections ; ( F 4 , 26 ) = 16 , 0 and p = 0 . 005 for the total dendritic length ; ( F 1 , 20 ) = 17 , 24 and p = 0 , 005 for the maximal dendritic length ; ( F 1 , 22 ) = 18 , 76 and p < 0 . 003 for polarization ) . Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point . The dotted line in panel P represents the cut - off above which a culture is defined polarized ( Horton et al , 2006 ) . Values are represented as mean ± SEM ; * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * * : p - value < 0 . 0001 . Sample size : n ≥ 3 wells deriving from three independent preparations ; for each well we measured at least 15 cells .

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

L: Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points. The binary mask is representative of a neuron used to study morphology. Scale bars: 30 µm. M-Q: Each graph represents a different morphological parameter analysed at selected time point. Two-way ANOVA indicated both a significant time effect ((F 4,20)=282,5 and p<0,0001 for the number of intersections; (F 4,26)=104,5 and p<0,0001 for the total dendritic length; (F 4,20)=58,51 and p<0,0001 for the maximal dendritic length; (F 3,22)=38,32 and p<0,0001 for polarization) and genotype effect ((F 1,20)=25,1 and p<0,0001 for the number of intersections; (F 4,26)=16,0 and p=0.005 for the total dendritic length; (F 1,20)=17,24 and p=0,005 for the maximal dendritic length; (F 1,22)=18,76 and p<0.003 for polarization). Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point. The dotted line in panel P represents the cut-off above which a culture is defined polarized (Horton et al, 2006). Values are represented as mean ± SEM; *: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001. Sample size: n≥3 wells deriving from three independent preparations; for each well we measured at least 15 cells.


B , C : Five morphological parameters were measured in untreated and treated null neurons at DIV14 . One - way ANOVA indicated a significant treatment effect for the maximal dendritic length ( F = 6 , 306 ; p = 0 , 0039 ) , total dendritic length ( F = 4 , 880 ; p = 0 , 0126 ) , number of intersections ( F = 10 , 70 ; p = 0 , 0003 ) and soma area ( F = 7 , 721 ; p = 0 , 0015 ) . Tukey ' s multiple comparison test was used to assess differences between null neurons and early and late treated neurons . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 . The variation of each of the selected parameters is summarized in the radar plot ( C ) . Sample size ( panels B , C ) : n ≥ 18 wells for ctrl group and n ≥ 10 wells for the treatment groups deriving from three independent preparations ; for each well we measured at least 15 cells . Each point is representative of a single well . Values are represented as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C: Five morphological parameters were measured in untreated and treated null neurons at DIV14. One-way ANOVA indicated a significant treatment effect for the maximal dendritic length (F=6,306;p=0,0039), total dendritic length (F=4,880;p=0,0126), number of intersections (F=10,70; p=0,0003) and soma area (F=7,721;p=0,0015). Tukey's multiple comparison test was used to assess differences between null neurons and early and late treated neurons. *: p-value<0.05; **: p-value<0.01; ***: p-value<0.001. The variation of each of the selected parameters is summarized in the radar plot (C). Sample size (panels B,C): n≥18 wells for ctrl group and n≥10 wells for the treatment groups deriving from three independent preparations; for each well we measured at least 15 cells. Each point is representative of a single well. Values are represented as mean ± SEM.


Bar graphs represent the expression values ( mean ± SEM ) of 27 genes , selected for their downregulation in null samples at DIV 14 ( p - value < 0 , 07 ; see Appendix Figure S2 ) , after CX546 administration in the two different time windows ( early in G and late in H ) . One - way ANOVA followed by Dunnett ' s multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls ( set to 1 , red dotted line ) . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 , * * * : p - value < 0 . 001 , * * * * : p - value < 0 . 0001 . Sample size : n ≥ 8 wells deriving from two independent preparations . Each point is representative of a single well .

O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graphs represent the expression values (mean ± SEM) of 27 genes, selected for their downregulation in null samples at DIV 14 (p-value <0,07; see Appendix Figure S2), after CX546 administration in the two different time windows (early in G and late in H). One-way ANOVA followed by Dunnett's multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls (set to 1, red dotted line). *: p-value<0.05; **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001. Sample size: n≥8 wells deriving from two independent preparations. Each point is representative of a single well.


F , G : Violin plots represent the average values of MQAE intensity for early ( F ) or late ( G ) treated null neurons and their relative controls . One - way ANOVA followed by Tukey ' s multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons . * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 . The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles . Sample size : n ≥ 300 cells from five different embryos per genotype .

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

F,G: Violin plots represent the average values of MQAE intensity for early (F) or late (G) treated null neurons and their relative controls. One-way ANOVA followed by Tukey's multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons. ***: p<0.001; ****: p <0.0001. The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles. Sample size: n≥300 cells from five different embryos per genotype.


I , J : In I One - way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons ( set to 100 ) , null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4 . * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 ( * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 ) ; # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine ( # : p - value < 0 . 05 ; # # : p - value < 0 . 01 ) . Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 ( expressed as 2 - ∆ ct ) . Box plot shows the median ( central band ) , the lower and upper quartiles ( boxes ) and the max and min values ( whiskers ) from at least 3 wells obtained from four different embryos . One - way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones . * * : p - value < 0 . 01 . Sample size : n ≥ 3 wells from four different embryos .

O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I,J: In I One-way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons (set to 100), null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4. * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 (*: p-value<0.05; **: p-value<0.01; ***: p-value<0.001); # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine (#: p-value<0.05; ##: p-value<0.01). Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 (expressed as 2-∆ct). Box plot shows the median (central band), the lower and upper quartiles (boxes) and the max and min values (whiskers) from at least 3 wells obtained from four different embryos. One-way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones. **: p-value<0.01. Sample size: n≥3 wells from four different embryos.


B : Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals . Sample size : wt = 11 , ko = 16 , ko treated with CX546 = 11 . Gehan - Breslow - Wilcoxon test was used to compare the two groups . * * : p - value < 0 . 01 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B: Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals. Sample size: wt=11, ko=16, ko treated with CX546=11. Gehan-Breslow-Wilcoxon test was used to compare the two groups. **: p-value<0.01.


F , G : Line graph ( F ) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials . Two - way ANOVA indicated a significant effect of time ( F ( 2 , 21 ) = 5 , 490 and p = 0 , 0121 ) and genotype ( F ( 2 , 33 ) = 16 , 33 and p < 0 . 0001 ) . Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546 . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 . The scatter plot ( G ) shows latency to fall ( in seconds ) on the accelerating rotarod in the last trial . One - way ANOVA was used to compare ko mice and ko treated mice to the wt group . The Tukey ' s multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine ; * * : p - value < 0 . 01 . Values are represented as average ± SEM . Sample size : n = 6 wt treated with vehicle and n = 14 ko ( 6 treated with vehicle and 8 treated with CX546 ) . Each dot represents a single animal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O

F,G: Line graph (F) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials. Two-way ANOVA indicated a significant effect of time (F(2,21)=5,490 and p=0,0121) and genotype (F(2,33)=16,33 and p<0.0001). Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546. *: p-value<0.05; **: p-value<0.01. The scatter plot (G) shows latency to fall (in seconds) on the accelerating rotarod in the last trial. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine; **: p-value<0.01. Values are represented as average ± SEM. Sample size: n=6 wt treated with vehicle and n=14 ko (6 treated with vehicle and 8 treated with CX546). Each dot represents a single animal.


( A ) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAKΔmyeloid mice .

(A) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAKΔmyeloid mice.


( B ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 0 - 5 hours before immunoblotting with the indicated antibodies .

O O O O O O 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) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-5 hours before immunoblotting with the indicated antibodies.


( C , E and G ) WT PEMs expressing GFP - FAK were incubated with either ΔinvG Salmonella ( C ) , LPS - coated beads ( E ) or E . coli ( G ) for a total of 5 hours before analysis by immunofluorescence . Cells were co - stained with antibodies recognizing LAMP1 ( red ) . Dapi was used to visualize nuclei and bacteria ( blue ) . Bars represent 10 µm . Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm . ( D , F and H ) The percent of LAMP1 - positive ( gray bars ) and LAMP1 + GFP + ( black bars ) ΔinvG Salmonella ( D ) , LPS - coated beads ( F ) and E . coli ( H ) was quantified . At least 100 bacteria were counted per condition ; N = 3 .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, E and G) WT PEMs expressing GFP-FAK were incubated with either ΔinvG Salmonella (C), LPS-coated beads (E) or E. coli (G) for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). Dapi was used to visualize nuclei and bacteria (blue). Bars represent 10 µm. Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm. (D, F and H) The percent of LAMP1-positive (gray bars) and LAMP1+GFP+ (black bars) ΔinvG Salmonella (D), LPS-coated beads (F) and E. coli (H) was quantified. At least 100 bacteria were counted per condition; N = 3.


A ) TLR4 − / − PEMs were incubated with ΔinvG Salmonella for 0 - 5 hours before immunoblotting with the indicated antibodies .

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

A) TLR4−/− PEMs were incubated with ΔinvG Salmonella for 0-5 hours before immunoblotting with the indicated antibodies.


( B ) WT PEMs were incubated with LPS - coated beads for 0 - 5 hours before immunoblotting for the indicated antibodies .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

(B) WT PEMs were incubated with LPS-coated beads for 0-5 hours before immunoblotting for the indicated antibodies.


( C ) Levels of phosphorylated proteins were quantified by densitometry , normalized to the amount of total protein present in each sample , and expressed relative to the basal level in uninfected cells . Values are means ± SEM , N = 3 , * p < 0 . 05 . Dashed line drawn at 1 indicates basal levels of activation .

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

(C) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.


( D ) WT PEMs were incubated with the orgA : : tet ; spiA : : kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs ( ΔSPI1ΔSPI2 ) for 0 - 5 hours before immunoblotting with the indicated antibodies .

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

(D) WT PEMs were incubated with the orgA::tet; spiA::kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs (ΔSPI1ΔSPI2) for 0-5 hours before immunoblotting with the indicated antibodies.


( E ) WT PEMs expressing GFP - FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence . Cells were co - stained with antibodies recognizing LAMP1 ( red ) . DAPI was used to visualize nuclei and bacteria ( blue ) . Bars represent 10 µm or 2 µm in enlarged inset .

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

(E) WT PEMs expressing GFP-FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). DAPI was used to visualize nuclei and bacteria (blue). Bars represent 10 µm or 2 µm in enlarged inset.


( A ) WT and FAK − / − PEMs were incubated for 0 - 5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies .

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

(A) WT and FAK−/− PEMs were incubated for 0-5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies.


( B ) WT PEMs were pretreated for 1 hour with the FAK - kinase inhibitor PF - 228 ( 0 . 5 µm ) before incubation with ΔinvG Salmonella for 5 hours . Lysates were immunoblotted with the indicated antibodies .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(B) WT PEMs were pretreated for 1 hour with the FAK-kinase inhibitor PF-228 (0.5 µm) before incubation with ΔinvG Salmonella for 5 hours. Lysates were immunoblotted with the indicated antibodies.


( C - D ) WT or FAK − / − macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain ( C ) or LPS - coated beads ( D ) for 0 - 5 hours before immunoblotting with the indicated antibodies . In panels C and D , WT PEMs were also incubated with ΔinvG ( 5 h ) for comparison of Akt activation . Vertical white lines in panel D indicate non - contiguous lanes generated from a single exposure .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) WT or FAK−/− macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain (C) or LPS-coated beads (D) for 0-5 hours before immunoblotting with the indicated antibodies. In panels C and D, WT PEMs were also incubated with ΔinvG (5 h) for comparison of Akt activation. Vertical white lines in panel D indicate non-contiguous lanes generated from a single exposure.


E ) Levels of phosphorylated proteins were quantified by densitometry , normalized to the amount of total protein present in each sample , and expressed relative to the basal level in uninfected cells . Values are means ± SEM , N = 3 , * p < 0 . 05 . Dashed line drawn at 1 indicates basal levels of activation .

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

E) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.


( F ) WT PEMs expressing HA - Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours . Lysates were subjected to immunoprecipitation ( IP ) with anti - HA antibodies before immunoblotting for FAK and HA . In parallel , lysates were immunoblotted for FAK , HA and ERK1 / 2 to control for the level of expression .

(F) WT PEMs expressing HA-Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours. Lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies before immunoblotting for FAK and HA. In parallel, lysates were immunoblotted for FAK, HA and ERK1/2 to control for the level of expression.


G ) WT and FAK − / − macrophages expressing HA - Akt were stained with anti - HA and anti - LAMP1 before analysis by confocal microscopy . White boxes show enlarged regions in lower panel . Arrows indicate HA + LAMP + Salmonella evident in WT PEMs . ( H ) The percentage of ΔΣΠΙ1ΔΣΠΙ2 ( first grouping ) or ΔinvG ( second grouping ) Salmonella co - localizing with HA and LAMP - 1 5 h post - infection . At least 100 bacteria were counted per condition . Values are means ± SEM , N = 3 , * p < 0 . 05 .

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

G) WT and FAK−/− macrophages expressing HA-Akt were stained with anti-HA and anti-LAMP1 before analysis by confocal microscopy. White boxes show enlarged regions in lower panel. Arrows indicate HA+LAMP+ Salmonella evident in WT PEMs. (H) The percentage of ΔΣΠΙ1ΔΣΠΙ2 (first grouping) or ΔinvG (second grouping) Salmonella co-localizing with HA and LAMP-1 5 h post-infection. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05.


( A ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 0 - 24 hours before immunoblotting with the indicated antibodies .

(A) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-24 hours before immunoblotting with the indicated antibodies.


( B ) WT PEMs were incubated with Akt inhibitor AKTV / triciribine ( 10 µm ) for 30 minutes before infection with S . typhimurium strain ΔinvG for 5 hours . Lysates were immunoblotted with the indicated antibodies . (

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O 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) WT PEMs were incubated with Akt inhibitor AKTV/triciribine (10 µm) for 30 minutes before infection with S. typhimurium strain ΔinvG for 5 hours. Lysates were immunoblotted with the indicated antibodies. (


. ( C ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for a total of 5 hours before analysis with antibodies recognizing LC3 . DAPI was used to visualize nuclei and bacteria . Bars represent 10 µm . White boxes show enlarged regions in inset panels , bars represent 2 µm . White arrowheads indicate LC3 - negative bacteria , yellow arrow heads indicate LC3 - positive bacteria ( panels c , f ) . Yellow arrowhead in panel f denotes an LC3 + cluster of bacteria . ( D ) The percentage of ΔinvG ( first grouping ) or ΔΣΠΙ1ΔΣΠΙ2 ( second grouping ) Salmonella co - localizing with LC3 at the indicated time points is displayed . At least 100 bacteria were counted per condition per time point . Values are means ± SEM , N = 3 , * p < 0 . 05 .

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

. (C) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for a total of 5 hours before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei and bacteria. Bars represent 10 µm. White boxes show enlarged regions in inset panels, bars represent 2 µm. White arrowheads indicate LC3-negative bacteria, yellow arrow heads indicate LC3-positive bacteria (panels c, f). Yellow arrowhead in panel f denotes an LC3+ cluster of bacteria. (D) The percentage of ΔinvG (first grouping) or ΔΣΠΙ1ΔΣΠΙ2 (second grouping) Salmonella co-localizing with LC3 at the indicated time points is displayed. At least 100 bacteria were counted per condition per time point. Values are means ± SEM, N = 3, *p<0.05.


( E ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 ( 300 ng ) before immunoblotting with the indicated antibodies . Vertical white line indicate non - contiguous lanes generated from a single exposure .

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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(E) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 (300 ng) before immunoblotting with the indicated antibodies. Vertical white line indicate non-contiguous lanes generated from a single exposure.


( F ) FAK − / − PEMs were incubated with DQ - BSA - red for 1 hour before infection with S . typhimurium strain ΔinvG for 5 h . Cells were co - stained for LC3 and DAPI was used to visualize nuclei and bacteria . White boxes show enlarged regions in inset panel .

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

(F) FAK−/− PEMs were incubated with DQ-BSA-red for 1 hour before infection with S. typhimurium strain ΔinvG for 5 h. Cells were co-stained for LC3 and DAPI was used to visualize nuclei and bacteria. White boxes show enlarged regions in inset panel.


G ) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin ( 4 µm ) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours . Cells were lysed before immunoblotting with the indicated antibodies .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin (4 µm) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours. Cells were lysed before immunoblotting with the indicated antibodies.