Figure 5E , F . Abundance of ARF19 ( E ) and LBD16 ( F ) transcripts in RNAseq samples . mRNA accumulation in response to auxin is comparable for both , whether TOR is knocked down . n = 3 biological replicates . G , H . Relative expression levels ( normalized to ACTIN ) of ARF19 ( G ) and LBD16 ( H ) measured by RT - qPCR upon TOR activity inhibition with AZD8055 . Comparison between samples was performed by one - way ANOVA . Different letters indicate significant differences based on a post - hoc Tukey HSD Test ( n = 5 biological replicates , α = 0 . 05 ) . L , M . Total lysates prepared from lateral roots treated or not with IAA and AZD were fractionated through sucrose gradients , and the relative redistribution ( percentage of total ) of ACTIN , ARF7 , ARF19 , and LARP1 mRNAs in every 8 fractions were studied by RT - qPCR analysis . ( L ) Polysome profiles . 40S , small ribosomal subunit ; 60S , large ribosomal subunit ; 80S , mono - ribosome ; polysomes , polyribosomes . AU is arbitrary units of RNA absorbance at A260 nanometers . ( M ) RT - qPCR analysis of mRNA redistribution through a sucrose gradient ( 8 fractions collected ) . Translation efficiency was computed as a percentage of mRNA in non - polysome fractions ( 40 / 60 / 80S ; fractions 1 - 3 ) against both lights ( fractions 4 - 5 ) and heavy polysomes ( fractions 6 - 8 ) . The plot is representative of three independently performed experiments with similar results .

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

Figure 5E, F. Abundance of ARF19 (E) and LBD16 (F) transcripts in RNAseq samples. mRNA accumulation in response to auxin is comparable for both, whether TOR is knocked down. n=3 biological replicates.G, H. Relative expression levels (normalized to ACTIN) of ARF19 (G) and LBD16 (H) measured by RT-qPCR upon TOR activity inhibition with AZD8055. Comparison between samples was performed by one-way ANOVA. Different letters indicate significant differences based on a post-hoc Tukey HSD Test (n=5 biological replicates, α = 0.05).L, M. Total lysates prepared from lateral roots treated or not with IAA and AZD were fractionated through sucrose gradients, and the relative redistribution (percentage of total) of ACTIN, ARF7, ARF19, and LARP1 mRNAs in every 8 fractions were studied by RT-qPCR analysis. (L) Polysome profiles. 40S, small ribosomal subunit; 60S, large ribosomal subunit; 80S, mono-ribosome; polysomes, polyribosomes. AU is arbitrary units of RNA absorbance at A260 nanometers. (M) RT-qPCR analysis of mRNA redistribution through a sucrose gradient (8 fractions collected). Translation efficiency was computed as a percentage of mRNA in non-polysome fractions (40/60/80S; fractions 1-3) against both lights (fractions 4-5) and heavy polysomes (fractions 6-8). The plot is representative of three independently performed experiments with similar results.


Figure 6A . Abundance of WOX11 transcripts in RNAseq samples . mRNA accumulation in response to auxin is elevated when TOR is knocked down . n = 3 biological replicates . B . Distribution of GUS - staining in UB10pro > > amiR - TOR / WOX11pro : : GUS seedlings 6 days after transfer to 10µM Est . The proportion of root bends with the depicted phenotype is indicated . C . The arf7 / arf19 mutant shows defective LR initiation . D . Formation of roots ( arrows ) close to the wound site of arf7 / arf19 primary roots 6 days after excision ( DAE ) . E . Blocking TOR - activity via exposure to 10µM AZD8055 ( AZD ) resulted in the loss of rooting capability on the wound site of arf7 / arf19 primary roots at 6 DAE . SF , G . Close - ups indicate root formation location or absence of root formation on ctrl media and AZD - containing media , respectively , in arf7 / arf19 primary roots at 6 DAE . The proportion of root wound sites with the depicted phenotype is indicated .

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

Figure 6A. Abundance of WOX11 transcripts in RNAseq samples. mRNA accumulation in response to auxin is elevated when TOR is knocked down. n=3 biological replicates.B. Distribution of GUS-staining in UB10pro>>amiR-TOR/WOX11pro::GUS seedlings 6 days after transfer to 10µM Est. The proportion of root bends with the depicted phenotype is indicated.C. The arf7/arf19 mutant shows defective LR initiation. D. Formation of roots (arrows) close to the wound site of arf7/arf19 primary roots 6 days after excision (DAE). E. Blocking TOR-activity via exposure to 10µM AZD8055 (AZD) resulted in the loss of rooting capability on the wound site of arf7/arf19 primary roots at 6 DAE. SF, G. Close-ups indicate root formation location or absence of root formation on ctrl media and AZD-containing media, respectively, in arf7/arf19 primary roots at 6 DAE. The proportion of root wound sites with the depicted phenotype is indicated.


Figure 1Growth analysis of wild type Xcc8004 , EPS deficient mutants ( B5 and B12 ) , ndvB and ndvB ( pHMJ ) grown in PS medium ( A ) ; PS medium containing 50 µm extracellularion chelator , diethylenetriaminepentaacetic acid ( DETAPAC ) ( B ) ; or supplemented with either with ( C ) secreted glucan ( 0 . 5 mg / ml ) ; ( D ) xanthan ( 0 . 5 mg / ml ) ; ( E ) FeSO4 ( 40µM ) ; and ( F ) MnCl2 ( 40µM ) . Data shown are mean ± SD ( n = 3 ) . From here onward , until not mentioned , the ' n ' indicates three biological replicates .

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

Figure 1Growth analysis of wild type Xcc8004, EPS deficient mutants (B5 and B12), ndvB and ndvB (pHMJ) grown in PS medium (A); PS medium containing 50 µm extracellularion chelator, diethylenetriaminepentaacetic acid (DETAPAC) (B); or supplemented with either with (C) secreted glucan (0.5 mg/ml); (D) xanthan (0.5 mg/ml); (E) FeSO4 (40µM); and (F) MnCl2 (40µM). Data shown are mean ± SD (n=3). From here onward, until not mentioned, the 'n' indicates three biological replicates.


Figure 2 ( A ) MALDI - MS analysis on the 2 . 1 RT eluted fraction from the wild type Xcc8004 EPS . Shown one quasimolecular ion at m / z ( mass - to - charge ratio ) 2632 . 8 which matched with the calculated mass for an [ M + Na ] + ion based on an unsubstituted 16 - member cyclic glucan ( inset ) of Xanthomonas campestris . Peaks with m / z of below 1100 were matrix derived peaks after comparing the MALDI - MS of matrix only control ( See also Fig . EV3A - B ; Fig . EV4D ) . ( B ) Top : GC - MS analysis of acid hydrolyzed and BSTFA - derivatized , 2 . 1 RT eluted fraction from the EPS of wild type Xcc strain . Bottom : matching spectrum from NIST database , corresponding to glucopyranose pentakis - O - trimethyl silyl ( See also Fig . EV3C ) . ( C ) Quantification of total EPS , secreted ( S ) and periplasmic ( P ) glucan by phenol - sulfuric acid colorimetric assay from 2 - day - old cultures . Secreted xanthan and glucan was isolated from the cell - free culture supernatant by addition of KCl [ 1 % ( w / v ) ] and 2 volumes of ethanol , to precipitate xanthan . Supernatant containing cyclic - β - ( 1 , 2 ) was purified by BioGel - P4 size - exclusion chromatography . To isolate periplasmic glucan , cell pellet was treated with 1 % trichloroacetic acid ( TCA ) to release periplasmic glucans , purified by BioGel - P4 size - exclusion chromatography . Data shown are mean ± SD ( n = 3 ) .

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

Figure 2(A) MALDI-MS analysis on the 2.1 RT eluted fraction from the wild type Xcc8004 EPS. Shown one quasimolecular ion at m/z (mass-to-charge ratio) 2632.8 which matched with the calculated mass for an [M+Na] + ion based on an unsubstituted 16-member cyclic glucan (inset) of Xanthomonas campestris. Peaks with m/z of below 1100 were matrix derived peaks after comparing the MALDI-MS of matrix only control (See also Fig. EV3A-B; Fig. EV4D).(B) Top: GC-MS analysis of acid hydrolyzed and BSTFA-derivatized, 2.1 RT eluted fraction from the EPS of wild type Xcc strain. Bottom: matching spectrum from NIST database, corresponding to glucopyranose pentakis-O-trimethyl silyl (See also Fig. EV3C).(C) Quantification of total EPS, secreted (S) and periplasmic (P) glucan by phenol-sulfuric acid colorimetric assay from 2-day-old cultures. Secreted xanthan and glucan was isolated from the cell-free culture supernatant by addition of KCl [1% (w/v)] and 2 volumes of ethanol, to precipitate xanthan. Supernatant containing cyclic-β-(1, 2) was purified by BioGel-P4 size-exclusion chromatography. To isolate periplasmic glucan, cell pellet was treated with 1% trichloroacetic acid (TCA) to release periplasmic glucans, purified by BioGel-P4 size-exclusion chromatography. Data shown are mean ± SD (n=3).


Figure 3 ( A ) Iron ( Fe2 + ) - binding potential of different polysaccharide [ used at 5 . 56 micromole ( glucose equivalent ) ] using the Fe2 + - ferrozine colorimetric assay . Lipopolysacchrides ( LPS ; E . coli 0111 : B4 ) , mannan ( Saccharomyces cerevisiae ) , isolated and commercial ( purchased from Sigma Aldrich ) xanthan , extracellular glucan from Xcc . Data shown are mean ± SD ( n = 3 ) . ( B ) Colorimetric iron - binding assay . Glucan from diverse source binds Fe2 + iron . Extracellular ( ECG ) or periplasmic ( PPG ) glucan isolated from the wild type Xcc8004 or the ndvB ( pHM1J ) ; commercial glucan from yeast , barley and Euglena purchased from Sigma Aldrich . Glucans from various sources were incubated with FeSO4 at room temperature for 15 min , amount of free iron was determined using the standard concentration of ferrozine - Fe2 + complex . ( C ) Iron - binding measured by Inductively Coupled Plasma - Optical Emission Spectrometry ( ICP - OES ) . Xanthan and various glucans were mixed with 2 mM FeSO4 , dialysed against deionized water before determination of Fe2 + content by ICP - OES . Data shown are mean ± SD ( n ≥ 3 ) . * * indicates p - value < 0 . 01 and * * * indicates p - value < 0 . 001 by paired student t - test . ( D ) Specificity of glucan - Fe2 + interaction . Purified Xcc β - ( 1 , 2 ) - glucan was incubated with 100 µM FeSO4 and increasing concentrations of Ca2 + , Co2 + , Ni2 + , Mn2 + , Mg2 + or Zn2 + , and free or unbound iron was measured by Fe2 + - ferrozine colorimetric assay . Data shown are mean ± SD ( n = 3 ) .

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

Figure 3(A) Iron (Fe2+)-binding potential of different polysaccharide [used at 5.56 micromole (glucose equivalent)] using the Fe2+-ferrozine colorimetric assay. Lipopolysacchrides (LPS; E. coli 0111:B4), mannan (Saccharomyces cerevisiae), isolated and commercial (purchased from Sigma Aldrich) xanthan, extracellular glucan from Xcc. Data shown are mean ± SD (n=3).(B) Colorimetric iron-binding assay. Glucan from diverse source binds Fe2+ iron. Extracellular (ECG) or periplasmic (PPG) glucan isolated from the wild type Xcc8004 or the ndvB (pHM1J); commercial glucan from yeast, barley and Euglena purchased from Sigma Aldrich. Glucans from various sources were incubated with FeSO4 at room temperature for 15 min, amount of free iron was determined using the standard concentration of ferrozine-Fe2+ complex.(C) Iron-binding measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Xanthan and various glucans were mixed with 2 mM FeSO4, dialysed against deionized water before determination of Fe2+ content by ICP-OES. Data shown are mean ± SD (n≥3). ** indicates p-value < 0.01 and *** indicates p-value < 0.001 by paired student t-test.(D) Specificity of glucan-Fe2+ interaction. Purified Xcc β-(1, 2)-glucan was incubated with 100 µM FeSO4 and increasing concentrations of Ca2+, Co2+, Ni2+, Mn2+, Mg2+ or Zn2+, and free or unbound iron was measured by Fe2+-ferrozine colorimetric assay. Data shown are mean ± SD (n=3).


Figure 4 ( A ) Analysis of total iron levels measured by Inductively Coupled Plasma - Optical Emission Spectrometry ( ICP - OES ) . Cells were grown under either rich - media ( PS ) , iron - restricted ( PS + 150 μM DP ) or glucan supplemented iron - restricted media ( PS + 150 μM DP + 0 . 5 mg / ml glucan ) till the late exponential growth phase . Bacterial cells were harvested , washed and then iron content was determined using ICP - OES . Data shown are mean ± SD ( n = 3 ) . * * indicates p value < 0 . 01 determined by paired student t - test ( two tailed with equal variance ) . ( B ) Co - elution of periplasmic glucan and radiolabellediron - glucan complex . Periplasmic glucan was isolated from the 55Fe2 + - loaded cells , and analyzed by BioGel - P4 size - exclusion chromatography . Fractions ( 1 . 5 ml ) were collected at a flow rate of 20 ml / h and assayed for glucan using the phenol - sulphuric acid colorimetric assay , and radioactivity was measured to detect the presence of 55Fe . ( C ) Iron localization assay using atomic absorption spectroscopy . Two sets of bacterial cells were grown in iron - restricted media ( PS + 150 μM DP ) till late exponential phase . Cold iron ( 50 μM FeSO4 ) was added to the one set of bacterial cultures and incubated for one hour at 28 ° C . Bacterial cells were harvested , washed from the both sets of bacterial cultures . Periplasmic and cytoplasmic fractions were collected , freeze dried , and then iron content in each fraction was determined by Inductively Coupled Plasma - Optical Emission Spectrometry ( ICP - OES ) . Iron content is represented on Y - axis as microgram of iron present per milligram of proteins in respective fractions . Data shown are mean ± S . E . ( n = 3 ) . * indicates p - value < 0 . 05 and * * indicates p - value < 0 . 01 by paired student t - test .

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

Figure 4(A) Analysis of total iron levels measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Cells were grown under either rich-media (PS), iron-restricted (PS + 150 μM DP) or glucan supplemented iron-restricted media (PS + 150 μM DP + 0.5 mg/ml glucan) till the late exponential growth phase. Bacterial cells were harvested, washed and then iron content was determined using ICP-OES. Data shown are mean ± SD (n=3). ** indicates p value < 0.01 determined by paired student t-test (two tailed with equal variance).(B) Co-elution of periplasmic glucan and radiolabellediron-glucan complex. Periplasmic glucan was isolated from the 55Fe2+-loaded cells, and analyzed by BioGel-P4 size-exclusion chromatography. Fractions (1.5 ml) were collected at a flow rate of 20 ml/h and assayed for glucan using the phenol-sulphuric acid colorimetric assay, and radioactivity was measured to detect the presence of 55Fe.(C) Iron localization assay using atomic absorption spectroscopy. Two sets of bacterial cells were grown in iron-restricted media (PS + 150 μM DP) till late exponential phase. Cold iron (50 μM FeSO4) was added to the one set of bacterial cultures and incubated for one hour at 28°C. Bacterial cells were harvested, washed from the both sets of bacterial cultures. Periplasmic and cytoplasmic fractions were collected, freeze dried, and then iron content in each fraction was determined by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Iron content is represented on Y-axis as microgram of iron present per milligram of proteins in respective fractions. Data shown are mean ± S.E. (n = 3). * indicates p-value < 0.05 and ** indicates p-value < 0.01 by paired student t-test.


Figure 5Xanthomonas campestris : ( A ) wild type Xcc 8004 ; ( B ) B5 ( EPS deficient ) ( C ) B12EPS deficient ) ; ( D ) ndvB ( glucan deficient ) ; and ( E ) ndvB ( pHM1J ) + ( complementing strain ) ; cells were treated in the presence or absence of either FeSO4 , H2O2 or FeSO4 + Dp ( dipyridyl ) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry .

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Figure 5Xanthomonas campestris: (A) wild type Xcc 8004; (B) B5 (EPS deficient) (C) B12EPS deficient); (D) ndvB (glucan deficient); and (E) ndvB(pHM1J)+ (complementing strain); cells were treated in the presence or absence of either FeSO4, H2O2 or FeSO4+Dp (dipyridyl) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry.


Figure 6 ( A ) Xanthomonas campestris cells treated with FeSO4 , H2O2 and FeSO4 + Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry . Data shown are mean ± S . E . ( n = 3 ) . ( B ) The percent of ROS positive Xanthomonas campestris cells treated with FeSO4 , H2O2 and FeSO4 + Dp determined by flow cytometry at 3 h after treatment . Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99 % of untreated cells . Data shown are mean ± SE ( n = 3 ) . * * * indicates p - value < 0 . 001 compared with data obtained from iron treated wild type and mutant by paired student t - test . ( C ) Iron sensitivity assay . Cells were grown overnight in PS medium and cultures were diluted in fresh media at an density of 1 x 106 cells / ml , treated in the absence or presence of 500 µM FeSO4 . Viability of cultures was measured at the indicated time point by dilution plating . Percent survival was determined as the number of viable cells at each time divided by the number of viable cells before exposure to FeSO4 . Data shown are mean ± SE ( n = 3 ) . * indicates p - value < 0 . 05 and * * indicates p - value < 0 . 01 compared with data obtained from iron treated wild type and mutant by paired student t - test .

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

Figure 6(A) Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry. Data shown are mean ± S.E. (n = 3).(B) The percent of ROS positive Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp determined by flow cytometry at 3 h after treatment. Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99% of untreated cells. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.(C) Iron sensitivity assay. Cells were grown overnight in PS medium and cultures were diluted in fresh media at an density of 1 x 106 cells/ml, treated in the absence or presence of 500 µM FeSO4. Viability of cultures was measured at the indicated time point by dilution plating. Percent survival was determined as the number of viable cells at each time divided by the number of viable cells before exposure to FeSO4. Data shown are mean ± SE (n=3). * indicates p-value < 0.05 and ** indicates p-value < 0.01 compared with data obtained from iron treated wild type and mutant by paired student t-test.


Figure 7 ( A ) Wild type E . coli ( MG1655 ) ; ΔopgG ( ΔmdoG ; JW1035 ) and ΔopgH ( ΔmdoH ; JW1037 ) , mutants blocked in periplasmic glucan synthesis were grown in LOS medium , and cells were treated in the presence or absence of either FeSO4 , H2O2 or FeSO4 + DP ( dipyridyl ) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry . ( B ) The percent of ROS positive E . coli cells treated with FeSO4 , H2O2 and FeSO4 + DP determined by flow cytometry at 3 h after treatment . Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99 % of untreated cells . Data shown are mean ± SE ( n = 3 ) . * * * indicates p - value < 0 . 001 compared with data obtained from iron treated wild type and mutant by paired student t - test . ( C ) E . coli cells treated with FeSO4 , H2O2 and FeSO4 + Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry . Data shown are mean ± SE ( n = 3 ) . * * * indicates p - value < 0 . 001 compared with data obtained from iron treated wild type and mutant by paired student t - test .

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

Figure 7(A) Wild type E. coli (MG1655); ΔopgG (ΔmdoG; JW1035) and ΔopgH (ΔmdoH; JW1037), mutants blocked in periplasmic glucan synthesis were grown in LOS medium, and cells were treated in the presence or absence of either FeSO4, H2O2 or FeSO4+DP (dipyridyl) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry.(B) The percent of ROS positive E. coli cells treated with FeSO4, H2O2 and FeSO4+DP determined by flow cytometry at 3 h after treatment. Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99% of untreated cells. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.(C) E. coli cells treated with FeSO4, H2O2 and FeSO4+ Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.


Figure 1 ( D ) Stretching of the PDMS sandwich concomitantly stretches the wing disc inside the microchannel ( top - down view ) ; wing disc resting on a stretching device ( anchor ) and 10 min after stretch ; M = microchannel . ( E ) zipper RNAi clones ( zip iR , in red ) in Arm : : GFP expressing anchored and stretched third instar wing disc ; lower panel shows color coded cell aspect ratio in anchored and stretched discs ( bright colors indicate high elongation ) ; zip RNAi clone is outlined in white . ( F ) Box plot showing distribution of cell aspect ratio for control ( wild - type ) and zipper RNAi cells in anchored and stretched ( 20 min ) wing disc ; median represented by horizontal line , 75th and 25th centiles are represented by top and bottom of the boxes respectively ; n = 8 wing discs . ( G ) Color - coded aspect ratio in control and zip RNAi cells ( zip RNAi clone outlined in white ) subjected to anchoring , stretching ( 20 min ) and relaxation ( 10 min , post stretch ) . ( H ) Box plot showing distribution of cell aspect ratio in anchored , stretched ( 20 min ) and relaxed ( 10 min ) wing disc for control ( wild - type ) and zipper RNAi cells ; median represented by horizontal line ; 75th and 25th centiles are represented by top and bottom of the boxes respectively , n = 3 wing discs . ( I ) zip - RNAi clone in Arm : : GFP wing disc at the beginning ( 5 min ) and at the end ( 40 min ) of stretching experiment . Yellow and red arrows indicate the control and zip RNAi cells respectively . ( J ) Change of zip - RNAi aspect ratio ( a . r . ) relative to change in control cells aspect ratio ( a . r . ) in the course of stretch , blue line shows fitted regression ; dotted line represents 95 % confidence bounds of the best fit line ; n = 3 wing discs .

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Figure 1(D) Stretching of the PDMS sandwich concomitantly stretches the wing disc inside the microchannel (top-down view); wing disc resting on a stretching device (anchor) and 10 min after stretch; M=microchannel.(E) zipper RNAi clones (zip iR, in red) in Arm::GFP expressing anchored and stretched third instar wing disc; lower panel shows color coded cell aspect ratio in anchored and stretched discs (bright colors indicate high elongation); zip RNAi clone is outlined in white.(F) Box plot showing distribution of cell aspect ratio for control (wild-type) and zipper RNAi cells in anchored and stretched (20 min) wing disc; median represented by horizontal line, 75th and 25th centiles are represented by top and bottom of the boxes respectively; n=8 wing discs.(G) Color-coded aspect ratio in control and zip RNAi cells (zip RNAi clone outlined in white) subjected to anchoring, stretching (20 min) and relaxation (10 min, post stretch).(H) Box plot showing distribution of cell aspect ratio in anchored, stretched (20 min) and relaxed (10 min) wing disc for control (wild-type) and zipper RNAi cells; median represented by horizontal line; 75th and 25th centiles are represented by top and bottom of the boxes respectively, n=3 wing discs.(I) zip-RNAi clone in Arm::GFP wing disc at the beginning (5 min) and at the end (40 min) of stretching experiment. Yellow and red arrows indicate the control and zip RNAi cells respectively.(J) Change of zip-RNAi aspect ratio (a.r.) relative to change in control cells aspect ratio (a.r.) in the course of stretch, blue line shows fitted regression; dotted line represents 95% confidence bounds of the best fit line; n=3 wing discs.


Figure 2 ( A ) Wing disc expressing E - cad : : GFP and Sqh : : mCherry prior to stretch ( anchor ) and after stretch ( 15 min ) . ( A " ) , Insets of discs shown in ( A ) . ( B ) Schematics demonstrating change in Sqh concentration before and after stretch on vertical ( V ) and horizontal ( H ) junctions ( C ) Quantification of mean fluorescent intensity of E - cad : : GFP and Sqh : : mCherry on horizontal ( H ) junctions relative to the intensity on vertical ( V ) junctions ( referred to as " polarity " ) prior to and after 15 min stretch . The detailed rationale of intensity determination is described in Fig . S3 and Experimental Procedures . ( D - E ) , Quantification of E - cad : : GFP ( D ) and Sqh : : mCherry ( E ) concentration ( i . e . mean intensity per junctional unit area ) prior and after stretch on both horizontal and vertical junctions normalized to starting concentration on unstretched ( anchored ) vertical junctions . ( F ) Quantification of total amount of Sqh : : mCherry and E - cad : : GFP fluorescence on vertical and horizontal junctions in anchored and stretched discs . ( G ) Tissue expressing E - cad : : GFP and Sqh : : mCherry subjected to increasing levels of stretch . ( H ) Quantification of Sqh : : mCherry polarity ( as in c ) of experiment described in ( G ) . The level of stretch is measured as a change in tissue strain ( deformation relative to unstretched tissue ) . ( I - K ) Sqh : : mCherry polarity emergence as a function of cell aspect ratio change ( I ) , stress ( J ) , stress applied on average cell area of 10 μm2 ( K ) . Yellow asterisks indicate equivalent regions in the tissue . N = 8 wing discs ( A - F ) , N = 3 wing discs ( G - K ) ;

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Figure 2(A) Wing disc expressing E-cad::GFP and Sqh::mCherry prior to stretch (anchor) and after stretch (15 min).
(A"), Insets of discs shown in (A).
(B) Schematics demonstrating change in Sqh concentration before and after stretch on vertical (V) and horizontal (H) junctions(C) Quantification of mean fluorescent intensity of E-cad::GFP and Sqh::mCherry on horizontal (H) junctions relative to the intensity on vertical (V) junctions (referred to as "polarity") prior to and after 15 min stretch. The detailed rationale of intensity determination is described in Fig. S3 and Experimental Procedures.(D-E), Quantification of E-cad::GFP (D) and Sqh::mCherry (E) concentration (i.e. mean intensity per junctional unit area) prior and after stretch on both horizontal and vertical junctions normalized to starting concentration on unstretched (anchored) vertical junctions.(F) Quantification of total amount of Sqh::mCherry and E-cad::GFP fluorescence on vertical and horizontal junctions in anchored and stretched discs.(G) Tissue expressing E-cad::GFP and Sqh::mCherry subjected to increasing levels of stretch.(H) Quantification of Sqh::mCherry polarity (as in c) of experiment described in (G). The level of stretch is measured as a change in tissue strain (deformation relative to unstretched tissue).(I-K) Sqh::mCherry polarity emergence as a function of cell aspect ratio change (I), stress (J), stress applied on average cell area of 10 μm2 (K). Yellow asterisks indicate equivalent regions in the tissue. N=8 wing discs (A- F), N=3 wing discs (G-K);


Figure 3 ( A ) Short ( 15 min ) stretch and relaxation ( 10 min ) of E - cad : : GFP and Sqh : : mCherry expressing discs . ( B ) Quantification of Sqh : : mCherry polarity , E - cad : : GFP polarity and aspect ratio change of experiment described in ( A ) ; schematics below the graph illustrates quantified changes in cell aspect ratio ( yellow ) and Sqh polarity ( red ) . Statistical test compares Sqh polarity between anchored and stretched or stretched and relaxed disc . ( C ) Long ( 200 min ) stretch and relaxation ( 10 min ) of E - cad : : GFP and Sqh : : mCherry expressing discs . ( D ) Quantification of Sqh : : mCherry polarity , E - cad : : GFP polarity and aspect ratio change of experiment described in ( C ) ; schematics below the graph illustrates quantified changes in aspect ratio ( yellow ) and Sqh polarity ( red ) .

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Figure 3(A) Short (15 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs.(B) Quantification of Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (A); schematics below the graph illustrates quantified changes in cell aspect ratio (yellow) and Sqh polarity (red). Statistical test compares Sqh polarity between anchored and stretched or stretched and relaxed disc.(C) Long (200 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs.(D) Quantification of Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (C); schematics below the graph illustrates quantified changes in aspect ratio (yellow) and Sqh polarity (red).


Figure 4 ( A ) Overview of the E - cad : : GFP disc prior to ( anchor ) and after compression . ( B ) E - cad : : GFP and Sqh : : mCherry localization in anchored and compressed wing disc . ( C ) Laser cuts ( perpendicular to the line of stretch ) across circa 10 cells in E - cad : : GFP expressing wing disc tissue subjected to anchoring , short ( < 20 min ) stretching and short ( < 20 min ) compression . Plot shows increase in distance between recoiling short axis [ μm ] of the fitted ellipse ( see Fig . S5A for details ) against time [ sec . ] ; data is plotted as mean ± S . E . M , n = 7 - 10 discs / condition . ( D ) Activated Myosin ( anti - phosphorylated - Sqh ; in red ) and anti - E - cad ( green ) staining in compressed discs . ( D " ) Close up of compression experiment described in ( D ) ; yellow arrow points towards an example of polarized phosphorylated - Sqh cable in compressed discs .

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Figure 4(A) Overview of the E-cad::GFP disc prior to (anchor) and after compression.(B) E-cad::GFP and Sqh::mCherry localization in anchored and compressed wing disc.(C) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) stretching and short (<20 min) compression. Plot shows increase in distance between recoiling short axis [μm] of the fitted ellipse (see Fig. S5A for details) against time [sec.]; data is plotted as mean ± S.E.M, n= 7-10 discs/condition.(D) Activated Myosin (anti-phosphorylated-Sqh; in red) and anti-E-cad (green) staining in compressed discs. (D") Close up of compression experiment described in (D); yellow arrow points towards an example of polarized phosphorylated-Sqh cable in compressed discs.


Figure 5 ( A ) Laser cuts ( perpendicular to the line of stretch ) across circa 10 cells in E - cad : : GFP expressing wing disc tissue subjected to anchoring , short ( < 20 min ) and long ( > 120 min ) stretching and stretch retraction ( tissue forced back to the original , pre - stretch shape . ( C ) An overlay of a junction prior to ( red ) and 15 . 44 sec . after ablation ( green ) . ( E ) Laser cuts ( perpendicular or parallel to the line of stretch ) across circa 10 cells in E - cad : : GFP expressing wing disc tissue subjected to anchoring or short stretching ( < 30 min ) . . ( H ) An overlay of anchored and stretched discs prior to ablation ( red ) and 15 . 44 seconds post ablation ( green ) . Inset zooms in on the area next to ablation site ( marked with yellow asterisk ) .

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Figure 5(A) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) and long (>120 min) stretching and stretch retraction (tissue forced back to the original, pre-stretch shape.(C) An overlay of a junction prior to (red) and 15.44 sec. after ablation (green).(E) Laser cuts (perpendicular or parallel to the line of stretch) across circa 10 cells in E-cad::GFP expressing wing disc tissue subjected to anchoring or short stretching (< 30 min)..(H) An overlay of anchored and stretched discs prior to ablation (red) and 15.44 seconds post ablation (green). Inset zooms in on the area next to ablation site (marked with yellow asterisk).


Figure 6 ( A ) RokK116a : : Venus localization in anchored , stretched , relaxed third instar imaginal discs . discs . ( B ) Drok2 mitotic clones ( RFP - ) in Sqh : : GFP third instar imaginal disc . Inset demonstrates Sqh : : GFP polarity inside Drok2 clone . ( C ) Sqh : : GFP , enGal4 > UAS - rok RNAi , UAS - Act5CRFP discs subjected to stretching ; inset demonstrates Sqh : : GFP polarity in rok RNAi cells ( marked by red . ( D ) zip RNAi clones ( labeled as zip iR , indicated in red ) in stretched third instar RokK116a : : Venus discs .

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Figure 6(A) RokK116a::Venus localization in anchored, stretched, relaxed third instar imaginal discs.discs.
(B) Drok2 mitotic clones (RFP-) in Sqh::GFP third instar imaginal disc. Inset demonstrates Sqh::GFP polarity inside Drok2 clone.(C) Sqh::GFP, enGal4>UAS-rok RNAi, UAS-Act5CRFP discs subjected to stretching; inset demonstrates Sqh::GFP polarity in rok RNAi cells (marked by red.(D) zip RNAi clones (labeled as zip iR, indicated in red) in stretched third instar RokK116a::Venus discs.


Figure 7 ( A ) Discs expressing Sqh : : GFP and enGal4 > UAS - dia RNAi , UAS - Act5c - RFP that were stretched ; UAS - Act5C : : RFP marks dia iR ( posterior side ) in red . ( A " ) Insets comparing Sqh : : GFP polarity in stretchedenGal4 > dia iR , UAS - Act5c - RFP discs ; panel 1 refers to anterior ( ctrl ) side ; panel 2 refers to posterior ( dia iR ) side . ( B ) Tissue marked with E - cad : : GFP and Sqh : : mCherry , expressing dia - RNAi ( dia iR ) with hhGal4 driver and subjected to stretching . Dotted line indicates anterior - posterior ( A - P ) compartment boundary . ( B " ) Inset demonstrates lack of Sqh : : GFP polarity in dia - RNAi cells . ( C - D ) , Quantification of E - cad : : GFP polarity ( C ) and Sqh : : mCherry polarity ( D ) in experiment described in ( B ) ; polarity is measured as mean fluorescent intensity on horizontal junctions relative to vertical junctions ( see Experimental Procedures for details ) ; n = 4 wing discs . ( E ) Stretched discs expressing Sqh : : GFP and enGal4 > UAS - cofilin ( upper panel ) or UAS - AIP1 ( lower panel ) RNAi and UAS - Act5c - RFP ( marks posterior side in red ) . ( E " ) , Insets comparing Sqh : : GFP polarity in stretched discs described in ( E ) ; panel 1 refers to anterior ( ctrl ) side ; panel 2 refers to posterior ( dia iR ) side . ( F ) Sqh : : GFP polarity in enGal4 > dia iR , UAS - Act5C : : RFP discs labeled with Phalloidin - 647 ; Inset ( Phall - 647 ) zooms in on cell shape in anterior and posterior disc compartments . ( G ) Discs expressing nubGal4 and Cd8 : : GFP ( control ) or dia iR ( KK ) labeled with DAPI ; ellipse is fitted into the pouch region ( red dotted line ) and aspect ratio determined as long ellipse axis ( b ) divided by short ellipse axis ( a ) ( white lines ) . ( H ) Box plot showing distribution of wing disc pouch aspect ratios in conditions described in ( G ) , n = 19 ( ctrl ) and 16 ( dia iR ) wing pouches ; horizontal line indicates median .

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Figure 7(A) Discs expressing Sqh::GFP and enGal4>UAS-dia RNAi, UAS-Act5c-RFP that were stretched; UAS-Act5C::RFP marks dia iR (posterior side) in red. (A") Insets comparing Sqh::GFP polarity in stretchedenGal4>dia iR, UAS- Act5c-RFP discs; panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side. (B) Tissue marked with E-cad::GFP and Sqh::mCherry, expressing dia-RNAi (dia iR) with hhGal4 driver and subjected to stretching. Dotted line indicates anterior-posterior (A-P) compartment boundary. (B") Inset demonstrates lack of Sqh::GFP polarity in dia-RNAi cells. (C-D), Quantification of E-cad::GFP polarity (C) and Sqh::mCherry polarity (D) in experiment described in (B); polarity is measured as mean fluorescent intensity on horizontal junctions relative to vertical junctions (see Experimental Procedures for details); n=4 wing discs.(E) Stretched discs expressing Sqh::GFP and enGal4>UAS-cofilin (upper panel) or UAS-AIP1 (lower panel) RNAi and UAS-Act5c-RFP (marks posterior side in red).
(E"), Insets comparing Sqh::GFP polarity in stretched discs described in (E); panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side.(F) Sqh::GFP polarity in enGal4> dia iR, UAS-Act5C::RFP discs labeled with Phalloidin-647; Inset (Phall-647) zooms in on cell shape in anterior and posterior disc compartments.(G) Discs expressing nubGal4 and Cd8::GFP (control) or dia iR (KK) labeled with DAPI; ellipse is fitted into the pouch region (red dotted line) and aspect ratio determined as long ellipse axis (b) divided by short ellipse axis (a) (white lines).(H) Box plot showing distribution of wing disc pouch aspect ratios in conditions described in (G), n=19 (ctrl) and 16 (dia iR) wing pouches; horizontal line indicates median.


Figure 1Immunofluorescence images of U2OS cells expressing FLAG - HA - FAM134 after 24hrs doxycycline induction stained for HA ( green ) and endogenous calnexin ( CANX ; red ) . Scale bar : 20μm .

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Figure 1Immunofluorescence images of U2OS cells expressing FLAG-HA-FAM134 after 24hrs doxycycline induction stained for HA (green) and endogenous calnexin (CANX; red). Scale bar: 20μm.


Figure 2Lysates of HEK293T cells transiently transfected with HA - tagged wild - type FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) purified GST , wild type GST - LC3s , GST - GABARAPs , GST - Ubiquitin ( Ub ) , GST - Tetra - Ubiquitin , A representative Ponceau staining is shown . Lysates of HEK293T cells transiently transfected with HA - tagged wild - type and wild - type or ∆ LIR mutant FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) A representative Ponceau staining is shown . Lysates of HEK293T cells transiently transfected with HA - tagged wild - type FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) using GST - LC3B and GST - LC3B F52A - V53A as baits . A representative Ponceau staining is shown . Immunofluorescence images of U2OS expressing wild - type or ∆ LIR mutant FLAG - HA - FAM134 proteins after 24hrs doxycycline induction under different conditions ( basal = DMSO , Baf . A1 = 2h 200nM Bafilomycin A1 , EBSS = 2h starvation in EBSS ) Scale bar : 10μm ; staining against FAM134 ( HA ; green ) and endogenous LC3B ( red ) .

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

Figure 2Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) purified GST, wild type GST-LC3s, GST-GABARAPs, GST-Ubiquitin (Ub), GST-Tetra-Ubiquitin, A representative Ponceau staining is shown.Lysates of HEK293T cells transiently transfected with HA-tagged wild-type and wild-type or ∆LIR mutant FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) A representative Ponceau staining is shown.Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) using GST-LC3B and GST- LC3B F52A-V53A as baits. A representative Ponceau staining is shown.Immunofluorescence images of U2OS expressing wild-type or ∆LIR mutant FLAG-HA-FAM134 proteins after 24hrs doxycycline induction under different conditions (basal=DMSO, Baf.A1=2h 200nM Bafilomycin A1, EBSS=2h starvation in EBSS) Scale bar: 10μm; staining against FAM134 (HA; green) and endogenous LC3B (red).


Figure 3Small sections of sample pictures of U2OS cells stably expressing the ER - phagy reporter ssRFP - GFP - KDEL . Pictures were obtained by the IncuCyte ® S3 ( 10x ) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time . Scale bar 25µm . Time lapse of representative total image views can be found in Movie EV7 and Movie EV8 . Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction . Autophagy flux was induced and blocked by treatment with Torin1 ( 8hrs ) and Bafilomycin A1 ( Baf . A1 , 8hrs ) , respectively . n =

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

Figure 3Small sections of sample pictures of U2OS cells stably expressing the ER-phagy reporter ssRFP-GFP-KDEL. Pictures were obtained by the IncuCyte® S3 (10x) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time. Scale bar 25µm. Time lapse of representative total image views can be found in Movie EV7 and Movie EV8.Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction. Autophagy flux was induced and blocked by treatment with Torin1 (8hrs) and Bafilomycin A1 (Baf.A1, 8hrs), respectively. n=3


Figure 4Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts ( MEFs ) . Western blot for Fam134 proteins in indicated MEFs , expressing the ER - phagy reporter ssRFP - GFP - KDEL and being reconstituted with the respective wild - type or ∆ LIR mutant Fam134 protein .

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Figure 4Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts (MEFs).Western blot for Fam134 proteins in indicated MEFs, expressing the ER-phagy reporter ssRFP-GFP-KDEL and being reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein.


Figure 5Profile plots showing the Log2 LFQ intensity ( G ) and representative immunofluorescence ( H ) of Climp63 ( green ) in wild - type and Fam134 knockout MEFs . Profile plots showing the Log2 LFQ intensity ( I ) and representative immunofluorescence ( J ) for collagen I ( red ) expression in wild - type and Fam134 knockout MEFs . Scale bar : 10μm . Inset scale bar : 5μm . Nuclei were stained with Hoechst 33342 .

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Figure 5Profile plots showing the Log2 LFQ intensity (G) and representative immunofluorescence (H) of Climp63 (green) in wild-type and Fam134 knockout MEFs.Profile plots showing the Log2 LFQ intensity (I) and representative immunofluorescence (J) for collagen I (red) expression in wild-type and Fam134 knockout MEFs. Scale bar: 10μm. Inset scale bar: 5μm. Nuclei were stained with Hoechst 33342.


Figure 6Electron microscopy of wild - type and Fam134 single knockout MEFs ( ER = endoplasmic reticulum ) . Scale bar : 500nm . Immunofluorescence images stained for endogenous Calnexin ( Canx ) or Climp63 in wild - type ( WT ) , Fam134 knockout MEFs , and Fam134 knockout MEFs reconstituted with the respective wild - type or ∆ LIR mutant Fam134 protein . Scale bar : 10μm . Inset scale bar : 5μm . Representative Western blots of total cell lysate derived from MEFs used in ( B ) and detected for endogenous Fam134 and Actin , respectively .

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

Figure 6Electron microscopy of wild-type and Fam134 single knockout MEFs (ER=endoplasmic reticulum). Scale bar: 500nm.Immunofluorescence images stained for endogenous Calnexin (Canx) or Climp63 in wild-type (WT), Fam134 knockout MEFs, and Fam134 knockout MEFs reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. Scale bar: 10μm. Inset scale bar: 5μm.Representative Western blots of total cell lysate derived from MEFs used in (B) and detected for endogenous Fam134 and Actin, respectively.


Figure 7Representative Western Blot of Collagen I in wild - type MEFs and Fam134 knockout MEFs reconstituted for the respective wild - type ( WT ) or ∆ LIR mutant Fam134 protein . Vinculin has been used as reference for ratio calculation . The statistical significance was estimated by unpaired Student ' s t test . All data are represented as mean ± s . e . m . of n = 3 biological experiments and the statistical significance is defined as * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Representative Western Blot ( D ) and the relative bar plot ( E ) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆ LIR Fam134a protein . Representative Western Blot ( F ) and the relative bar plot ( G ) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c . Representative Western Blot ( H ) and the relative bar plot ( I ) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b . Representative Western Blot ( J ) and the relative bar plot ( K ) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b . Representative Western Blot ( L ) and the relative bar plot ( M ) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c .

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

Figure 7Representative Western Blot of Collagen I in wild-type MEFs and Fam134 knockout MEFs reconstituted for the respective wild-type (WT) or ∆LIR mutant Fam134 protein. Vinculin has been used as reference for ratio calculation. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.Representative Western Blot (D) and the relative bar plot (E) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆LIR Fam134a protein.Representative Western Blot (F) and the relative bar plot (G) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c. Representative Western Blot (H) and the relative bar plot (I) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b.Representative Western Blot (J) and the relative bar plot (K) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b. Representative Western Blot (L) and the relative bar plot (M) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c.


Figure 1 ( A ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 12 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 16 ) mice ( age 9 - 12 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles . Mice were monitored for up to 200 days post - treatment with tamoxifen . Data are presented as % survival post - treatment with tamoxifen and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test ; * * * * p < 0 . 0001 . ( B ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 24 ) or , as controls , Bcl - xfl / fl ( n = 9 ) and RosaCreERT2 + / Ki ( n = 6 ) mice ( males and females , aged 8 - 14 weeks , numbers also indicated in the figure legend ) were lethally γ - irradiated ( 2 x 5 . 5 Gy , 3 h apart ) and reconstituted with bone marrow from UBC - GFP mice ( referred to as GFP - Chimeras ) . After 8 weeks , reconstituted mice were treated with tamoxifen ( 200 mg / kg body weight administered in 3 daily doses oral gavage ) and monitored for up to 200 days ( termination of the experiment ) . Data are presented as % survival post - treatment with tamoxifen and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test ; * * * * p < 0 . 0001 . ( C - F ) Total counts of ( C ) platelets , ( D ) red blood cells ( RBC ) , ( E ) hemoglobin ( HGB ) content and ( F ) hematocrit ( HCT ) of tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 12 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimera ( n = 21 ) or , as controls , Bcl - xfl / fl ; GFP - Chimera ( n = 6 ) and RosaCreERT2 + / Ki ; GFP - Chimera ( n = 6 ) mice were determined by ADVIA . Data are presented as mean ± SEM . Each data point represents an individual mouse . Statistical significance was assessed using the Student ' s t - test ; * * * * p < 0 . 0001 . ( G ) Spleen weights were measured in sick mice ( at sacrifice ) or , for the healthy control mice , at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents an individual mouse and numbers are indicated . Statistical significance was assessed using the Student ' s t - test ; * * * * p < 0 . 0001 . ( H ) Representative image of enlarged spleens from two Bcl - xfl / fl ; RosaCreERT2Ki / + mice and age - matched control Bcl - xfl / fl and RosaCreERT2 + / Ki mice ( 34 days after treatment ) . ( I ) Histological analysis of H & E - stained sections of spleens of tamoxifen - treated mice of the indicated genotypes .

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

Figure 1 (A) Bcl-xfl/fl;RosaCreERT2+/Ki (n=12) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=16) mice (age 9-12 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. Mice were monitored for up to 200 days post-treatment with tamoxifen. Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.  (B) Bcl-xfl/fl;RosaCreERT2+/Ki (n=24) or, as controls, Bcl-xfl/fl (n=9) and RosaCreERT2+/Ki (n=6) mice (males and females, aged 8-14 weeks, numbers also indicated in the figure legend) were lethally γ-irradiated (2 x 5.5 Gy, 3 h apart) and reconstituted with bone marrow from UBC-GFP mice (referred to as GFP-Chimeras). After 8 weeks, reconstituted mice were treated with tamoxifen (200 mg/kg body weight administered in 3 daily doses oral gavage) and monitored for up to 200 days (termination of the experiment). Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.  (C-F) Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n=21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n=6) and RosaCreERT2+/Ki;GFP-Chimera (n=6) mice were determined by ADVIA. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; ****p<0.0001. (G) Spleen weights were measured in sick mice (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse and numbers are indicated. Statistical significance was assessed using the Student's t-test; ****p<0.0001.  (H) Representative image of enlarged spleens from two Bcl-xfl/fl;RosaCreERT2Ki/+ mice and age-matched control Bcl-xfl/fl and RosaCreERT2+/Ki mice (34 days after treatment).  (I) Histological analysis of H&E-stained sections of spleens of tamoxifen-treated mice of the indicated genotypes.


Figure 2Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 12 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , or as controls , Bcl - xfl / fl ; GFP - Chimeras ( n = 6 ) and RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( age 8 - 14 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles . ( A ) Total white blood cell counts ( WBC ) were analyzed by ADVIA in sick mice or at the termination of the experiment ( healthy control mice ) . Data are presented as mean ± SEM . Each data point represents an individual mouse and n numbers are indicated above . Statistical significance was assessed using the Student ' s t - test . No statistically significant differences were observed . ( B ) The neutrophil / lymphocyte ratio ( NLR ) is presented as mean ± SEM for sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) or healthy control RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents an individual mouse . Statistical significance was assessed using the Student ' s t - test ; * p < 0 . 05 . ( C ) Histological analysis of H & E - stained sections of the sternum of sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras or healthy wild - type and RosaCreERT2 + / Ki ; GFP - Chimera control mice at the indicated time points post - treatment with tamoxifen ( dpt = days post - treatment ) . ( D ) Histological analysis of H & E - stained sections of the livers of sick mice or age - matched healthy control wild - type mice or healthy RosaCreERT2 + / Ki ; GFP - Chimeras at the termination of the experiment ( dpt = days post - treatment ) . ( E ) ALT ( left panel ) , AST ( middle panel ) and bilirubin levels ( right panel ) in the serum were determined in sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 18 ) or in healthy RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 4 ) at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test ; * * p < 0 . 01 . nd = not detected .

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

Figure 2Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), or as controls, Bcl-xfl/fl;GFP-Chimeras (n=6) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Total white blood cell counts (WBC) were analyzed by ADVIA in sick mice or at the termination of the experiment (healthy control mice). Data are presented as mean ±SEM. Each data point represents an individual mouse and n numbers are indicated above. Statistical significance was assessed using the Student's t-test. No statistically significant differences were observed.  (B) The neutrophil/lymphocyte ratio (NLR) is presented as mean ±SEM for sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21) or healthy control RosaCreERT2+/Ki;GFP-Chimeras (n=6) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; *p<0.05.  (C) Histological analysis of H&E-stained sections of the sternum of sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras or healthy wild-type and RosaCreERT2+/Ki;GFP-Chimera control mice at the indicated time points post-treatment with tamoxifen (dpt=days post-treatment).  (D) Histological analysis of H&E-stained sections of the livers of sick mice or age-matched healthy control wild-type mice or healthy RosaCreERT2+/Ki;GFP-Chimeras at the termination of the experiment (dpt=days post-treatment).  (E) ALT (left panel), AST (middle panel) and bilirubin levels (right panel) in the serum were determined in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=18) or in healthy RosaCreERT2+/Ki;GFP-Chimeras (n=4) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; **p<0.01. nd=not detected.


Figure 3 Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 7 ) or as controls , Bcl - xfl / fl ( n = 8 ) and RosaCreERT2 + / Ki ( n = 16 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 5 ) , Bcl - xfl / fl ; GFP - Chimeras ( n = 8 ) and RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( age 8 - 14 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles . ( A ) Kidney weights were measured in sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( at sacrifice ) or , for the healthy control mice , at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test ; * p < 0 . 05 , * * * p < 0 . 001 . ( B ) Picture showing both kidneys from age - matched healthy control wild - type mice ( left ) and sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( right ) . ( C ) Histological analysis of H & E - stained sections of the kidneys of age - matched healthy control mice or sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras as indicated , showing from left to right , shrunken scarred kidney , segmental chronic tubulo - interstitial disease , accumulation of cellular debris and amorphous material within the collecting ducts of the papilla . The higher magnification shows tubular epithelial degeneration , apoptosis and segmental secondary glomerular sclerosis . Pictures are representative of at least 5 mice for each genotype and treatment . ( D ) Multiphoton analysis of fixed sections of the kidneys of age - matched healthy RosaCreERT2 + / Ki ; GFP - Chimeras ( left ) or sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( right ) . Pictures are representative of at least 3 mice for each genotype and treatment . green = second harmony , blue = short wavelength autofluorescence .

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

Figure 3 Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Kidney weights were measured in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; *p<0.05, ***p<0.001.  (B) Picture showing both kidneys from age-matched healthy control wild-type mice (left) and sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right).  (C) Histological analysis of H&E-stained sections of the kidneys of age-matched healthy control mice or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras as indicated, showing from left to right, shrunken scarred kidney, segmental chronic tubulo-interstitial disease, accumulation of cellular debris and amorphous material within the collecting ducts of the papilla. The higher magnification shows tubular epithelial degeneration, apoptosis and segmental secondary glomerular sclerosis. Pictures are representative of at least 5 mice for each genotype and treatment.  (D) Multiphoton analysis of fixed sections of the kidneys of age-matched healthy RosaCreERT2+/Ki;GFP-Chimeras (left) or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right). Pictures are representative of at least 3 mice for each genotype and treatment. green=second harmony, blue=short wavelength autofluorescence.


Figure 4 ( A ) TUNEL staining of kidney sections from control RosaCreERT2 + / Ki , Bcl - xfl / fl ; RosaCreERT2 + / Ki , RosaCreERT2 + / Ki ; GFP - Chimeras or Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras at the indicated time points post - tamoxifen treatment ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) or γ - irradiation ( 2 x 550 Rad ) , as indicated . Slides were counterstained with hematoxylin . Arrow heads indicate examples of TUNEL + ( apoptotic ) cells . ( B ) TUNEL + cells were quantified using a personalized script for the Image J software . Kidney sections were divided into ~ 40 microscopic images and the total numbers of blue and brown ( TUNEL + ) nuclei were determined . The percentages of TUNEL + nuclei were calculated for each picture . The average value for each kidney section was then determined and plotted as a single data point . Error bars represent SEM from at least 3 independent samples for each genotype and treatment . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test ( comparing control RosaCreERT2 + / Ki ( 24 h post - tamoxifen n = 2 , 24 h post - γ - irradiation n = 9 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ( 24 h post - tamoxifen n = 6 , 24 h post - γ - irradiation n = 5 ) and control RosaCreERT2 + / Ki ; GFP - Chimeras ( 24 h post - tamoxifen n = 7 , End point n = 8 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( 24 h post - tamoxifen n = 8 , End point n = 6 ) ( * * * p < 0 . 001 ) .

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

Figure 4 (A) TUNEL staining of kidney sections from control RosaCreERT2+/Ki, Bcl-xfl/fl;RosaCreERT2+/Ki, RosaCreERT2+/Ki;GFP-Chimeras or Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras at the indicated time points post-tamoxifen treatment (200 mg/kg/body weight administered in 3 daily doses by oral gavage) or γ-irradiation (2 x 550 Rad), as indicated. Slides were counterstained with hematoxylin. Arrow heads indicate examples of TUNEL+ (apoptotic) cells. (B) TUNEL+ cells were quantified using a personalized script for the Image J software. Kidney sections were divided into ~40 microscopic images and the total numbers of blue and brown (TUNEL+) nuclei were determined. The percentages of TUNEL+ nuclei were calculated for each picture. The average value for each kidney section was then determined and plotted as a single data point. Error bars represent SEM from at least 3 independent samples for each genotype and treatment. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test (comparing control RosaCreERT2+/Ki (24 h post-tamoxifen n=2, 24 h post-γ-irradiation n=9) with Bcl-xfl/fl;RosaCreERT2+/Ki (24 h post-tamoxifen n=6, 24 h post-γ-irradiation n=5) and control RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=7, End point n=8) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=8, End point n=6) (***p<0.001).


Figure 5 ( A , B ) Data are presented as % survival and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test comparing tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 25 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 25 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 18 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 15 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 22 ) . * p < 0 . 05 , * * * * p < 0 . 0001 . The survival curves for the Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( C ) Histological analysis of H & E - stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points ( dpt = days post - tamoxifen treatment ) . Pictures are representative of at least 3 mice for each genotype and treatment . ( D - F ) ( D ) Total red blood cell ( RBC ) count , ( E ) hematocrit ( HCT ) and ( F ) hemoglobin ( HGB ) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test comparing tamoxifen - treated RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 20 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 12 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 15 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 10 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 17 ) ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Data for the Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras and RosaCreERT2 + / Ki ; GFP - Chimeras

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

Figure 5 (A, B) Data are presented as % survival and statistical significance was assessed using the Mantel-Cox (Log-rank) test comparing tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=25) with Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=25), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=18), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=15) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=22). *p<0.05, ****p<0.0001. The survival curves for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras  (C) Histological analysis of H&E-stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points (dpt = days post-tamoxifen treatment). Pictures are representative of at least 3 mice for each genotype and treatment.  (D-F) (D) Total red blood cell (RBC) count, (E) hematocrit (HCT) and (F) hemoglobin (HGB) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test comparing tamoxifen-treated RosaCreERT2+/Ki;GFP-Chimeras (n=6) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=20), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=12), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=15), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=10) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=17) ; ***p<0.001; ****p<0.0001. Data for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras and RosaCreERT2+/Ki;GFP-Chimeras


Figure 6 ( A ) GFP - Chimeras were treated with the BCL - XL inhibitor A1331852 ( n = 8 , 5 doses by oral gavage , 25 mg / kg body weight each dose ) . Control groups ( n = 8 each group ) include untreated GFP - Chimeras and un - irradiated C57BL / 6 - Ly5 . 1 ( wild - type ) mice treated with the BCL - XL inhibitor A1331852 or left untreated . Mice were monitored for up to 200 days post - treatment ( dpt ) . Data are presented as % survival . ( B ) C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) were treated with Cyclophosphamide ( n = 7 , 150 mg / kg body weight , 1 dose i . v . ) or 5 - Fluorouracil ( 5 - FU , n = 7 , 100 mg / kg body weight , 1 dose i . v . ) and after 5 days additionally treated with the BCL - XL inhibitor A1331852 ( 5 doses by oral gavage , 100 mg / kg body weight each dose ) . Control groups ( n = 7 each group ) include C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) treated with A1331852 alone , Cyclophosphamide alone , 5 - FU alone or left untreated . Mice were monitored for up to 150 days post - treatment ( dpt ) . Data are presented as % survival . ( C ) C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) were treated with the BCL - XL inhibitor A1331852 ( n = 7 , 5 doses by oral gavage , 25 or 100 mg / kg body weight each dose ) . Total platelet counts were determined by ADVIA at the indicated time points . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test comparing untreated and drug - treated mice within each group ( * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p > 0 . 0001 ) . Total red blood cell count ( RBC ) , platelet count , hemoglobin ( HGB ) content and hematocrit ( HCT ) were determined by ADVIA as indicated in the blood of drug - treated GFP - Chimeras or control mice ( n = 8 each group ) at the termination of the experiment Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test comparing untreated and drug - treated mice within each group ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p > 0 . 0001 ) . Total red blood cell count ( RBC ) , platelet count , hemoglobin ( HGB ) content and hematocrit ( HCT ) were determined by ADVIA as indicated in the blood of in drug - treated C57BL / 6 - Ly5 . 1 ( wild - type ) mice or control mice at the indicated time points ( n = 7 each group ) . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test comparing untreated and drug - treated mice within each group ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p > 0 . 0001 ) .

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

Figure 6 (A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimeras and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Mice were monitored for up to 200 days post-treatment (dpt). Data are presented as % survival.  (B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Mice were monitored for up to 150 days post-treatment (dpt). Data are presented as % survival.  (C) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with the BCL-XL inhibitor A1331852 (n=7, 5 doses by oral gavage, 25 or 100 mg/kg body weight each dose). Total platelet counts were determined by ADVIA at the indicated time points. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (**p<0.01, ***p<0.001, ****p>0.0001).  Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of drug-treated GFP-Chimeras or control mice (n=8 each group) at the termination of the experiment Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).  Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of in drug-treated C57BL/6-Ly5.1 (wild-type) mice or control mice at the indicated time points (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).


Figure 7 ( A ) GFP - Chimeras were treated with the BCL - XL inhibitor A1331852 ( n = 8 , 5 doses by oral gavage , 25 mg / kg body weight each dose ) . Control groups ( n = 8 each group ) include untreated GFP - Chimera and un - irradiated C57BL / 6 - Ly5 . 1 ( wild - type ) mice treated with the BCL - XL inhibitor A1331852 or left untreated . Kidney ( left panel ) and spleen weights ( right panel ) were measured in drug - treated GFP - Chimeras or control mice at the termination of the experiment ( n = 8 each group ) . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test . No statistically significant differences were observed . ( B ) C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) were treated with Cyclophosphamide ( n = 7 , 150 mg / kg body weight , 1 dose i . v . ) or 5 - Fluorouracil ( 5 - FU , n = 7 , 100 mg / kg body weight , 1 dose i . v . ) and after 5 days additionally treated with the BCL - XL inhibitor A1331852 ( 5 doses by oral gavage , 100 mg / kg body weight each dose ) . Control groups ( n = 7 each group ) include C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) treated with the BCL - XL inhibitor A1331852 alone , Cyclophosphamide alone , 5 - FU alone or left untreated . Kidney ( left panel ) and spleen weights ( right panel ) were measured in drug - treated mice or control mice at the termination of the experiment ( n = 7 each group ) . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test . No statistically significant differences were observed . ( C ) Histological analysis of H & E - stained sections of the kidneys of drug - treated GFP - Chimeras or control mice at the termination of the experiment . Pictures are representative of at least 3 mice for each treatment group . ( D ) Histological analysis of H & E - stained sections of the kidneys of drug - treated mice or control untreated mice at the termination of the experiment . Pictures are representative of at least 3 mice for each treatment group .

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

Figure 7 (A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimera and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated GFP-Chimeras or control mice at the termination of the experiment (n=8 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed.  (B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with the BCL-XL inhibitor A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated mice or control mice at the termination of the experiment (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed.  (C) Histological analysis of H&E-stained sections of the kidneys of drug-treated GFP-Chimeras or control mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group.  (D) Histological analysis of H&E-stained sections of the kidneys of drug-treated mice or control untreated mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group.


Figure 1 C , Dose - response curves of BEAS - 2B and BEAS - 2B - KRAS cells treated for 72 hours with inhibitors of FGFR ( AZD4547 , BGJ398 ) and PLK1 ( BI2536 , BI6727 ) , alone or combination . Values of Fa and CI are shown underneath , with CI < 1 . 0 indicating synergistic effect . Data were from three independent experiments ( n = 3 ) , error bar : SD D , BEAS - 2B and BEAS - 2B - KRAS treated with the indicated drugs , alone or combination for 72 hours , were cultured in drug - free medium for additional 7 - 21 days . Surviving cells after the treatment were fixed and visualized by crystal violet staining .

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

Figure 1 C, Dose-response curves of BEAS-2B and BEAS-2B-KRAS cells treated for 72 hours with inhibitors of FGFR (AZD4547, BGJ398) and PLK1 (BI2536, BI6727), alone or combination. Values of Fa and CI are shown underneath, with CI<1.0 indicating synergistic effect. Data were from three independent experiments (n=3), error bar: SD  D, BEAS-2B and BEAS-2B-KRAS treated with the indicated drugs, alone or combination for 72 hours, were cultured in drug-free medium for additional 7-21 days. Surviving cells after the treatment were fixed and visualized by crystal violet staining.


Figure 2 C , Clongenic assay of KRAS - mutant lung and pancreatic cells ( MIA PaCa - 2 , H2122 , H2009 , H23 , H358 and A549 ) treated with control ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or combination . D , Apoptotic assay of KRAS - mutant cancer cells ( H358 , H441 , A549 , and SW620 ) and KRAS - WT lung cancer cells ( EBC - 1 ) treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or in combination for 48h . Data are presented as mean ± SD ( n = 3 ) . * * * P < 0 . 001 and * * * * P < 0 . 0001 by two - way ANOVA with Tukey ' s multiple comparisons test . E , KRAS mutant lung ( A549 , H358 ， H441 ) , pancreatic ( MIAPaCa - 2 ) and colon ( SW620 ) cancer cells were transfected with control siRNAs , FGFR1 - and PLK1 - specific siRNAs , alone or combination . Cell viability was determined 72 h post transfection . Data are presented as mean ± SD ( n = 3 ) . * * p < 0 . 01 , * * * * p < 0 . 0001 , and ns P ＞ 0 . 05 by two - way ANOVA with Tukey ' s multiple comparisons test . ns , no significance .

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

Figure 2 C, Clongenic assay of KRAS-mutant lung and pancreatic cells (MIA PaCa-2, H2122, H2009, H23, H358 and A549) treated with control (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination.  D, Apoptotic assay of KRAS-mutant cancer cells (H358, H441, A549, and SW620) and KRAS-WT lung cancer cells (EBC-1) treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 48h. Data are presented as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.  E, KRAS mutant lung (A549, H358，H441), pancreatic (MIAPaCa-2) and colon (SW620) cancer cells were transfected with control siRNAs, FGFR1- and PLK1-specific siRNAs, alone or combination. Cell viability was determined 72 h post transfection. Data are presented as mean ± SD (n=3). **p<0.01, ****p<0.0001, and ns P＞0.05 by two-way ANOVA with Tukey's multiple comparisons test. ns, no significance.


Figure 3 D , Clonogenic assay of H358 , A549 and MIA PaCa - 2 cells treated with AZD4547 , BI2536 , and NAC , alone or in combination as indicated . E , Immunoblots of H358 and A549 cells treated for 24 h with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , in the presence or absence of NAC ( 5 mM ) . clonogenic assay of H358 cells treated for 48 h with vehicle , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , in the presence or absence of Q - VD - OPh ( 20 µM ) . Data shown as mean ± SD ( n = 3 ) . * * * P < 0 . 001 and * * * * P < 0 . 0001 by two - way ANOVA with Tukey ' s multiple comparisons test .

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

Figure 3 D, Clonogenic assay of H358, A549 and MIA PaCa-2 cells treated with AZD4547, BI2536, and NAC, alone or in combination as indicated.  E, Immunoblots of H358 and A549 cells treated for 24 h with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of NAC (5 mM).  clonogenic assay of H358 cells treated for 48 h with vehicle, AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of Q-VD-OPh (20 µM). Data shown as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.