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 B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SUBCELLULAR 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 B-SUBCELLULAR 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 B-SUBCELLULAR 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 B-SUBCELLULAR 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 B-SUBCELLULAR 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 B-SUBCELLULAR 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 ) mRNA expressions of PINK1 in CSCs and non - CSCs . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) mRNA expressions of PINK1 in CSCs and non-CSCs. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( B - C ) Immunoblot analysis of PINK1 protein in non - CSCs and CSCs . Representative and mean ± SEM from 9 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(B-C) Immunoblot analysis of PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 9 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D - E ) Immunoblot analysis of p - PINK1 protein in non - CSCs and CSCs . Representative and mean ± SEM from 7 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(D-E) Immunoblot analysis of p-PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 7 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Immunoblot analysis of Parkin protein in non - CSCs and CSCs .

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

Immunoblot analysis of Parkin protein in non-CSCs and CSCs.


analysis of Parkin protein in non - CSCs and CSCs . Representative and mean ± SEM from 8 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

analysis of Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 8 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( H - I ) PINK1 and Parkin protein levels in mitochondrial fractions and whole cell lyses of CSCs and non - CSCs . Representative and mean ± SEM from 8 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(H-I) PINK1 and Parkin protein levels in mitochondrial fractions and whole cell lyses of CSCs and non-CSCs. Representative and mean±SEM from 8 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( J - K ) Intracellular levels of p - Parkin protein in non - CSCs and CSCs were analyzed using flow cytometry . Representative histograms and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(J-K) Intracellular levels of p-Parkin protein in non-CSCs and CSCs were analyzed using flow cytometry. Representative histograms and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Immunoblot analysis of p - Parkin protein in non - CSCs and CSCs .

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

Immunoblot analysis of p-Parkin protein in non-CSCs and CSCs.


analysis of p - Parkin protein in non - CSCs and CSCs . Representative and mean ± SEM from 11 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

analysis of p-Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 11 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( N - P ) CSCs were treated with Mdivi - 1 ( 50 μM ) and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Scale bar 20 mm . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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 B-EXP_ASSAY I-EXP_ASSAY 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

(N-P) CSCs were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Scale bar 20 mm. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( Q ) CSCs transfected with PINK1 shRNA or the control were analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(Q) CSCs transfected with PINK1 shRNA or the control were analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( R ) CSCs were treated with DAPT ( 10 μM ) and analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(R) CSCs were treated with DAPT (10 μM) and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( S ) CSCs were transfected with PINK1 shRNA or control , and analysed for activated Notch1 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-GENEPROD 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

(S) CSCs were transfected with PINK1 shRNA or control, and analysed for activated Notch1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( T ) CSCs with or without transfection of Notch1 ICD were treated with Mdivi - 1 ( 50 μM ) and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(T) CSCs with or without transfection of Notch1 ICD were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A - B ) Lysosomal DNA was identified with anti - LAMP1 and anti - DNA antibodies in non - CSCs and CSCs . Scale bar 5μm . Representative and mean ± SEM with 50 cells from 5 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A-B) Lysosomal DNA was identified with anti-LAMP1 and anti-DNA antibodies in non-CSCs and CSCs. Scale bar 5μm. Representative and mean±SEM with 50 cells from 5 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( C ) Lysosomal content of mtDNA in CSCs and non - CSCs was determined with qPCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C) Lysosomal content of mtDNA in CSCs and non-CSCs was determined with qPCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs were treated with Mdivi - 1 ( 50 μM ) and analysed for lysosomal mtDNA . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(D) CSCs were treated with Mdivi-1 (50 μM) and analysed for lysosomal mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs with or without transfection of PINK1 shRNA were analysed for lysosomal mtDNA . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E) CSCs with or without transfection of PINK1 shRNA were analysed for lysosomal mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F - G ) Protein expressions of TLR9 and MyD88 in CSCs and non - CSCs . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(F-G) Protein expressions of TLR9 and MyD88 in CSCs and non-CSCs. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( H ) TLR9 proteins were immunoprecipitated from CSCs and non - CSCs and analyzed for the binding load of mtDNA . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(H) TLR9 proteins were immunoprecipitated from CSCs and non-CSCs and analyzed for the binding load of mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( I ) CSCs were transfected with mtDNA and analysed for expressions of TLR9 plus MyD88 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(I) CSCs were transfected with mtDNA and analysed for expressions of TLR9 plus MyD88. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( J - K ) CSCs treated with EtBr ( 50 ng / ml ) were analysed for lysosomal mtDNA , TLR9 , and MyD88 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(J-K) CSCs treated with EtBr (50 ng/ml) were analysed for lysosomal mtDNA, TLR9, and MyD88. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( L ) CSCs transfected with DNase II expression vector or the control were analyzed for lysosomal DNA plus MyD88 protein . Mean ± SEM from 6 patients . ( M ) CSCs transfected with DNase II shRNA or the control were analyzed for lysosomal DNA plus MyD88 protein . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(L) CSCs transfected with DNase II expression vector or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients. (M) CSCs transfected with DNase II shRNA or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) or control and analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) CSCs were stimulated with CpG ODN (10 μg/ml) or control and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


CSCs transfected with mtDNA were analyzed for sphere formation Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-SUBCELLULAR 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 O O O O O O O O O O O O O O

CSCs transfected with mtDNA were analyzed for sphere formation Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


CSCs transfected with mtDNA were analyzed for tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-SUBCELLULAR 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

CSCs transfected with mtDNA were analyzed for tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs were transfected with 2 different TLR9 shRNAs or the control , and analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(D) CSCs were transfected with 2 different TLR9 shRNAs or the control, and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E - F ) CSCs transfected with DNase II expression vector , DNase II shRNA , or the control was analyzed for sphere formation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E-F) CSCs transfected with DNase II expression vector, DNase II shRNA, or the control was analyzed for sphere formation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( G ) CpG promoted tumor growth of PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O 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) CpG promoted tumor growth of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( H ) TLR9 inhibitor chloroquine abrogated the effect of CpG on tumor growth in PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O 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) TLR9 inhibitor chloroquine abrogated the effect of CpG on tumor growth in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( I ) CpG treatment increased the frequency of CD133 - expressing CSCs in PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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-ORGANISM O O O O O 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) CpG treatment increased the frequency of CD133-expressing CSCs in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( J ) Chloroquine impeded the function of CpG in elevating CD133 - expressing CSCs of PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(J) Chloroquine impeded the function of CpG in elevating CD133-expressing CSCs of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) CSCs transfected with TLR9 shRNA or control were analysed for protein expressions of activated Notch1 plus HES1 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) CSCs transfected with TLR9 shRNA or control were analysed for protein expressions of activated Notch1 plus HES1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( B ) CSCs treated with or without EtBr ( 50 ng / ml ) were analyzed for activated Notch1 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-SMALL_MOLECULE 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

(B) CSCs treated with or without EtBr (50 ng/ml) were analyzed for activated Notch1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( C ) CSCs transfected with DNase II expression vector or control were analyzed for activated Notch1 expression . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C) CSCs transfected with DNase II expression vector or control were analyzed for activated Notch1 expression. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs with or without CpG treatment were analysed for interaction between TLR9 and Notch1 . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(D) CSCs with or without CpG treatment were analysed for interaction between TLR9 and Notch1. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs were treated with CpG ODN ( 10 μg / ml ) in the presence or absence of DAPT ( 10 μM ) , and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E) CSCs were treated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F ) CSCs were transfected with 2 different Notch1 shRNAs or control , treated with CpG ODN ( 10 μg / ml ) , and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(F) CSCs were transfected with 2 different Notch1 shRNAs or control, treated with CpG ODN (10 μg/ml), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


PDX chimeras treated by CpG with or without DAPT were analysed for tumor growth Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

PDX chimeras treated by CpG with or without DAPT were analysed for tumor growth Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


PDX chimeras treated by CpG with or without DAPT were analysed for CD133 - expressing CSCs . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

PDX chimeras treated by CpG with or without DAPT were analysed for CD133-expressing CSCs. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) CSCs and non - CSCs were analyzed for mitochondrial metabolism using seahorse assay . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method ( A , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) CSCs and non-CSCs were analyzed for mitochondrial metabolism using seahorse assay. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method (A, **p < 0.01, ***p < 0.001.


( B ) CSCs were treated with 2 - DG ( 10 mM ) or Malonate ( 10 mM ) , and assayed for sphere formation plus tumorgenicity . Mean ± SEM from 6 patients . Data information : ANOVA test with Turkey ' s method ( B ) . * * p < 0 . 01 , * * * p < 0 . 001 .

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

(B) CSCs were treated with 2-DG (10 mM) or Malonate (10 mM), and assayed for sphere formation plus tumorgenicity. Mean±SEM from 6 patients. Data information: ANOVA test with Turkey's method (B). **p < 0.01, ***p < 0.001.


( C ) CSCs treated with DAPT ( 10 μM ) were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C) CSCs treated with DAPT (10 μM) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( D ) CSCs transfected with Notch1 shRNA or control shRNA were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

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

(D) CSCs transfected with Notch1 shRNA or control shRNA were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( E ) CSCs treated with EtBr ( 50 ng / ml ) were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E) CSCs treated with EtBr (50 ng/ml) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( F ) CSCs transfected with DNase II expression vector or control were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

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

(F) CSCs transfected with DNase II expression vector or control were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( G ) CSCs treated with chloroquine ( 50 μM ) were analyzed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

(G) CSCs treated with chloroquine (50 μM) were analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( A ) CSCs stimulated with or without CpG ODN ( 10 μg / ml ) were analyzed for expression of phosphor - AMPKα . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(A) CSCs stimulated with or without CpG ODN (10 μg/ml) were analyzed for expression of phosphor-AMPKα. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( B ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in the presence or absence of DAPT ( 10 μM ) , and analyzed for phosphor - AMPKα . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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-SMALL_MOLECULE 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 O O O O O 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) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( C ) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN ( 10 μg / ml ) and analyzed for phosphor - AMPKα . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O 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 O O O O O 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) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN (10 μg/ml) and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( D ) CSCs were treated with DAPT ( 10 μM ) in the presence or absence of A769662 ( 10 μM ) , and analysed for mitochondrial OCR . Ctrl represents CSCs without any treatment . Mean ± SEM from 6 patients . Data information : ANOVA with Turkey method ( D ) , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(D) CSCs were treated with DAPT (10 μM) in the presence or absence of A769662 (10 μM), and analysed for mitochondrial OCR. Ctrl represents CSCs without any treatment. Mean±SEM from 6 patients. Data information: ANOVA with Turkey method (D), **p < 0.01, ***p < 0.001, ****p < 0.0001.


( E ) CSCs were stimulated with or without CpG ODN ( 10 μg / ml ) and analyzed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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-SUBCELLULAR 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

(E) CSCs were stimulated with or without CpG ODN (10 μg/ml) and analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( F ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in the presence or absence of Compound C ( 10 μM ) and analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(F) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of Compound C (10 μM) and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( G ) CSCs were transfected with 2 different AMPKα shRNA or the control , stimulated with CpG ODN ( 10 μg / ml ) , and analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(G) CSCs were transfected with 2 different AMPKα shRNA or the control, stimulated with CpG ODN (10 μg/ml), and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( A ) CSCs transfected with or without Notch1 ICD were analyzed for AMP / ATP ratio plus LKB1 phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) CSCs transfected with or without Notch1 ICD were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( B ) CSCs treated with or without DAPT ( 10 μM ) were analyzed for AMP / ATP ratio plus LKB1 phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(B) CSCs treated with or without DAPT (10 μM) were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( C ) CSCs treated with or without CpG ODN ( 10 μg / ml ) were analysed for interactions between Notch1 and AMPK . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C) CSCs treated with or without CpG ODN (10 μg/ml) were analysed for interactions between Notch1 and AMPK. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs transfected with LKB1 shRNA （ right ） or the control ( left ) were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O 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 B-GENEPROD 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

(D) CSCs transfected with LKB1 shRNA（right）or the control (left) were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs transfected with TAK1 shRNA or the control were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E) CSCs transfected with TAK1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F ) CSCs transfected with LAMTOR1 shRNA or the control were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

(F) CSCs transfected with LAMTOR1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( G ) CSCs transfected with AXIN1 shRNA or the control were treated with CpG ODN ( 10 μg / ml ) and analysed for AMPK phosphorylation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

(G) CSCs transfected with AXIN1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) NSCLC PDX models treated with or without chemotherapy for the first 2 weeks were analyzed for tumor growth at the indicated time . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O B-DISEASE 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 O O O O O 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) NSCLC PDX models treated with or without chemotherapy for the first 2 weeks were analyzed for tumor growth at the indicated time. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( B ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 weeks . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O B-GENEPROD O O O O B-DISEASE 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

(B) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( C - E ) Flow cytometric analyses of protein expressions of TLR9 , activated Notch1 , and phosphor - AMPKα in cancer cells from NSCLC xenografts treated with or without chemotherapy after 4 weeks . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(C-E) Flow cytometric analyses of protein expressions of TLR9, activated Notch1, and phosphor-AMPKα in cancer cells from NSCLC xenografts treated with or without chemotherapy after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( F ) NSCLC PDX models were treated with chemotherapy alone or together with the indicated inhibitors , respectively , for the first 2 weeks . Tumor volume was determined at the indicated time . Mean ± SEM from 6 experiments . ( G ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with the indicated inhibitors after 4 weeks . Mean ± SEM from 6 experiments . Data information : ANOVA test with Turkey ' s method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O B-DISEASE 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 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 B-DISEASE 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

(F) NSCLC PDX models were treated with chemotherapy alone or together with the indicated inhibitors, respectively, for the first 2 weeks. Tumor volume was determined at the indicated time. Mean±SEM from 6 experiments. (G) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with the indicated inhibitors after 4 weeks. Mean±SEM from 6 experiments. Data information: ANOVA test with Turkey's method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


C - F ( C , E ) Immunofluorescence labelling with an antibody that recognizes both full - length and cleaved Xkr8 in developing ( P0 , P8 ) and adult S1 cortex and hippocampus . ( D , F ) Immunofluorescence labelling with an antibody recognizing full - length uncleaved Xkr8 in developing ( P0 , P8 ) and adult S1 cortex and hippocampus . Loss of Xkr8 immunosignal in double transgenic Xkr8flx / flx ; Emx1 : : Cre animals ( Xkr8 cKO ) is observed with both Xkr8 antibodies ( C - F ) .

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C-F (C, E) Immunofluorescence labelling with an antibody that recognizes both full-length and cleaved Xkr8 in developing (P0, P8) and adult S1 cortex and hippocampus. (D, F) Immunofluorescence labelling with an antibody recognizing full-length uncleaved Xkr8 in developing (P0, P8) and adult S1 cortex and hippocampus. Loss of Xkr8 immunosignal in double transgenic Xkr8flx/flx;Emx1::Cre animals (Xkr8 cKO) is observed with both Xkr8 antibodies (C-F).


A , B Organotypic slices of Xkr8 WT and cKO hippocampus with Thy1 : : GFP neurons ( green ) were labelled with PtdSer - binding fluorescently tagged Annexin V ( magenta ) at 16 - 19 DIV . The fluorescence of bound Annexin V was quantified on ImageJ by measuring the integrated density of fluorescent structures within the same volume for each slice and expressing the value as fluorescence units ( f . u . ; two - tailed Student ' s t - test , each dot represents an individual slice preparation , n = 4 ) .

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A, B Organotypic slices of Xkr8 WT and cKO hippocampus with Thy1::GFP neurons (green) were labelled with PtdSer-binding fluorescently tagged Annexin V (magenta) at 16-19 DIV. The fluorescence of bound Annexin V was quantified on ImageJ by measuring the integrated density of fluorescent structures within the same volume for each slice and expressing the value as fluorescence units (f.u.; two-tailed Student's t-test, each dot represents an individual slice preparation, n = 4).


C Immunofluorescence labelling of active caspase - 3 in developing brain from P8 to P28 in Xkr8 WT and Xkr8 cKO brains was quantified and compared by nested design and mixed model ANOVA ( F ( 2 , 30 ) = 0 . 86 , p = 0 . 433 ) , each dot represents an individual mouse ( n = 6 per age group )

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C Immunofluorescence labelling of active caspase-3 in developing brain from P8 to P28 in Xkr8 WT and Xkr8 cKO brains was quantified and compared by nested design and mixed model ANOVA (F(2,30) = 0.86, p = 0.433), each dot represents an individual mouse (n = 6 per age group)


A - C The size and density of axonal boutons on Thy1 : : GFP axons ( green ) in Xkr8 WT and cKO animals ( nested design and mixed model ANOVA , each dot represents an individual mouse , n = 6 per age and genotype group ) .

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A-C The size and density of axonal boutons on Thy1::GFP axons (green) in Xkr8 WT and cKO animals (nested design and mixed model ANOVA, each dot represents an individual mouse, n = 6 per age and genotype group).


D , E Dendritic spine morphology ( D ) and density ( E ) of Thy1 : : GFP + CA1 neurons in Xkr8 WT and cKO animals from P8 to P28 ( nested design and mixed model ANOVA , each dot represents an individual mouse , n = 5 - 6 per age and genotype group ) .

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D, E Dendritic spine morphology (D) and density (E) of Thy1::GFP+ CA1 neurons in Xkr8 WT and cKO animals from P8 to P28 (nested design and mixed model ANOVA, each dot represents an individual mouse, n = 5-6 per age and genotype group).


J - L The density of cortical ( vGluT1 + , J ) and thalamic ( vGluT2 + , K ) and the ratio of vGluT1 + and vGluT2 + inputs ( L ) in Xkr8 WT and cKO brains ( nested design and mixed model ANOVA , each dot represents an individual mouse , n = 6 per age and genotype group ) .

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J-L The density of cortical (vGluT1+, J) and thalamic (vGluT2+, K) and the ratio of vGluT1+ and vGluT2+ inputs (L) in Xkr8 WT and cKO brains (nested design and mixed model ANOVA, each dot represents an individual mouse, n = 6 per age and genotype group).


A - F The immunolabelling of internalized pan - axonal material ( A , B ) , pre - synaptic vGluT1 particles ( C , D ) and post - synaptic PSD95 particles ( E , F ) within Iba1 + microglia in P8 cortex of Xkr8 WT and Xkr8 cKO mice was quantified per individual 3D reconstructed microglial cell . The total volume of internalized particles was normalized to microglial cell volume and compared by nested design and mixed model ANOVA , each dot represents an individual mouse ( n = 3 per genotype group ) . Data presented as mean ± SEM ; * * p < 0 . 01 , * * * * p < 0 . 0001 ; scale bar 10 µm , grid cell 10 µm .

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A-F The immunolabelling of internalized pan-axonal material (A, B), pre-synaptic vGluT1 particles (C, D) and post-synaptic PSD95 particles (E, F) within Iba1+ microglia in P8 cortex of Xkr8 WT and Xkr8 cKO mice was quantified per individual 3D reconstructed microglial cell. The total volume of internalized particles was normalized to microglial cell volume and compared by nested design and mixed model ANOVA, each dot represents an individual mouse (n = 3 per genotype group). Data presented as mean ± SEM; ** p < 0.01, **** p < 0.0001; scale bar 10 µm, grid cell 10 µm.


A , B Corticospinal axons of the axonal bundles of pyramidal tracts in medulla of P8 and P28 Xkr8 WT and cKO mice were visualized by Palmgren staining and quantified per each bundle , delineated by a dashed line ( Mann - Whitney test , n = 6 mice per genotype , the data are presented as median and quartiles ) .

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A, B Corticospinal axons of the axonal bundles of pyramidal tracts in medulla of P8 and P28 Xkr8 WT and cKO mice were visualized by Palmgren staining and quantified per each bundle, delineated by a dashed line (Mann-Whitney test, n = 6 mice per genotype, the data are presented as median and quartiles).


To label callosal projections in vivo , cholera toxin subunit B ( CTB ) was injected stereotactically into corpus callosum of Xkr8 WT and cKO brains at P30 - 32 . Back - labelled neurons were quantified in L4 of primary ( S1 ) and secondary ( S2 ) somatosensory cortex .

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To label callosal projections in vivo, cholera toxin subunit B (CTB) was injected stereotactically into corpus callosum of Xkr8 WT and cKO brains at P30-32. Back-labelled neurons were quantified in L4 of primary (S1) and secondary (S2) somatosensory cortex.


Xkr8 cKO neurons had increased mean sEPSC amplitude Data were analyzed by two - tailed Student ' s t - test ; n = 7 - 14 neurons per genotype .

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Xkr8 cKO neurons had increased mean sEPSC amplitude Data were analyzed by two-tailed Student's t-test ; n = 7-14 neurons per genotype.


F - H Evoked excitatory postsynaptic currents ( eEPSC ) of Xkr8 cKO neurons showed a trend to decrease ( p = 0 . 08 ) ( F , G ) and the ratio of eEPSC and sEPSC amplitudes was lower in Xkr8 knockout neurons ( H ) . two - way repeated measures ANOVA ( H ) ; n = 7 - 14 neurons per genotype .

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F-H Evoked excitatory postsynaptic currents (eEPSC) of Xkr8 cKO neurons showed a trend to decrease (p = 0.08) (F, G) and the ratio of eEPSC and sEPSC amplitudes was lower in Xkr8 knockout neurons (H). two-way repeated measures ANOVA (H); n = 7-14 neurons per genotype.


A Brain regions with significantly increased functional connectivity in Xkr8 cKO mice compared to Xkr8 WT mice as identified by seed - based mapping of rsfMRI networks ( t - test , p < 0 . 05 FWE cluster - corrected , with cluster - defining threshold of t24 > 2 . 07 , p < 0 . 05 ; Colored regions represent the areas of the brain exhibiting increased functional connectivity in Xkr8 cKO compared to Xkr8 WT brains ; color scale represents the level of functional correlation ; abbreviations specify anatomical designations of identified regions . ACA : anterior cingulate cortex , AI : agranular insular area , AUD : auditory areas MOp : primary motor area , MOs : secondary motor area , PL : prelimbic area , SSp : primary somatosensory area , STR : striatum , STRd : striatum dorsal region , SUB : subiculum , TH : thalamus , VIS : visual areas , VISC : visceral area .

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A Brain regions with significantly increased functional connectivity in Xkr8 cKO mice compared to Xkr8 WT mice as identified by seed-based mapping of rsfMRI networks (t-test, p < 0.05 FWE cluster-corrected, with cluster-defining threshold of t24 > 2.07, p < 0.05; Colored regions represent the areas of the brain exhibiting increased functional connectivity in Xkr8 cKO compared to Xkr8 WT brains; color scale represents the level of functional correlation; abbreviations specify anatomical designations of identified regions. ACA: anterior cingulate cortex, AI: agranular insular area, AUD: auditory areas MOp: primary motor area, MOs: secondary motor area, PL: prelimbic area, SSp: primary somatosensory area, STR: striatum, STRd: striatum dorsal region, SUB: subiculum, TH: thalamus, VIS: visual areas, VISC: visceral area.


( B ) Quantification by micro - CT of mean tumor volumes in KP mice derived from 50 randomly - selected tumors at 8 , 12 , 16 , and 20 weeks post - infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 18 mice , 21 mice , 21 mice , and 20 mice , respectively ) .

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(B) Quantification by micro-CT of mean tumor volumes in KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 18 mice, 21 mice, 21 mice, and 20 mice, respectively).


( C ) Quantification by micro - CT of total tumor volume per mouse in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 at 8 , 12 , 16 , and 20 weeks post - infection .

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(C) Quantification by micro-CT of total tumor volume per mouse in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection.


( D ) Depictions and quantitation of total tumor burden ( total tumor area / total lung area ) in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 at 21 weeks after infection . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(D) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 21 weeks after infection. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( E ) Distribution of histological tumor grades in KP mice after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 50 tumors each ) at 21 weeks after infection . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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(E) Distribution of histological tumor grades in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each) at 21 weeks after infection. For bar charts, data presented as means with error bars representing standard deviations (SDs).


( F ) Assessment of the mitotic index of tumor cells by phosphorylated - histone H3 ( pHH3 ) - positive nuclei density in KP mice LUAD tumors at 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 ( n = 50 tumors each ) . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(F) Assessment of the mitotic index of tumor cells by phosphorylated-histone H3 (pHH3)-positive nuclei density in KP mice LUAD tumors at 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( G ) Nf1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(G) Nf1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( H ) Representative hematoxylin and eosin ( H & E ) and p - Fak1 immunohistochemical ( IHC ) staining of LUAD tumor sections 21 weeks after infection with control sgTom ( grade 1 depicted ) , sgNf1 . 1 ( grade 3 depicted ) , sgNf1 . 2 ( grade 3 depicted ) , or sgNf1 . 3 ( grade 3 depicted ) ( n = 50 tumors each ) . H & E scale bars ( low - magnification top row = 100 μm , high - magnification bottom row = 250 μm ) ; p - Fak1 IHC scale bars ( low - magnification top row = 250 μm , high - magnification bottom row = 500 μm ) .

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(H) Representative hematoxylin and eosin (H&E) and p-Fak1 immunohistochemical (IHC) staining of LUAD tumor sections 21 weeks after infection with control sgTom (grade 1 depicted), sgNf1.1 (grade 3 depicted), sgNf1.2 (grade 3 depicted), or sgNf1.3 (grade 3 depicted) (n = 50 tumors each). H&E scale bars (low-magnification top row = 100 μm, high-magnification bottom row = 250 μm); p-Fak1 IHC scale bars (low-magnification top row = 250 μm, high-magnification bottom row = 500 μm).


( I ) Quantification of p - Fak1 IHC signals in LUAD tumor sections 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(I) Quantification of p-Fak1 IHC signals in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( J ) Quantification of p - Fak1 IHC signals in sgNf1 . 3 LUAD tumor sections analyzed by tumor grade . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(J) Quantification of p-Fak1 IHC signals in sgNf1.3 LUAD tumor sections analyzed by tumor grade. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( K ) Quantification of Psat1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom , sgNf1 . 1 , sgNf1 . 2 , or sgNf1 . 3 . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(K) Quantification of Psat1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( L ) Quantification of Psat1 mRNA expression in sgNf1 . 3 LUAD tumor sections analyzed by tumor grade . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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(L) Quantification of Psat1 mRNA expression in sgNf1.3 LUAD tumor sections analyzed by tumor grade. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( A ) Western blotting analysis of p53 expression in various p53 / TP53 - WT cell lines incubated with the DNA - intercalating agent doxorubicin ( DOXO , 0 . 2 µg / ml for 6 hours ) to induce p53 stabilization ( n = 3 biological replicates ) .

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(A) Western blotting analysis of p53 expression in various p53/TP53-WT cell lines incubated with the DNA-intercalating agent doxorubicin (DOXO, 0.2 µg/ml for 6 hours) to induce p53 stabilization (n = 3 biological replicates).


( B , C ) Western blotting analysis confirming NF1 upregulation and p - FAK1 downregulation in ( B ) DOXO - treated PDKN1 cells and ( C ) DOXO - treated PDKN2 cells transduced with PGK - NF1 ( n = 3 biological replicates ) .

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(B, C) Western blotting analysis confirming NF1 upregulation and p-FAK1 downregulation in (B) DOXO-treated PDKN1 cells and (C) DOXO-treated PDKN2 cells transduced with PGK-NF1 (n = 3 biological replicates).


( D ) Western blotting analysis confirming NF1 downregulation and p - FAK1 upregulation in DOXO - treated SW1573 cells transduced with sgNf1 . 3 ( n = 3 biological replicates ) .

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

(D) Western blotting analysis confirming NF1 downregulation and p-FAK1 upregulation in DOXO-treated SW1573 cells transduced with sgNf1.3 (n = 3 biological replicates).


( E , F ) Western blotting analysis confirming Nf1 downregulation and p - Fak1 upregulation in ( E ) DOXO - treated LKR10 and ( F ) DOXO - treated LKR13 clones transduced with sgNf1 . 3 ( n = 3 biological replicates ) .

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

(E, F) Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates).


( G ) FAK1 target gene expression in DOXO - treated PDKN1 cells transduced with PGK - control or PGK - NF1 ( n = 3 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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

(G) FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( H , I ) Subcutaneous tumor volumes of DOXO - treated PDKN1 and PDKN2 cells transduced with PGK - control or PGK - NF1 ( n = 18 each ) .

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

(H, I) Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n=18 each).


( J - L ) Subcutaneous tumor volumes of ( J ) SW1573 , ( K ) LKR10 , and ( L ) LKR13 cells transfected with sgTom or sgNf1 . 3 ( n = 18 each ) .

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

(J-L) Subcutaneous tumor volumes of (J) SW1573, (K) LKR10, and (L) LKR13 cells transfected with sgTom or sgNf1.3 (n=18 each).


( M ) Depictions and quantitation of KrasG12D / + ; p53 + / + ( K - only ) autochthonous tumor burden ( total tumor area / total lung area ) in pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

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

(M) Depictions and quantitation of KrasG12D/+; p53+/+ (K-only) autochthonous tumor burden (total tumor area/total lung area) in pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( N ) Analysis of tumor grades in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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

(N) Analysis of tumor grades in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( O ) Quantification of Ki - 67 - positive nuclei per mm2 in K - only autochthonous tumors derived from pSECC - sgTom ( n = 18 mice ) or pSECC - sgNf1 . 3 mice ( n = 21 mice ) . For boxplots , whiskers indicate the minimum and maximum values , the upper and lower perimeters represent the first and third quartiles , the midline represents the median value , and the x symbol represents the mean .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(O) Quantification of Ki-67-positive nuclei per mm2 in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.


( A ) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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

(A) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( B ) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count ( n = 4 biological replicates ) . For bar charts , data presented as means with error bars representing standard deviations ( SDs ) .

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

(B) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).


( C ) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell - titer glo ( relative luminescent units ) ( n = 4 biological replicates ) .

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

(C) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units) (n = 4 biological replicates).


( D ) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2 . 0 or 0 . 5 mM glutamine ( n = 4 biological replicates ) .

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 B-SMALL_MOLECULE O O O O O O O O

(D) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates).


( E ) Two patient - derived KRAS ; NF1 - mutant LUAD cell lines ( PDKN1 , PDKN2 ) , as well as the control patient - derived KRAS - mutant / NF1 - WT LUAD cell line ( PDK ) , were passaged for 14 population doublings prior to collection . The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines ( relative to the PDK control ) , with the degree of upregulation depicted by color as indicated in the legend .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-DISEASE O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-DISEASE 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 O O O O O O O O O O O O O O O O O O O O

(E) Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of upregulation depicted by color as indicated in the legend.