f , Kaplan - Meier analysis comparing PDAC - free survival of KrasG12D / + p53 − / − mice that were chloroquine - treated ( CQ , red ) , vehicle - treated ( PBS , black ) or untreated ( blue ) . a , e , f , The blue curve in all Kaplan - Meier plots represents the same colony of mice ( KrasG12D / + , p53 − / − , Atg7 + / + , Atg5 + / + ) . A Mantel - Cox test was used for statistics . b , d , Scale bars : 100 µm ( b ) , 200 µm ( d ) . * P 0 . 05 , * * P 0 . 01 .

O O O O O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-ORGANISM O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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, Kaplan-Meier analysis comparing PDAC-free survival of KrasG12D/+ p53−/− mice that were chloroquine-treated (CQ, red), vehicle-treated (PBS, black) or untreated (blue). a, e, f, The blue curve in all Kaplan-Meier plots represents the same colony of mice (KrasG12D/+, p53−/−, Atg7+/+, Atg5+/+). A Mantel-Cox test was used for statistics. b, d, Scale bars: 100 µm (b), 200 µm (d). *P 0.05, **P 0.01.


a , LC3 staining of representative pancreatic tumours from mice of indicated genotypes . Scale bars : 50 µm .

O O B-GENEPROD O O O B-TISSUE O O B-ORGANISM O O O O O O O O O O

a, LC3 staining of representative pancreatic tumours from mice of indicated genotypes. Scale bars: 50 µm.


b , Western blotting for LC3 , p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O

b, Western blotting for LC3, p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53.


c , d , Average oxygen consumption rate ( OCR , c ) and extracellular acidification rate ( ECAR ,

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR,


c , d , Average oxygen consumption rate ( OCR , c ) and extracellular acidification rate ( ECAR , d ) of eight Atg7 + / + and seven Atg7 − / − cell lines revealed increased ECAR in autophagy - deficient cells . Error bars are s . e . m .

O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD 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, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR, d) of eight Atg7+/+ and seven Atg7−/− cell lines revealed increased ECAR in autophagy-deficient cells. Error bars are s.e.m.


e , Treatment with 2 - deocyglucose ( 2 - DG ) reduces ECAR and abrogates the difference between cell lines from Atg7 + / + and Atg7 − / − tumours . Values are the average of three biological replicates . Error bars are s . e . m .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

e, Treatment with 2-deocyglucose (2-DG) reduces ECAR and abrogates the difference between cell lines from Atg7+/+ and Atg7−/− tumours. Values are the average of three biological replicates. Error bars are s.e.m.


, g , LC - MS analysis revealed increased glucose consumption from medium ( f ) and extracellular lactate accumulation ( g ) in Atg7 - null cell lines . Values are the average of eight ( Atg7 + / + ) and seven ( Atg7 − / − ) biological replicates .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O

, g, LC-MS analysis revealed increased glucose consumption from medium (f) and extracellular lactate accumulation (g) in Atg7-null cell lines. Values are the average of eight (Atg7+/+) and seven (Atg7−/−) biological replicates.


h , LC - MS analysis shows increased accumulation of 2 - DG and its metabolite 2 - DG - 6 - phosphate in Atg7 - deficient cell lines . Values are averages of five biological replicates for each genotype .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

h, LC-MS analysis shows increased accumulation of 2-DG and its metabolite 2-DG-6-phosphate in Atg7-deficient cell lines. Values are averages of five biological replicates for each genotype.


i , Background levels of 18F - FDG uptake in brain and heart can be seen in sagittal and coronal views ( middle and right panels , respectively ) of wild - type Atg7 mice ( top row ) . In contrast , intense 18F - FDG uptake is seen in pancreatic tumours of Atg7 deficient mice ( bottom row ) .

O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O B-GENEPROD O B-ORGANISM O O O O O

i, Background levels of 18F-FDG uptake in brain and heart can be seen in sagittal and coronal views (middle and right panels, respectively) of wild-type Atg7 mice (top row). In contrast, intense 18F-FDG uptake is seen in pancreatic tumours of Atg7 deficient mice (bottom row).


j , LC - MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7 − / − tumour cell lines . Values are average of eight ( Atg7 + / + ) and six ( Atg7 − / − ) biological replicates . f - h , j , Error bars are s . e . m . a , f - h , j , A Mann - Whitney U - test was used to ascertain significance . * P 0 . 05 , * * P 0 . 01 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

j, LC-MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7−/− tumour cell lines. Values are average of eight (Atg7+/+) and six (Atg7−/−) biological replicates. f-h, j, Error bars are s.e.m. a, f-h, j, A Mann-Whitney U-test was used to ascertain significance. *P  0.05, **P  0.01.


( f ) A549 - ACE2 or control A549 - Venus cells were pretreated with Tubercidin or vehicle ( DMSO ) 3h prior to infection with SARS - CoV - 2 at MOI 0 . 1 . After 24 hours , abundances of SARS - CoV - 2 nucleoprotein ( N ) , ACE2 , Venus and β - actin ( ACTB , loading control ) were visualized using Western blotting . Presented data is representative of 3 independent repeats .

O O O B-CELL_LINE O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

(f) A549-ACE2 or control A549-Venus cells were pretreated with Tubercidin or vehicle (DMSO) 3h prior to infection with SARS-CoV-2 at MOI 0.1. After 24 hours, abundances of SARS-CoV-2 nucleoprotein (N), ACE2, Venus and β-actin (ACTB, loading control) were visualized using Western blotting. Presented data is representative of 3 independent repeats.


( i ) NHBEs derived from three independent donors were pretreated for 6 h with indicated concentrations of DZNep or vehicle and infected with SARS - CoV - 2 for 24 h . Cells were fixed and the abundance of SARS - CoV - 2 N was quantified by immunofluorescent staining . Shown are vehicle - normalized integrated anti - N fluorescent intensity and cell confluence , error bars represent mean + / - sd of n = 3 donors .

O O O B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

(i) NHBEs derived from three independent donors were pretreated for 6 h with indicated concentrations of DZNep or vehicle and infected with SARS-CoV-2 for 24 h. Cells were fixed and the abundance of SARS-CoV-2 N was quantified by immunofluorescent staining. Shown are vehicle-normalized integrated anti-N fluorescent intensity and cell confluence, error bars represent mean +/- sd of n=3 donors.


Mass spectrometry based analysis of cells treated with DZNep and infected with SARS - CoV - 2 and SARS - CoV . ( b ) Number of significantly up - or downregulated proteins in indicated comparisons according to ( a ) .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O

Mass spectrometry based analysis of cells treated with DZNep and infected with SARS-CoV-2 and SARS-CoV. (b) Number of significantly up- or downregulated proteins in indicated comparisons according to (a).


Mass spectrometry based analysis of cells treated with DZNep and infected with SARS - CoV - 2 and SARS - CoV . ( c ) Donor - normalized LFQ abundances of viral nucleoprotein ( N ) and spike ( S ) in the indicated conditions . Error bars represent mean + / - sd of n = 4 donors ( NHBE ) or n = 4 independently infected A549 - ACE2 cultures . Statistics were calculated using Student ' s two - sided t - test as indicated .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O

Mass spectrometry based analysis of cells treated with DZNep and infected with SARS-CoV-2 and SARS-CoV. (c) Donor-normalized LFQ abundances of viral nucleoprotein (N) and spike (S) in the indicated conditions. Error bars represent mean +/- sd of n=4 donors (NHBE) or n=4 independently infected A549-ACE2 cultures. Statistics were calculated using Student's two-sided t-test as indicated.


( d ) Expression patterns according to ( a ) of a selection of genes related to the disease - relevant pathways as annotated .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(d) Expression patterns according to (a) of a selection of genes related to the disease-relevant pathways as annotated.


( e ) NHBEs ( 6 independent donors ) were pretreated for 6 h with 0 . 75 μM DZNep or vehicle and infected with SARS - CoV or SARS - CoV - 2 at MOI 3 . 24 h later , accumulation of IL - 6 and IP - 10 was measured in the supernatant by ELISA . Donor - wise IL - 6 and IP - 10 secretion , normalized to vehicle treated uninfected controls ( as further described in materials and methods ) is shown .

O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(e) NHBEs (6 independent donors) were pretreated for 6 h with 0.75 μM DZNep or vehicle and infected with SARS-CoV or SARS-CoV-2 at MOI 3. 24 h later, accumulation of IL-6 and IP-10 was measured in the supernatant by ELISA. Donor-wise IL-6 and IP-10 secretion, normalized to vehicle treated uninfected controls (as further described in materials and methods) is shown.


B An Integrative Genomics Viewer ( IGV ) screenshot of the Notch de novo TE insertion site from sample P47 ( clonal neoplasia ) and its head control , sample P48 . Bars represent sequencing reads . Reads supporting the TE insertion are colored according to homology to a specific TE insertion sequence . Multiple colors at a putative insert site frequently indicate homology to different reference copies of the same TE family . Two types of supporting reads can be seen : soft - clipped reads - spanning the insertion site and mapping partially to the reference genome and partially to the TE , and mate pair support reads - flanking the insertion site and mapping to the reference genome but with mates ( not seen ) mapping to the TE .

O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-DISEASE O O O B-TISSUE O O O O O O O B-EXP_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 An Integrative Genomics Viewer (IGV) screenshot of the Notch de novo TE insertion site from sample P47 (clonal neoplasia) and its head control, sample P48. Bars represent sequencing reads. Reads supporting the TE insertion are colored according to homology to a specific TE insertion sequence. Multiple colors at a putative insert site frequently indicate homology to different reference copies of the same TE family. Two types of supporting reads can be seen: soft-clipped reads - spanning the insertion site and mapping partially to the reference genome and partially to the TE, and mate pair support reads - flanking the insertion site and mapping to the reference genome but with mates (not seen) mapping to the TE.


D PCR validation of 4 somatic , neoplasia - specific TE insertions . Primers were designed to target regions flanking the insertion sites . Yellow arrowheads indicate PCR products containing an insertion amplified in the clonal DNA but not in the neighboring gut tissue ( non - clonal ) , head or thorax for the same fly . Short wild - type amplicon was detectable in all samples . Thorax DNA sample was not available for sample P15 .

O B-EXP_ASSAY O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-TISSUE O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O O O O O O O O O

D PCR validation of 4 somatic, neoplasia-specific TE insertions. Primers were designed to target regions flanking the insertion sites. Yellow arrowheads indicate PCR products containing an insertion amplified in the clonal DNA but not in the neighboring gut tissue (non-clonal), head or thorax for the same fly. Short wild-type amplicon was detectable in all samples. Thorax DNA sample was not available for sample P15.


D TSD length distribution for somatic insertions of most frequent TE families . Insertions from both genotypes were pooled .

D TSD length distribution for somatic insertions of most frequent TE families. Insertions from both genotypes were pooled.


C The distribution of somatic insertions with estimated cell fraction higher or lower than Notch - inactivating mutations for the most represented LTR - elements ( rover and copia ) and LINE - like I - elements . ( p - values were calculated with Fisher ' s exact test , two - tailed , n = number of insertions )

O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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 The distribution of somatic insertions with estimated cell fraction higher or lower than Notch-inactivating mutations for the most represented LTR-elements (rover and copia) and LINE-like I-elements. (p-values were calculated with Fisher's exact test, two-tailed, n= number of insertions)


A Heatmaps representing normalized TE expression levels ( in log2 ( TPM ) , transcripts per million ) and mobility ( log2 ( insertion counts ) ) in Pros > 2xGFP midguts . TEs with TPM values below 0 . 05 ( log2 ( TPM ) < - 4 . 3 ) are not depicted . Crossed out cells represent no somatic insertions of that family identified .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O 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 Heatmaps representing normalized TE expression levels (in log2(TPM), transcripts per million) and mobility (log2(insertion counts)) in Pros>2xGFP midguts. TEs with TPM values below 0.05 (log2(TPM)< -4.3) are not depicted. Crossed out cells represent no somatic insertions of that family identified.


B Normalized read coverage over the full - length canonical sequence of selected TE families .

B Normalized read coverage over the full-length canonical sequence of selected TE families.


C Normalized expression levels of siRNA pathway genes .

C Normalized expression levels of siRNA pathway genes.


D The size distribution of sense and antisense reads from gut small RNA fractions mapping to all TEs ( upper panel ) or selected TE families mobilizing in the gut . R1 and R2 are two biological replicates .

O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O

D The size distribution of sense and antisense reads from gut small RNA fractions mapping to all TEs (upper panel) or selected TE families mobilizing in the gut. R1 and R2 are two biological replicates.


E Scatter plot of normalized transcript ( mRNA ) levels and antisense , 21nt siRNA levels for all TE families . Transposons generating somatic insertions are highlighted in red ( LTR - elements ) or blue ( LINE - like ) , with a symbol size reflecting the somatic insertion counts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Scatter plot of normalized transcript (mRNA) levels and antisense, 21nt siRNA levels for all TE families. Transposons generating somatic insertions are highlighted in red (LTR-elements) or blue (LINE-like), with a symbol size reflecting the somatic insertion counts.


B TE families with tissue - specific , singleton TSD - bearing insertions detected in pooled gut or head DNA .

O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O

B TE families with tissue-specific, singleton TSD-bearing insertions detected in pooled gut or head DNA.


B Somatic TE insertion sites of rover LTR elements were depleted from intergenic and exonic sequences and enriched in introns and 3 ' UTR regions of the fly genome . Insertions of copia LTR elements were also enriched in introns .

O O O 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-ORGANISM O O O O O O O O O O O O O

B Somatic TE insertion sites of rover LTR elements were depleted from intergenic and exonic sequences and enriched in introns and 3'UTR regions of the fly genome. Insertions of copia LTR elements were also enriched in introns.


D Correlations of somatic insertion sites of the three most represented TE families ( rover , copia and I - element ) with modENCODE tracks for adult fly tissues .

O O O 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-ORGANISM O O

D Correlations of somatic insertion sites of the three most represented TE families (rover, copia and I-element) with modENCODE tracks for adult fly tissues.


E Correlations of somatic insertion sites of the three most represented TE families ( rover , copia and I - element ) with DamID tracks for adult fly intestinal stem cells ( ISC ) .

O O O 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-ORGANISM B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O

E Correlations of somatic insertion sites of the three most represented TE families (rover, copia and I-element) with DamID tracks for adult fly intestinal stem cells (ISC).


[ Ca2 + ] i changes in individual BMDMs were measured ( A ) using fura - 2 during frustrated phagocytosis ( n = 46 - 113 ) and ( B ) by dropping beads onto adherent cells loaded with Calbryte 520 ( n = 64 - 159 ) , in 1 . 8 mM extracellular Ca2 + ( + Ca2 + o ) or Ca2 + - free medium containing 100 μM EGTA ( - Ca2 + o ) with or without prior loading with 25 μM EGTA / AM or 25 μM BAPTA / AM . The maximum fura - 2 ratio changes ( ∆ 350 / 380 ) or fluorescence changes normalized to initial fluorescence ( ∆ F / F0 ) are plotted .

O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

[Ca2+]i changes in individual BMDMs were measured (A) using fura-2 during frustrated phagocytosis (n = 46-113) and (B) by dropping beads onto adherent cells loaded with Calbryte 520 (n = 64-159), in 1.8 mM extracellular Ca2+ (+Ca2+o) or Ca2+-free medium containing 100 μM EGTA (-Ca2+o) with or without prior loading with 25 μM EGTA/AM or 25 μM BAPTA/AM. The maximum fura-2 ratio changes (∆ 350/380) or fluorescence changes normalized to initial fluorescence (∆ F/F0) are plotted.


FcγR - mediated phagocytosis was monitored using beads conjugated to Alexa Fluor 488 ( in green ) . External beads were differentially labelled with anti - IgG ( in red ) and distinguished by their dual labelling ( in yellow ) . Cytochalasin D ( 10 μM , Cyt D ) prevented bead internalization .

B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

FcγR-mediated phagocytosis was monitored using beads conjugated to Alexa Fluor 488 (in green). External beads were differentially labelled with anti-IgG (in red) and distinguished by their dual labelling (in yellow). Cytochalasin D (10 μM, Cyt D) prevented bead internalization.


Bead uptake in 15 min , under conditions as in ( A , B ) , n = 80 - 91 cells with representative images of individual cells delineated by a dotted line .

B-EXP_ASSAY I-EXP_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 O O

Bead uptake in 15 min, under conditions as in (A, B), n = 80-91 cells with representative images of individual cells delineated by a dotted line.


Experiments conducted in - Ca2 + o : ( F ) 1 μM thapsigarin ( Tg ) or 10 μM CPA pre - treatment , all but eliminated the 1 μM ionomycin ( iono ) response ; single - cell Ca2 + traces and mean ∆ F / F0 , n = 450 - 603 cells . ( G ) FcγR activation ( as in B ) evoked Ca2 + oscillations that were blocked with Tg or CPA treatment , whilst bead phagocytosis was not affected ( H ) , n = 312 - 428 cells . ( I ) 1 μM bafilomycin A1 ( Baf ) for 3h , or Ca2 + - binding dextran ( Cal520 - dx ) and non - Ca2 + - binding dextrans ( FITC - dx and Texas red - dx ) trafficked to lysosomes , minimally affected global Ca2 + signals evoked by FcγR activation ( J ) ; in parallel , bead uptake was only reduced by bafilomycin A1 or by chelating Ca2 + within the lysosome with Cal520 - dx ( K ) , n = 100 - 162 cells . All cells are WT BMDM and all beads are 3 - μm IgG - opsonized .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O

Experiments conducted in -Ca2+o: (F) 1 μM thapsigarin (Tg) or 10 μM CPA pre-treatment, all but eliminated the 1 μM ionomycin (iono) response; single-cell Ca2+ traces and mean ∆ F/F0, n = 450-603 cells. (G) FcγR activation (as in B) evoked Ca2+ oscillations that were blocked with Tg or CPA treatment, whilst bead phagocytosis was not affected (H), n = 312-428 cells. (I) 1 μM bafilomycin A1 (Baf) for 3h, or Ca2+- binding dextran (Cal520-dx) and non-Ca2+-binding dextrans (FITC-dx and Texas red-dx) trafficked to lysosomes, minimally affected global Ca2+ signals evoked by FcγR activation (J); in parallel, bead uptake was only reduced by bafilomycin A1 or by chelating Ca2+ within the lysosome with Cal520-dx (K), n = 100-162 cells. All cells are WT BMDM and all beads are 3-μm IgG-opsonized.


The Ca2 + signal elicited by frustrated phagocytosis was significantly reduced in Ned - 19 ( 10 μM ) - treated WT and in all TPC - KO BMDMs ; maximum Ca2 + amplitude , expressed as a percentage of WT control ( n = 314 - 641 cells ) . Phagocytosis of opsonized 3 - µm ( B and C ) and 6 - μm ( D ) beads was inhibited by Ned - 19 ( B ) and in BMDMs from Tpcn - / - mice compared to WT ( n = 134 - 280 cells ) .

O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O

The Ca2+ signal elicited by frustrated phagocytosis was significantly reduced in Ned-19 (10 μM)-treated WT and in all TPC-KO BMDMs; maximum Ca2+ amplitude, expressed as a percentage of WT control (n = 314-641 cells). Phagocytosis of opsonized 3-µm (B and C) and 6-μm (D) beads was inhibited by Ned-19 (B) and in BMDMs from Tpcn-/- mice compared to WT (n = 134-280 cells).


( E ) Phagocytosis of 3 - µm beads was unchanged in MCOLN1 - / - BMDMs , but was impaired using 6 - µm beads ( F ) , n = 89 - 157 .

O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(E) Phagocytosis of 3-µm beads was unchanged in MCOLN1-/- BMDMs, but was impaired using 6-µm beads (F), n = 89-157.


Heterologous expression of mouse TPCs tagged with TagRFP - T ( in red ) rescues the phagocytic defect of Tpcn1 - / - and Tpcn2 - / - BMDM , but a pore - dead TPC2 - mutant ( N257A ) does not , n = 39 - 94 . Images taken 40 min post - 3 - μm bead addition .

O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

Heterologous expression of mouse TPCs tagged with TagRFP-T (in red) rescues the phagocytic defect of Tpcn1-/- and Tpcn2-/- BMDM, but a pore-dead TPC2-mutant (N257A) does not, n = 39-94. Images taken 40 min post- 3-μm bead addition.


Raising cytosolic Ca2 + with ionomycin ( 1 μM ) rescues the Tpcn1 - / - and Tpcn2 - / - phagocytic defect , whilst UTP ( 100 μM ) and ML - SA1 ( 50 μM ) only weakly rescue ( n = 84 - 192 cells ) .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O

Raising cytosolic Ca2+ with ionomycin (1 μM) rescues the Tpcn1-/- and Tpcn2-/- phagocytic defect, whilst UTP (100 μM) and ML-SA1 (50 μM) only weakly rescue (n = 84-192 cells).


Internalization of bacteria is reduced in Tpcn - / - BMDM , as assessed by single - cell imaging of BMDM allowed to internalise M . smegmatis tagged with mCherry ( in red ) for 1 h . Internalization of bacteria assessed discontinuously in BMDM populations : ( B ) The resultant growth of bacteria colonies on agar plates following lysis of BMDM infected with M . smegmatis and ( C ) quantification of colony forming units ( CFU ) expressed as a percentage of WT control ( n = 12 ) ;

O O O O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O

Internalization of bacteria is reduced in Tpcn-/- BMDM, as assessed by single-cell imaging of BMDM allowed to internalise M. smegmatis tagged with mCherry (in red) for 1 h. Internalization of bacteria assessed discontinuously in BMDM populations: (B) The resultant growth of bacteria colonies on agar plates following lysis of BMDM infected with M. smegmatis and (C) quantification of colony forming units (CFU) expressed as a percentage of WT control (n = 12);


Real - time internalization of fluorescent bacteria into BMDM populations : ( D ) Relative Fluorescence Units ( RFU ) of internalised mCherry - tagged M . smegmatis ( E ) and mCherry - tagged BCG over time , read using a plate reader ( n = 8 ) .

O O O O O O O O B-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O

Real-time internalization of fluorescent bacteria into BMDM populations: (D) Relative Fluorescence Units (RFU) of internalised mCherry-tagged M. smegmatis (E) and mCherry-tagged BCG over time, read using a plate reader (n = 8).


BMDMs were co - loaded with 2 µM Calbryte590 / AM ( 50 min ) and 200 nM Lysotracker Green ( LTG , 5 min ) and , at room temperature , presented with 3 - µm opsonized beads labelled with Alexa Fluor 647 ( blue ) . A three - channel image was recorded every 10 s . The LTG brightness has been enhanced post hoc to visualize the dimmer , peripheral lysosomes . Time Zero is defined as the frame when the first Ca2 + signal was observed . ( A ) Images represent Ca2 + responses or an overlay of LTG and bead fluorescence . Times of each image are relative to the first Ca2 + spike . ( B ) Enlargement of the first contact point between lysosomes and bead ( red arrow ) as depicted by the dashed box , 80 s post - Ca2 + . ( C ) Whole - cell Ca2 + responses from the cell in A . ( D ) Collation of the post - Ca2 + contact time , displayed as a scatter plot of individual cells and the mean ± S . E . M . ( n = 27 cells ) .

B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BMDMs were co-loaded with 2 µM Calbryte590/AM (50 min) and 200 nM Lysotracker Green (LTG, 5 min) and, at room temperature, presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). A three-channel image was recorded every 10 s. The LTG brightness has been enhanced post hoc to visualize the dimmer, peripheral lysosomes. Time Zero is defined as the frame when the first Ca2+ signal was observed. (A) Images represent Ca2+ responses or an overlay of LTG and bead fluorescence. Times of each image are relative to the first Ca2+ spike. (B) Enlargement of the first contact point between lysosomes and bead (red arrow) as depicted by the dashed box, 80 s post-Ca2+. (C) Whole-cell Ca2+ responses from the cell in A. (D) Collation of the post-Ca2+ contact time, displayed as a scatter plot of individual cells and the mean ± S.E.M. (n = 27 cells).


RAW246 . 7 cells were transfected with either TPC1 - TagRFP - T or TPC2 - TagRFP - T ( red ) and presented with 3 - µm opsonized beads labelled with Alexa Fluor 647 ( blue ) . Acute time - series of bead engagement were collected . Contact between TPCs and beads are depicted in the time - stamped micrographs , where time is arbitrary after the start of the image stack ( E , F ) i . e . 110 s is still prior to engagement . The spatial relationship between TPCs and beads was quantified using a target graticule of 0 . 5 - µm bands centred on the engulfed bead ; the mean fluorescence within each band was normalized to the fluorescence of the maximum band and plotted against this radial distance ( G , H ) ; n = 35 - 36 cells .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RAW246.7 cells were transfected with either TPC1-TagRFP-T or TPC2-TagRFP-T (red) and presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). Acute time-series of bead engagement were collected. Contact between TPCs and beads are depicted in the time-stamped micrographs, where time is arbitrary after the start of the image stack (E,F) i.e. 110 s is still prior to engagement. The spatial relationship between TPCs and beads was quantified using a target graticule of 0.5-µm bands centred on the engulfed bead; the mean fluorescence within each band was normalized to the fluorescence of the maximum band and plotted against this radial distance (G,H); n = 35-36 cells.


RAW 247 . 6 macrophages loaded with a cytosolic chemical dye ( Cyto dye ) to detect global cytosolic Ca2 + , and heterologously expressing ( B ) TPC1 - G - GECO1 . 2 ( TPC1 - GG ) or ( C - E ) TPC2 - G - GECO1 . 2 ( TPC2 - GG ) to detect peri - endo - lysosomal Ca2 + domains , which were not detected by ( F ) a TPC2 mutant ( D276K ) - G - GECO1 . 2 that blocks ion permeation or ( A ) unfused cytosolic G - GECO1 . 2 ( Cyto GG ) . ( A - F ) Single - cell Ca2 + traces upon FcγR activation by opsonized 3 - μm beads in - Ca2 + o expressed as a percentage of the maximum fluorescence of the indicator , determined by adding 2 μM ionomycin and 10 mM CaCl2 at the end of each experiment ( % Fmax ) ( D ) TPC2 - G - GECO1 . 2 activity was inhibited by 10 µM Ned - 19 , but was unaffected by ( E ) chelating global Ca2 + with EGTA / AM ( 100 μM for 2 min prior to bead addition ) . Summary data of the first spike from cells A - F ( subsequent spikes show the same pattern ; Appendix Fig . S4B ) , n = 87 - 274 cells ; * * * P < 0 . 001 vs Cyto GG , # # # P < 0 . 001 vs TPC2 - GG ctrl , † † † P < 0 . 001 vs Cyto GG . Fusion of G - GECO1 . 2 to TPC2 did not alter the affinity of G - GECO1 . 2 for Ca2 + determined in permeabilized cells ( n = 34 - 71 cells ) .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

RAW 247.6 macrophages loaded with a cytosolic chemical dye (Cyto dye) to detect global cytosolic Ca2+, and heterologously expressing (B) TPC1-G-GECO1.2 (TPC1-GG) or (C-E) TPC2-G-GECO1.2 (TPC2-GG) to detect peri-endo-lysosomal Ca2+ domains, which were not detected by (F) a TPC2 mutant (D276K)-G-GECO1.2 that blocks ion permeation or (A) unfused cytosolic G-GECO1.2 (Cyto GG). (A-F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of the maximum fluorescence of the indicator, determined by adding 2 μM ionomycin and 10 mM CaCl2 at the end of each experiment (% Fmax) (D) TPC2-G-GECO1.2 activity was inhibited by 10 µM Ned-19, but was unaffected by (E) chelating global Ca2+ with EGTA/AM (100 μM for 2 min prior to bead addition). Summary data of the first spike from cells A-F (subsequent spikes show the same pattern; Appendix Fig. S4B), n = 87-274 cells; ***P<0.001 vs Cyto GG, ### P<0.001 vs TPC2-GG ctrl, ††† P<0.001 vs Cyto GG. Fusion of G-GECO1.2 to TPC2 did not alter the affinity of G-GECO1.2 for Ca2+ determined in permeabilized cells (n = 34-71 cells).


( I ) Single - cell Ca2 + traces upon FcγR activation by opsonized 3 - μm beads in - Ca2 + o expressed as a percentage of Fmax . ( I , J ) Pre - emptying the ER stores with CPA or thapsigargin ( Tg ) abolished the global cytosolic Ca2 + response evoked by beads , but a TPC2 - G ‑ GECO1 . 2 component remained , n = 17 - 121 cells .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

(I) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of Fmax. (I, J) Pre-emptying the ER stores with CPA or thapsigargin (Tg) abolished the global cytosolic Ca2+ response evoked by beads, but a TPC2-G‑GECO1.2 component remained, n = 17 - 121 cells.


No local Ca2 + signal was detected with TPC2 - G - GECO1 . 2 when Ca2 + spiking was evoked by ER - Ca2 + release with 100 μM UTP ( K , L ) or 10 μM CPA ( M ) ; only a bystander response equivalent to unfused cytosolic G - GECO1 . 2 was detected ( n = 121 - 182 cells ; * P < 0 . 05 ) .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O

No local Ca2+ signal was detected with TPC2-G-GECO1.2 when Ca2+ spiking was evoked by ER-Ca2+ release with 100 μM UTP (K, L) or 10 μM CPA (M); only a bystander response equivalent to unfused cytosolic G-GECO1.2 was detected (n = 121-182 cells; *P<0.05).


Translocation of CB , a non - Ca2 + binding CB mutant ( CB mut ) or a triple mTagBFP2 ( BFP ) to the lysosome ( LAMP1 ) , as indicated by the increase in the Pearson ' s correlation coefficient was induced by 250 nM rapalog ( 30 mins ) compared with ethanol ( EtOH ) control - treated RAW 247 . 6 cells . n = 11 - 25 cells ; * * * P < 0 . 001 .

B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O

Translocation of CB, a non-Ca2+ binding CB mutant (CB mut) or a triple mTagBFP2 (BFP) to the lysosome (LAMP1), as indicated by the increase in the Pearson's correlation coefficient was induced by 250 nM rapalog (30 mins) compared with ethanol (EtOH) control-treated RAW 247.6 cells. n = 11-25 cells; ***P<0.001.


Images of a single RAW 247 . 6 cell displaying yellow puncta indicative of successful re - positioning of CB ( in green ) to the lysosome ( in red ) in the presence of rapalog but not EtOH .

O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O

Images of a single RAW 247.6 cell displaying yellow puncta indicative of successful re-positioning of CB (in green) to the lysosome (in red) in the presence of rapalog but not EtOH.


When CB was brought to the lysosome ( + rapalog ) uptake of beads was inhibited , whilst the non - Ca2 + binding CB ( CB mut ; n = 46 - 187 cells ) or triple mTagBFP2 ( BFP ; n = 38 - 52 cells ) did not block phagocytosis ; * * * P < 0 . 001 .

O B-GENEPROD O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

When CB was brought to the lysosome (+rapalog) uptake of beads was inhibited, whilst the non-Ca2+ binding CB (CB mut; n = 46-187 cells) or triple mTagBFP2 (BFP; n = 38-52 cells) did not block phagocytosis;***P<0.001.


Bead uptake in BMDMs with and without loading of 25 µM EGTA / AM and 10 μM Ned - 19 . EGTA - insensitive phagocytosis of beads was inhibited by Ned - 19 ( n = 75 - 102 cells ) in WT BMDM ; * * * P < 0 . 001 .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O

Bead uptake in BMDMs with and without loading of 25 µM EGTA/AM and 10 μM Ned-19. EGTA-insensitive phagocytosis of beads was inhibited by Ned-19 (n= 75-102 cells) in WT BMDM; ***P<0.001.


RAW 247 . 6 macrophages expressing cytosolic G - GECO1 . 2 ( Cyto GG ) , TPC2 - G - GECO1 . 2 ( TPC2 - GG ) or TRPML1 - G - GECO1 . 2 ( TRPML1 - GG ) . ( F ) Single - cell Ca2 + traces upon FcγR activation by opsonized 3 - μm or 6 - μm beads in - Ca2 + o expressed as a percentage of the maximum indicator fluorescence ( % Fmax = 2 μM ionomycin and 10 mM CaCl2 ) . ( G ) Summary of first Ca2 + spike ; n = 69 - 121 ( 3 - μm bead ) , 22 - 46 ( 6 - μm beads ) cells ; * * * P < 0 . 001 , * * P < 0 . 01 vs Cyto GG , # # # P < 0 . 001 TPC2 - GG vs TRPML1 - GG .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O

RAW 247.6 macrophages expressing cytosolic G-GECO1.2 (Cyto GG), TPC2-G-GECO1.2 (TPC2-GG) or TRPML1-G-GECO1.2 (TRPML1-GG). (F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm or 6-μm beads in -Ca2+o expressed as a percentage of the maximum indicator fluorescence (% Fmax = 2 μM ionomycin and 10 mM CaCl2). (G) Summary of first Ca2+ spike; n = 69-121 (3-μm bead), 22-46 (6-μm beads) cells; ***P<0.001, **P<0.01 vs Cyto GG, ### P<0.001 TPC2-GG vs TRPML1-GG.


Dynamin inhibitors ( 80 μM Dynasore or 20 μM Dyngo - 4a ) inhibit phagocytosis of opsonized 3 - μm beads in WT BMDM ; n = 101 - 149 cells , * * * P < 0 . 001 .

B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O

Dynamin inhibitors (80 μM Dynasore or 20 μM Dyngo-4a) inhibit phagocytosis of opsonized 3-μm beads in WT BMDM; n = 101-149 cells, ***P<0.001.


Rescue of the TPC - KO dynamin defect by the dynamin activator , Ryngo - 1 - 23 ( 80 μM ) : mean number of internalised beads per cell as a percentage of the WT control ; n = 128 - 241 cells , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O B-GENEPROD O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Rescue of the TPC-KO dynamin defect by the dynamin activator, Ryngo-1-23 (80 μM): mean number of internalised beads per cell as a percentage of the WT control; n = 128-241 cells, **P<0.01, ***P<0.001.


In WT BMDM , expression of a dominant - negative dynamin - 2 mutant ( Dyn2 K44A ) reduced phagocytosis ( n = 68 - 184 ) . Expression of wildtype dynamin - 2 ( Dyn2 ) or constitutively active dynamin - 2 ( S764A mutant , mimicking the dephosphorylated state ) in Tpcn1 - / - macrophages restored phagocytosis of 3 - μm beads after 10 min , n = 10 - 76 cells ; * * * P < 0 . 001 vs WT control , # # # P < 0 . 001 vs Tpcn1 - / - control .

O O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O 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-GENEPROD O O O O O

In WT BMDM, expression of a dominant-negative dynamin-2 mutant (Dyn2 K44A) reduced phagocytosis (n = 68-184). Expression of wildtype dynamin-2 (Dyn2) or constitutively active dynamin-2 (S764A mutant, mimicking the dephosphorylated state) in Tpcn1-/- macrophages restored phagocytosis of 3-μm beads after 10 min, n = 10-76 cells; ***P<0.001 vs WT control, ### P<0.001 vs Tpcn1-/- control.


Single WT BMDM expressing HA - tagged dynamin - 2 ( immunolabelled with anti - HA , in red ) undergoing phagocytosis of IgG - 3 - μm beads ( blue ) . Images were taken 10 or 30 min post - addition of beads ; actin ( green ) labelled with Phalloidin Alexa 488 .

O O B-CELL_TYPE B-EXP_ASSAY B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

Single WT BMDM expressing HA-tagged dynamin-2 (immunolabelled with anti-HA, in red) undergoing phagocytosis of IgG-3-μm beads (blue). Images were taken 10 or 30 min post-addition of beads; actin (green) labelled with Phalloidin Alexa 488.


Calcineurin inhibitors , FK506 ( 10 μM ) or cyclosporin A ( CsA , 10 μM ) inhibited phagocytosis in WT cells , n = 65 - 159 cells . Ryngo ( 80 μM ) rescued phagocytosis defects in Tpcn1 - / - BMDM even in the presence of FK506 or CsA , n = 103 - 208 cells .

B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

Calcineurin inhibitors, FK506 (10 μM) or cyclosporin A (CsA, 10 μM) inhibited phagocytosis in WT cells, n = 65-159 cells. Ryngo (80 μM) rescued phagocytosis defects in Tpcn1-/- BMDM even in the presence of FK506 or CsA, n = 103-208 cells.


Calcineurin activity and cytosolic Ca2 + responses simultaneously monitored in single RAW 247 . 6 using CaNAR2 and jRGECO1a , respectively , upon addition of 3 - μm beads . CaNAR2 signals ( FRET Y / C ) were inhibited by FK506 ( 10 μM ) and Ned - 19 ( 10 μM ) ; ( H ) single - cell traces and ( I ) mean maximum responses . n = 65 - 169 ; * * * P < 0 . 001 vs control .

B-GENEPROD B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Calcineurin activity and cytosolic Ca2+ responses simultaneously monitored in single RAW 247.6 using CaNAR2 and jRGECO1a, respectively, upon addition of 3-μm beads. CaNAR2 signals (FRET Y/C) were inhibited by FK506 (10 μM) and Ned-19 (10 μM); (H) single-cell traces and (I) mean maximum responses. n = 65-169; ***P<0.001 vs control.


Phagocytosis induced dephosphorylation of dynamin - 2 ( ratio of phospho - dynamin to total dynamin - 2 expressed as a percentage of WT at time zero ) in WT BMDM , but not in Tpcn1 - / - BMDM ; * * P < 0 . 01 , * * * P < 0 . 001 ( time - point matched unpaired t - test ) n = 10 - 15 . Measured using in - cell western assay , representative wells of plate image : total dyn2 ( red ) and phospho - dyn2 ( green ) . See also Appendix Figures S6 and S7 .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O

Phagocytosis induced dephosphorylation of dynamin-2 (ratio of phospho-dynamin to total dynamin-2 expressed as a percentage of WT at time zero) in WT BMDM, but not in Tpcn1-/- BMDM; **P<0.01, ***P<0.001 (time-point matched unpaired t-test) n = 10-15. Measured using in-cell western assay, representative wells of plate image: total dyn2 (red) and phospho-dyn2 (green). See also Appendix Figures S6 and S7.


RAW 246 . 7 macrophages expressing the dynein - binding protein ( mTagBFP2 - BicD2 - FKBP12 ) for rapalog - induced lysosome repositioning to the MTOC , also co - expressed HsLAMP1 - mCherry with or without a C - terminal FRB * ( LAMP - FRB * or LAMP , in red ) . Cells were treated with 0 . 1 % ethanol or 250 nM rapalog for 2 h . Only the tripartite complex of FKBP12 - rapalog - FRB induced clustering of lysosomes at the MTOC ( A , B ) . This clustering was selective for lysosomes because ER and mitochondria ( KDEL - GFP and MitoTracker Green ) were not affected ( C ) . ( A , B ) Phagocytosis of IgG - 3 - μm beads was reduced by immobilizing lysosomes at the MTOC , n = 80 - 564 cells ; * * * P < 0 . 001 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

RAW 246.7 macrophages expressing the dynein-binding protein (mTagBFP2-BicD2-FKBP12) for rapalog-induced lysosome repositioning to the MTOC, also co-expressed HsLAMP1-mCherry with or without a C-terminal FRB* (LAMP-FRB* or LAMP, in red). Cells were treated with 0.1% ethanol or 250 nM rapalog for 2 h. Only the tripartite complex of FKBP12-rapalog-FRB induced clustering of lysosomes at the MTOC (A, B). This clustering was selective for lysosomes because ER and mitochondria (KDEL-GFP and MitoTracker Green) were not affected (C). (A, B) Phagocytosis of IgG-3-μm beads was reduced by immobilizing lysosomes at the MTOC, n = 80-564 cells; ***P<0.001.


Activation of dynamin - 2 with Ryngo 1 - 23 or expression of constitutively active dynamin - 2 ( S764A mutant , expression confirmed by the grayscale image ) restored phagocytosis of IgG - 3 - μm beads ( n = 34 - 51 cells ) , but had no effect on the phagocytosis of IgG - 6 - μm beads , n = 36 - 72 cells ( E ) ; * * * P < 0 . 001 EtOH Ctrl vs rapalog Ctrl , # # # P < 0 . 001 rapalog Ctrl vs rapalog Ryngo / Dyn2S764A .

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O

Activation of dynamin-2 with Ryngo 1-23 or expression of constitutively active dynamin-2 (S764A mutant, expression confirmed by the grayscale image) restored phagocytosis of IgG-3-μm beads (n = 34-51 cells), but had no effect on the phagocytosis of IgG-6-μm beads, n = 36-72 cells (E); ***P<0.001 EtOH Ctrl vs rapalog Ctrl, ###P<0.001 rapalog Ctrl vs rapalog Ryngo/Dyn2S764A.


( a ) Relative CK2α protein concentration over time following cytoplasmic microinjection of REF52 cells with the HA - tagged semisynthetic proteins bearing different modifications using immunocytochemistry with HA - specific antibody . Data represent mean values ± s . d . ( b ) Relative CK2α protein concentration following injection of β subunit together with CK2α proteins . Data represent mean values ± s . d . ( c ) Relative CK2α protein concentration following injection of proteasome inhibitor ( 1 μM MG132 ) together with unmodified CK2α protein . Data represent mean values ± s . d .

O O O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O

(a) Relative CK2α protein concentration over time following cytoplasmic microinjection of REF52 cells with the HA-tagged semisynthetic proteins bearing different modifications using immunocytochemistry with HA-specific antibody. Data represent mean values ± s.d. (b) Relative CK2α protein concentration following injection of β subunit together with CK2α proteins. Data represent mean values ± s.d. (c) Relative CK2α protein concentration following injection of proteasome inhibitor (1 μM MG132) together with unmodified CK2α protein. Data represent mean values ± s.d.


( d ) Western blot for total CK2α protein in HeLa cells following treatment with Cdk inhibitors . Numbers indicate relative quantification normalized for loading control . Mean values ( n = 3 ) shown ± s . d .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE O O O O O 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) Western blot for total CK2α protein in HeLa cells following treatment with Cdk inhibitors. Numbers indicate relative quantification normalized for loading control. Mean values (n = 3) shown ± s.d.


( e ) Western blot for total CK2α protein in HeLa cells following treatment with nocodazole to arrest cells in mitosis . Full gels corresponding to those in panels d and e are in Supplementary Figure 8 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

(e) Western blot for total CK2α protein in HeLa cells following treatment with nocodazole to arrest cells in mitosis. Full gels corresponding to those in panels d and e are in Supplementary Figure 8.


( a ) Pull - down experiments with semisynthetic HA - CK2α protein and recombinant Pin1 . CK2α was immobilized using HA - specific ( anti - HA ) antibodies bound to protein G beads . Pin1 was incubated with immobilized CK2α for 20 min at 4 ° C .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O

(a) Pull-down experiments with semisynthetic HA-CK2α protein and recombinant Pin1. CK2α was immobilized using HA-specific (anti-HA) antibodies bound to protein G beads. Pin1 was incubated with immobilized CK2α for 20 min at 4 °C.


( b ) Coimmunoprecipitation of endogenous Pin1 with CK2α . Pfa - containing and unmodified semisynthetic HA - CK2α proteins were spiked into REF52 cell lysates and immobilized using HA - specific antibodies . Input samples are shown for loading control .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O

(b) Coimmunoprecipitation of endogenous Pin1 with CK2α. Pfa-containing and unmodified semisynthetic HA-CK2α proteins were spiked into REF52 cell lysates and immobilized using HA-specific antibodies. Input samples are shown for loading control.


( c ) Pull - down experiments with Pfa344 HA - CK2α protein and recombinant Pin1 in the presence of Pin1 - specific or nonspecific IgY .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O

(c) Pull-down experiments with Pfa344 HA-CK2α protein and recombinant Pin1 in the presence of Pin1-specific or nonspecific IgY.


( d ) Relative CK2α protein concentration over time following microinjection of Pfa344 CK2α with Pin1 - specific IgY or nonspecific IgY ( control ) . Data represent mean values ± s . d . Full gels corresponding to those in panels a - c are in Supplementary Figures 10 and 11 .

O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O 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) Relative CK2α protein concentration over time following microinjection of Pfa344 CK2α with Pin1-specific IgY or nonspecific IgY (control). Data represent mean values ± s.d. Full gels corresponding to those in panels a-c are in Supplementary Figures 10 and 11.


( a ) Phosphorylation of C - terminal tail CK2α peptides ( residues 337 - 352 ) by Cdk1 - cyclin B . In vitro Cdk1 - cyclin B kinase assays were performed using the following peptide substrate : SSMPGGSTPVXSANMMK ( εBiotin ) , where X = serine or S - GlcNAc serine . After reactions were quenched , peptides were separated from the reaction mixture on Tris - tricine gels . Data represent mean values ± s . d .

O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O 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) Phosphorylation of C-terminal tail CK2α peptides (residues 337-352) by Cdk1-cyclin B. In vitro Cdk1-cyclin B kinase assays were performed using the following peptide substrate: SSMPGGSTPVXSANMMK(εBiotin), where X = serine or S-GlcNAc serine. After reactions were quenched, peptides were separated from the reaction mixture on Tris-tricine gels. Data represent mean values ± s.d.


( b ) Immunoprecipitation and western blot for O - GlcNAc CK2α in HeLa cells after treatment with the O - GlcNAcase inhibitor TMG for 12 h . Numbers indicate relative quantification normalized for total CK2α concentrations .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O

(b) Immunoprecipitation and western blot for O-GlcNAc CK2α in HeLa cells after treatment with the O-GlcNAcase inhibitor TMG for 12 h. Numbers indicate relative quantification normalized for total CK2α concentrations.


( c ) Western blot for total CK2α protein in HeLa cells following treatment with TMG for 12 h or 16 h . Numbers indicate relative quantification normalized for loading control , mean values ( n = 4 ) shown ± s . d . Full gels corresponding to those in panels b and c are in Supplementary Figures 14 and 15 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot for total CK2α protein in HeLa cells following treatment with TMG for 12 h or 16 h. Numbers indicate relative quantification normalized for loading control, mean values (n = 4) shown ± s.d. Full gels corresponding to those in panels b and c are in Supplementary Figures 14 and 15.


Phosphorimage analysis of in vitro CK2 kinase assays with protein substrates . All reactions were performed at 30 ° C . Numbers indicate the concentration ( nM ) of product for each kinase reaction . ( a ) AHCYL2 substrate ( 25 ng μl − 1 ) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min . ( b ) NAP1L3 substrate ( 15 ng μl − 1 ) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min . ( c ) NIPBL substrate ( 50 ng μl − 1 ) with 100 μM ATP and 5 nM CK2α ± 11 nM Pin1 for 5 min . Full gels corresponding to those in panels a - c are in Supplementary Figures 19 and 25 .

O O O O O B-SUBCELLULAR O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Phosphorimage analysis of in vitro CK2 kinase assays with protein substrates. All reactions were performed at 30 °C. Numbers indicate the concentration (nM) of product for each kinase reaction. (a) AHCYL2 substrate (25 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (b) NAP1L3 substrate (15 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (c) NIPBL substrate (50 ng μl−1) with 100 μM ATP and 5 nM CK2α ± 11 nM Pin1 for 5 min. Full gels corresponding to those in panels a-c are in Supplementary Figures 19 and 25.


( B ) Levels of PIN2 phosphopeptide in our experiments . Bars represent the mean plus standard error of replicates ( 2 biological replicates for nitrate treatments at 20 min ( Col - 0 roots ) and 3 biological replicates for all other experimental conditions ) . Each independent biological replicate consisted of a pool of 4 . 500 roots collected from Arabidopsis plants grown independently under the same experimental conditions . The asterisk indicates statistically significant differences in phosphoproteomic analysis ( multiple t - test comparison without testing corrections , assuming same variance , p < 0 . 05 ) .

O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE 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-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(B) Levels of PIN2 phosphopeptide in our experiments. Bars represent the mean plus standard error of replicates (2 biological replicates for nitrate treatments at 20 min (Col-0 roots) and 3 biological replicates for all other experimental conditions). Each independent biological replicate consisted of a pool of 4.500 roots collected from Arabidopsis plants grown independently under the same experimental conditions. The asterisk indicates statistically significant differences in phosphoproteomic analysis (multiple t-test comparison without testing corrections, assuming same variance, p < 0.05).


( C ) Detection of phosphorylation PIN2 by Phos - tag Western blotting . Arabidopsis plants ( Col - 0 ) were growth in ammonium as only nitrogen source , and treated with 5 mM KNO3 or 5mM KCl as control . Total protein from roots were analyzed in SDS PAGE using Phos - tag to detect changes in phosphorylation status . Immunoblotting was performed with PIN2 antibody . Total proteins isolated from eir1 . 1 roots were used as a negative control . White and red asterisks indicate a slow - or fast - mobility band corresponding to a more or less phosphorylated PIN2 , respectively .

O O O O O B-EXP_ASSAY B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE O O 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-EXP_ASSAY B-GENEPROD O O O

(C) Detection of phosphorylation PIN2 by Phos-tag Western blotting. Arabidopsis plants (Col-0) were growth in ammonium as only nitrogen source, and treated with 5 mM KNO3 or 5mM KCl as control. Total protein from roots were analyzed in SDS PAGE using Phos-tag to detect changes in phosphorylation status. Immunoblotting was performed with PIN2 antibody. Total proteins isolated from eir1.1 roots were used as a negative control. White and red asterisks indicate a slow- or fast-mobility band corresponding to a more or less phosphorylated PIN2, respectively.


( D ) Western blot against PIN2 protein comparing nitrate treated ( KNO3 ) and control ( KCl ) condition in Arabidopsis roots for all genotypes ( eir1 - 1 mutant background was complemented with PIN2 : : PIN2wt - GFP ( PIN2wt ) , PIN2 : : PIN2S439D - GFP ( PIN2S439D phospho - mimic point mutation ) or PIN2 : : PIN2S439A - GFP ( PIN2S439A , phospho - null point mutation ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-ORGANISM B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O

(D) Western blot against PIN2 protein comparing nitrate treated (KNO3) and control (KCl) condition in Arabidopsis roots for all genotypes (eir1-1 mutant background was complemented with PIN2::PIN2wt-GFP (PIN2wt), PIN2::PIN2S439D-GFP (PIN2S439D phospho-mimic point mutation) or PIN2::PIN2S439A-GFP (PIN2S439A, phospho-null point mutation).


( A ) Primary root length of Col - 0 wild type plants or eir1 - 1 mutant plants was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 10 - 15 roots each replicate ) . The box plot shows the data within the interquartile range ( 25th and 75th percentiles ) and a solid black line represents the median . Whiskers show maximum and minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicates statistically significant difference between analyzed by unpaired , two - tailed and assuming equal variance t - test ( * * p < 0 . 01 ) .

O O O O B-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary root length of Col-0 wild type plants or eir1-1 mutant plants was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).


( B ) Number of initiating and emerging lateral roots of Col - 0 or eir1 - 1 mutant plants were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 10 - 15 roots each replicate ) . The box plot shows the data within the interquartile range ( 25th and 75th percentiles ) and a solid black line represents the median . Whiskers show maximum and minimum values no further than 1 . 5x IQR ( interquartile range ) . Asterisk indicates statistically significant difference between analyzed by unpaired , two - tailed and assuming equal variance t - test ( * * p < 0 . 01 ) .

O O O O O O O O O B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Number of initiating and emerging lateral roots of Col-0 or eir1-1 mutant plants were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).


( A ) Primary root length of the different genotypes was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 8 - 10 roots each replicate ) . The box plot shows the data within the interquartile range ( 25th and 75th percentiles ) and a solid black line represents the median . Whiskers show maximum and minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicate statistically significant difference between means analyzed by unpaired , two - tailed and assuming equal variance t - test ( * p < 0 . 05 , * * p < 0 . 01 ) .

O O O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary root length of the different genotypes was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).


( B ) Number of initiating and emerging lateral roots for all genotypes were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 8 - 10 roots each replicate ) . The box plot shows the data within the interquartile range ( 25th and 75th percentiles ) and a solid black line represents the median . Whiskers show maximum and minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicate statistically significant difference between means analyzed by unpaired , two - tailed and assuming equal variance t - test ( * p < 0 . 05 , * * p < 0 . 01 ) .

O O O O O O O O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Number of initiating and emerging lateral roots for all genotypes were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).


( A ) Confocal microscopy using Zeiss Airyscan microscope and Zeiss - blue3 . 1 software was used to visualize PIN2 in Arabidopsis roots . " e " denotes epidermis and " c " cortex . Scale bar = 20 µm . ( B - D ) Tuckey box plots show PIN2 - GFP fluorescence intensity quantification ( number of roots analyzed per experimental conditions = 8 - 10 ; arbitrary units , a . u . ) at the total cell membrane in roots ( B ) , and from epidermal ( C ) and cortex ( D ) plasma membrane in the different genotypes . The box plot shows the data within the interquartile range ( 25th and 75th percentiles ) and a solid black line represents the median . Whiskers show maximum and minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Letters denote statistically significant difference between means as determined by one - way ANOVA analysis followed by Tukey HSD post hoc test ( p < 0 . 05 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE O O O O O O B-TISSUE O O O O B-TISSUE O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy using Zeiss Airyscan microscope and Zeiss-blue3.1 software was used to visualize PIN2 in Arabidopsis roots. "e" denotes epidermis and "c" cortex. Scale bar = 20 µm. (B-D) Tuckey box plots show PIN2-GFP fluorescence intensity quantification (number of roots analyzed per experimental conditions = 8-10; arbitrary units, a.u.) at the total cell membrane in roots (B), and from epidermal (C) and cortex (D) plasma membrane in the different genotypes. The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Letters denote statistically significant difference between means as determined by one-way ANOVA analysis followed by Tukey HSD post hoc test (p < 0.05).