Summary of the results obtained using the cytosolic CA activity detection method shown in A and quantified as the percentage of cells showing cytosolic CA activity . Data from CA II KO and CA II / VII KO neurons were obtained only at > P35 . The number of cells tested is indicated for each bar . The animal numbers for WT mice at the different age points was n = 3 ( P5 - 8 ) , n = 2 ( P10 - 11 ) , n = 3 ( P14 - 16 ) , n = 2 ( P17 - 18 ) , n = 2 ( P20 - 30 ) and n = 2 ( > P35 ) ; for CAVII KO mice the numbers were n = 3 ( P5 - 8 ) , n = 2 ( P10 - 11 ) , n = 5 ( P14 - 16 ) , n = 3 ( P17 - 18 ) , n = 4 ( P20 - 30 ) and n = 2 ( > P35 ) ; and for CA II KO and CA II / CAVII KO mice the number was n = 2 ( > P35 ) .

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

Summary of the results obtained using the cytosolic CA activity detection method shown in A and quantified as the percentage of cells showing cytosolic CA activity. Data from CA II KO and CA II/VII KO neurons were obtained only at >P35. The number of cells tested is indicated for each bar. The animal numbers for WT mice at the different age points was n=3 (P5-8), n=2 (P10-11), n=3 (P14-16), n=2 (P17-18), n=2 (P20-30) and n=2 (>P35); for CAVII KO mice the numbers were n=3 (P5-8), n=2 (P10-11), n=5 (P14-16), n=3 (P17-18), n=4 (P20-30) and n=2 (>P35); and for CA II KO and CA II/CAVII KO mice the number was n=2 (>P35).


Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2 / HCO3 − ( horizontal bar , HEPES ) in slices from > P35 WT , CAVII KO and CA II / VII KO mice . The rate of rise and the amplitude of the alkaline shift are identical in WT ( P39 , baseline pHi 7 . 01 ) and CAVII KO neurons ( P40 , 7 . 06 ) , whereas alkalinization develops much more slowly in the CA II / VII KO ( P46 , 7 . 04 )

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

Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2/HCO3− (horizontal bar, HEPES) in slices from >P35 WT, CAVII KO and CA II/VII KO mice. The rate of rise and the amplitude of the alkaline shift are identical in WT (P39, baseline pHi 7.01) and CAVII KO neurons (P40, 7.06), whereas alkalinization develops much more slowly in the CA II/VII KO (P46, 7.04)


Excitatory GABAergic response in WT , but not in CAVII KO neurons . Picrotoxin blocked the biphasic response ( PiTX 90 μM ; n = 3 / genotype ) . The mean initial slope of the depolarizing shift was faster in WT than in CAVII KO ( 13 . 53 ± 1 . 27 versus 6 . 39 ± 0 . 96 mV / s , P < 0 . 001 , Student ' s t - test ) . The resting membrane potential of WT and CAVII KO neurons did not differ ( − 71 . 0 ± 1 . 1 versus − 72 . 3 ± 1 . 4 mV , respectively ; n = 15 neurons for both genotypes , P = 0 . 47 , Student ' s t - test ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D )

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

Excitatory GABAergic response in WT, but not in CAVII KO neurons. Picrotoxin blocked the biphasic response (PiTX 90 μM; n=3/genotype). The mean initial slope of the depolarizing shift was faster in WT than in CAVII KO (13.53±1.27 versus 6.39±0.96 mV/s, P<0.001, Student's t-test). The resting membrane potential of WT and CAVII KO neurons did not differ (−71.0±1.1 versus −72.3±1.4 mV, respectively; n=15 neurons for both genotypes, P=0.47, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)


Microinjection of GABA ( 5 mM for 100 ms , sr / slm border in the presence of CGP55845 / TTX , arrowheads ) induced a pronounced depolarization in WT neurons ( black ) that was abolished in the absence of CO2 / HCO3 − ( grey ) . The depolarization was smaller in CA VII KO ( red ) . Bar diagram illustrates GABAergic mean peak depolarization in P12 - 16 WT and CAVII KO ( 13 . 3 ± 0 . 5 versus 8 . 3 ± 0 . 6 mV , P < 0 . 001 , Student ' s t - test ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D ) .

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

Microinjection of GABA (5 mM for 100 ms, sr/slm border in the presence of CGP55845/TTX, arrowheads) induced a pronounced depolarization in WT neurons (black) that was abolished in the absence of CO2/HCO3− (grey). The depolarization was smaller in CA VII KO (red). Bar diagram illustrates GABAergic mean peak depolarization in P12-16 WT and CAVII KO (13.3±0.5 versus 8.3±0.6 mV, P<0.001, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D).


In the presence of AP5 / CNQX / CGP55845 , microinjection of GABA ( 5 mM , sr / slm border ) triggered SR 95531 - sensitive field potential spikes in WT slices already upon 8 - ms injection , while even 16 - ms injection evoked hardly any spikes in CAVII KO ( left ) . Mean number of spikes was plotted against GABA puff duration in P12 - 16 WT and CAVII KO ( P < 0 . 01 and P < 0 . 001 , Student ' s t - test ; right ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D )

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

In the presence of AP5/CNQX/CGP55845, microinjection of GABA (5 mM, sr/slm border) triggered SR 95531-sensitive field potential spikes in WT slices already upon 8-ms injection, while even 16-ms injection evoked hardly any spikes in CAVII KO (left). Mean number of spikes was plotted against GABA puff duration in P12-16 WT and CAVII KO (P<0.01 and P<0.001, Student's t-test; right). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)


After P35 , depolarizations upon GABA microinjection ( arrowheads ) in the presence of CGP55845 / TTX were indistinguishable in WT ( black ) , CA VII KO ( red ) and CA II KO ( blue ) but smaller in CA II / VII KO ( green ) . The depolarization in WT was abolished in the absence of CO2 / HCO3 − ( grey , n = 5 ) . Bar diagram summarizes mean peak depolarization for each genotype ( right ; ANOVA , Bonferroni ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D )

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

After P35, depolarizations upon GABA microinjection (arrowheads) in the presence of CGP55845/TTX were indistinguishable in WT (black), CA VII KO (red) and CA II KO (blue) but smaller in CA II/VII KO (green). The depolarization in WT was abolished in the absence of CO2/HCO3− (grey, n=5). Bar diagram summarizes mean peak depolarization for each genotype (right; ANOVA, Bonferroni). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)


Representative raw traces of epidural EEG recordings during hyperthermia ( HT ) from a WT and a CA VII KO mouse . Seizures were reliably induced in WT ( n = 9 ) but not in CA VII KO ( n = 7 ) . Error bars denote s . e . m . , all P - values are based on Student ' s t - test ( * * * P < 0 . 001 ) , and animal number is given in bar diagrams . Rectal temperatures are given above the traces .

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

Representative raw traces of epidural EEG recordings during hyperthermia (HT) from a WT and a CA VII KO mouse. Seizures were reliably induced in WT (n=9) but not in CA VII KO (n=7). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Specimen recordings of breath rate from a WT and a CA VII KO mouse . The baseline breath rate ( WT 153 ± 11 breaths / min and CA VII KO 157 ± 8 breaths / min ) was increased during HT in both genotypes ( WT 272 ± 28 breaths / min and CA VII KO 279 ± 12 breaths / min ) . The bar diagram summarizes the mean increase in breath rate in WT ( 177 ± 12 % ) and CA VII KO mice ( 178 ± 6 % , P = 0 . 98 ) . Error bars denote s . e . m . , all P - values are based on Student ' s t - test ( * * * P < 0 . 001 ) , and animal number is given in bar diagrams . Rectal temperatures are given above the traces .

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

Specimen recordings of breath rate from a WT and a CA VII KO mouse. The baseline breath rate (WT 153±11 breaths/min and CA VII KO 157±8 breaths/min) was increased during HT in both genotypes (WT 272±28 breaths/min and CA VII KO 279±12 breaths/min). The bar diagram summarizes the mean increase in breath rate in WT (177±12%) and CA VII KO mice (178±6%, P=0.98).Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Hyperthermia caused a comparable alkalosis in blood pH in both WT and CA VII KO mice . The blood pH did not differ between the two genotypes under control conditions ( P = 0 . 37 ) or during HT ( P = 0 . 44 ) . Error bars denote s . e . m . , all P - values are based on Student ' s t - test ( * * * P < 0 . 001 ) , and animal number is given in bar diagrams . Rectal temperatures are given above the traces .

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

Hyperthermia caused a comparable alkalosis in blood pH in both WT and CA VII KO mice. The blood pH did not differ between the two genotypes under control conditions (P=0.37) or during HT (P=0.44). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Electrographic seizures were not detected in WT mice when hyperthermia was induced in the continuous presence of 5 % CO2 ( n = 5 ) . Error bars denote s . e . m . , all P - values are based on Student ' s t - test ( * * * P < 0 . 001 ) , and animal number is given in bar diagrams . Rectal temperatures are given above the traces .

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

Electrographic seizures were not detected in WT mice when hyperthermia was induced in the continuous presence of 5% CO2 (n=5). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Diazepam ( DZP , 150 μg / kg ) reduced the latency to seizure onset in P14 rat pups ( P < 0 . 05 ) . There was no statistical difference between 50 μg / kg and saline - injected animals . DZP 2 . 5 mg / kg completely blocked the seizures . DZP was given intraperitoneally 15 min before hyperthermia ( HT ) onset .

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

Diazepam (DZP, 150 μg/kg) reduced the latency to seizure onset in P14 rat pups (P<0.05). There was no statistical difference between 50 μg/kg and saline-injected animals. DZP 2.5 mg/kg completely blocked the seizures. DZP was given intraperitoneally 15 min before hyperthermia (HT) onset.


Diazepam given at 150 μg / kg did not affect the baseline breath rate , and HT induced an analogous increase in the breath rate in saline - and diazepam - injected rats ( P > 0 . 05 ) . At a higher concentration ( 2 . 5 mg / kg ) , DZP suppressed breathing significantly ( P < 0 . 01 ) and HT caused only a small increase in the breath rate .

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

Diazepam given at 150 μg/kg did not affect the baseline breath rate, and HT induced an analogous increase in the breath rate in saline- and diazepam-injected rats (P>0.05). At a higher concentration (2.5 mg/kg), DZP suppressed breathing significantly (P<0.01) and HT caused only a small increase in the breath rate.


In slice preparation , DZP ( 1 μM ) potentiated the high - frequency stimulation ( 40 pulses at 100 Hz , horizontal bar ) - evoked GABAAR - mediated depolarization . Sample trace from a slice from a P14 rat pup where stimulation to the border of stratum radiatum and stratum lacunosum - moleculare was given in the presence of AP5 , CNQX and CGP 55845 . The bar diagrams summarize the effects of DZP on the duration of the depolarization ( time to half - maximum ) and the number of action potentials associated with the depolarizing phase . P - values ( * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ) are based on ANOVA , Bonferroni ( A , B ) or Student ' s t - test ( C ) , and error bars denote s . e . m . The number of animals ( A , B ) or cells ( C ) is indicated in bar diagrams .

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

In slice preparation, DZP (1 μM) potentiated the high-frequency stimulation (40 pulses at 100 Hz, horizontal bar) -evoked GABAAR-mediated depolarization. Sample trace from a slice from a P14 rat pup where stimulation to the border of stratum radiatum and stratum lacunosum-moleculare was given in the presence of AP5, CNQX and CGP 55845. The bar diagrams summarize the effects of DZP on the duration of the depolarization (time to half-maximum) and the number of action potentials associated with the depolarizing phase. P-values (*P<0.05, **P<0.01 and ***P<0.001) are based on ANOVA, Bonferroni (A, B) or Student's t-test (C), and error bars denote s.e.m. The number of animals (A, B) or cells (C) is indicated in bar diagrams.


CA VII expression during human brain development . Line plots show the log2 - transformed exon array signal intensity in the neocortex ( NCX ) and hippocampal formation ( HIP ) from the early fetal period to late adulthood . The solid line between periods 7 and 8 separates the prenatal from the postnatal period .

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

CA VII expression during human brain development. Line plots show the log2-transformed exon array signal intensity in the neocortex (NCX) and hippocampal formation (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.


CA VII expression during human brain development . Line plots show the log . Line plots show the normalized ratio of absolute copy number of CA VII to Gapdh by droplet digital PCR ( ddPCR , right ) in the neocortex ( NCX ) and hippocampus ( HIP ) from the early fetal period to late adulthood . The solid line between periods 7 and 8 separates the prenatal from the postnatal period .

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

CA VII expression during human brain development. Line plots show the log. Line plots show the normalized ratio of absolute copy number of CA VII to Gapdh by droplet digital PCR (ddPCR, right) in the neocortex (NCX) and hippocampus (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.


( B ) Representative images of importin β cargo gradients detected by Rango - 3 FLIM / FRET imaging . Scale bars , 20µm .

O 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) Representative images of importin β cargo gradients detected by Rango-3 FLIM/FRET imaging. Scale bars, 20µm.


( C ) Line scans of Rango - 3 FRET efficiency ( E ) in 10 µm - wide

(C) Line scans of Rango-3 FRET efficiency (E) in 10 µm-wide areas


( D - F ) Quantification of the Rango - 3 E gradients ( top panels ) and average cellular Rango - 3 E ( bottom ) in involving untreated oocytes ( controls ) or oocytes injected with RanT24N mRNA , importin β ( 71 - 876 ) protein or treated with 20 µM IPZ . Data for each panel are from at least 2 separate experiments . Means ± SDs , t - test , oocyte numbers indicated in brackets .

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

(D-F) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) in involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20 µM IPZ. Data for each panel are from at least 2 separate experiments. Means ± SDs, t-test, oocyte numbers indicated in brackets.


( G ) Nonlinear single - exponential regression was used to fit the average of radial Rango - 3 τdonor line scans in control oocyte with the one - phase decay model . The dashed line corresponds to the average of spindle pole distance from chromosomes in the same cell .

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

(G) Nonlinear single-exponential regression was used to fit the average of radial Rango-3 τdonor line scans in control oocyte with the one-phase decay model. The dashed line corresponds to the average of spindle pole distance from chromosomes in the same cell.


( H ) Immunoblotting in biochemical pull - downs with recombinant Ran proteins added to human DL D1 cell lysates . A representative of at least 5 repeats .

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

(H) Immunoblotting in biochemical pull-downs with recombinant Ran proteins added to human DL D1 cell lysates. A representative of at least 5 repeats.


( A ) Quantification of chromosomal cargo gradients ( top ) and average Rango - 3 E in oocytes injected with increasing concentrations of Ran mutant mRNA concentrations . Data from oocytes imaged side - by - side within one experiment . Means ± SDs , one - side ANOVA , Tukey ' s test . Numbers of analyzed oocytes are indicated in the brackets in the top panel and are identical for both panels .

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

(A) Quantification of chromosomal cargo gradients (top) and average Rango-3 E in oocytes injected with increasing concentrations of Ran mutant mRNA concentrations. Data from oocytes imaged side-by-side within one experiment. Means ± SDs, one-side ANOVA, Tukey's test. Numbers of analyzed oocytes are indicated in the brackets in the top panel and are identical for both panels.


( C ) Linescans of the estimated Rango - 3 FRET efficiency ( E ) in 10 µm - wide

(C) Linescans of the estimated Rango-3 FRET efficiency (E) in 10 µm-wide areas


( D ) Quantification of the Rango - 3 E gradients ( top panels ) and average cellular Rango - 3 E ( bottom ) oocytes that were treated side - by - side by the injection of mRNA for Rango - 3 , alongside with buffer ( controls ) or 2000ng / µl mRNA for RanT24N or RanT24N , T42A . Means ± SDs , one - side ANOVA , Tukey ' s test , oocyte numbers indicated in brackets .

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

(D) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) oocytes that were treated side-by-side by the injection of mRNA for Rango-3, alongside with buffer (controls) or 2000ng/µl mRNA for RanT24N or RanT24N, T42A. Means ± SDs, one-side ANOVA, Tukey's test, oocyte numbers indicated in brackets.


( B ) Chromosomal gradients detected by RBP3 FLIM / FRET imaging . Scale bars , 20µm .

(B) Chromosomal gradients detected by RBP3 FLIM/FRET imaging. Scale bars, 20µm.


( C ) Line scans of the estimated RBP - 3 in 10 µm - wide

(C) Line scans of the estimated RBP-3 in 10 µm-wide areas


( D - F ) Quantification of the RBP - 3 gradients ( top panels ) and average cellular RBP - 3 E ( bottom ) in separate experiments involving untreated oocytes ( controls ) or oocytes injected with RanT24N mRNA , importin β ( 71 - 876 ) protein or treated with 20µM IPZ . Means ± SDs , t - test , oocyte numbers indicated in brackets .

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

(D-F) Quantification of the RBP-3 gradients (top panels) and average cellular RBP-3 E (bottom) in separate experiments involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20µM IPZ. Means ± SDs, t-test, oocyte numbers indicated in brackets.


( G ) Nonlinear single - exponential regression was used to fit the average of radial RBP - 3 τdonor line scans in control oocyte with the one - phase decay model .

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

(G) Nonlinear single-exponential regression was used to fit the average of radial RBP-3 τdonor line scans in control oocyte with the one-phase decay model.


( H ) Images of oocytes expressing GFP - Ran and RCC1 - mCherry from injected mRNAs before FRAP assays . Scale bars , 20 µm .

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

(H) Images of oocytes expressing GFP-Ran and RCC1-mCherry from injected mRNAs before FRAP assays. Scale bars, 20 µm.


( I ) RCC1 - mCherry half - time fluorescence recovery ( right , means ± SDs , t - test . ) and an immobile fraction ( left , means ± SDs , Mann - Whitney - test ) in control and IPZ - treated cells , oocyte numbers indicated in brackets .

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

(I) RCC1-mCherry half-time fluorescence recovery (right, means ± SDs, t-test.) and an immobile fraction (left, means ± SDs, Mann-Whitney-test) in control and IPZ-treated cells, oocyte numbers indicated in brackets.


( A ) Dose - dependent effects of IPZ on MI spindle assembly are shown by maximum z - projection images from confocal time - lapse imaging of oocytes obtained from transgenic histone H2B - EGFP mice stained with SiR - tubulin . Dashed circles indicate oocyte edges ; scale bars , 10 µm .

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(A) Dose-dependent effects of IPZ on MI spindle assembly are shown by maximum z-projection images from confocal time-lapse imaging of oocytes obtained from transgenic histone H2B-EGFP mice stained with SiR-tubulin. Dashed circles indicate oocyte edges; scale bars, 10 µm.


( B - C ) Live - cell imaging of oocytes was used to quantify the effects of IPZ on GVBD ( B ) and anaphase I entry ( C ) . Anaphase I was defined as the first time point when chromosome segregation was visible . Fisher ' s exact test , oocyte numbers are indicated in brackets .

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(B-C) Live-cell imaging of oocytes was used to quantify the effects of IPZ on GVBD (B) and anaphase I entry (C). Anaphase I was defined as the first time point when chromosome segregation was visible. Fisher's exact test, oocyte numbers are indicated in brackets.


( D ) Frequency and severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at the top of the bars .

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(D) Frequency and severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at the top of the bars.


( E ) MI spindle formation in oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry and stained with SiR - tubulin matured in the presence of 15 µM IPZ . Representative still images of maximum z - projections are shown . Time in hh : mm is relative to GVBD . Scale bar , 10 µm .

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(E) MI spindle formation in oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin matured in the presence of 15 µM IPZ. Representative still images of maximum z-projections are shown. Time in hh:mm is relative to GVBD. Scale bar, 10 µm.


( F ) MT intensity at MTOCs at the time of GVBD quantified from SiR - tubulin signal that was normalized to mean cytoplasmic SiR - tubulin signal . Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group ( means ± SDs , Mann - Whitney - test ) . Total of 184 MTOC in 17 control oocytes and a total of 170 MTOCs in 17 IPZ - treated oocytes were analyzed . ( G ) Time course of the MT intensity in the area of spindle after GVBD . Mean of MT intensity was arbitrarily centered to 1 in the control group ( means ± SDs ) . Oocyte numbers are indicated in brackets .

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(F) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group (means ± SDs, Mann-Whitney-test). Total of 184 MTOC in 17 control oocytes and a total of 170 MTOCs in 17 IPZ-treated oocytes were analyzed. (G) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group (means ± SDs). Oocyte numbers are indicated in brackets.


( H ) Oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry and stained with SiR - tubulin were treated with 15 µM IPZ after being cultured for 5h in control media . Maximum intensity z - projections from confocal live - cell imaging are shown . Time in hh : mm is relative to GVBD , DMSO or 15 uM IPZ was added at time 5 : 00 . Scale bars , 10 µm .

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(H) Oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin were treated with 15 µM IPZ after being cultured for 5h in control media. Maximum intensity z-projections from confocal live-cell imaging are shown. Time in hh:mm is relative to GVBD, DMSO or 15 uM IPZ was added at time 5:00. Scale bars, 10 µm.


( I ) . Spindle volume at 1h and metaphase I after IPZ addition . The metaphase time was defined as the last time point before anaphase or , in the IPZ - treated oocytes that failed anaphase entry , as the average control metaphase time . Means ± SDs , Mann - Whitney test , oocyte numbers are indicated in brackets .

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(I). Spindle volume at 1h and metaphase I after IPZ addition. The metaphase time was defined as the last time point before anaphase or, in the IPZ-treated oocytes that failed anaphase entry, as the average control metaphase time. Means ± SDs, Mann-Whitney test, oocyte numbers are indicated in brackets.


( J ) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media . Oocyte numbers are indicated in brackets .

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(J) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media. Oocyte numbers are indicated in brackets.


( A ) The rescue of the effects of 15 µM IPZ on MI spindles was examined in mouse oocytes expressing histone H2B - mCherry from microinjected mRNA , co - stained with SiR - tubulin and noninjected or injected with mRNAs coding for RanQ69L or hTPX2 . Maximum intensity z - stack projections from confocal live - cell movies 30 min after GVBD , at metaphase I and anaphase I . Time in hh : mm is relative to GVBD . Scale bars , 10 µm .

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(A) The rescue of the effects of 15 µM IPZ on MI spindles was examined in mouse oocytes expressing histone H2B-mCherry from microinjected mRNA, co-stained with SiR-tubulin and noninjected or injected with mRNAs coding for RanQ69L or hTPX2. Maximum intensity z-stack projections from confocal live-cell movies 30 min after GVBD, at metaphase I and anaphase I. Time in hh: mm is relative to GVBD. Scale bars, 10 µm.


( B - C ) The frequency of GVBD ( B ) and anaphase I entry ( C ) quantified by live confocal imaging of oocytes stained and treated Fisher ' s exact test , oocyte numbers are shown in brackets . ( D ) Changes in spindle volume over time after GVBD . Means ± SDs , mixed - design ANOVA , oocyte numbers are shown in brackets .

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(B-C) The frequency of GVBD (B) and anaphase I entry (C) quantified by live confocal imaging of oocytes stained and treated Fisher's exact test, oocyte numbers are shown in brackets. (D) Changes in spindle volume over time after GVBD. Means ± SDs, mixed-design ANOVA, oocyte numbers are shown in brackets.


( A ) The effects of RanT24N , T42A , and RanT24N on MI spindle assembly observed in maximum z - projection images from confocal time - lapse imaging of oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry and stained with SiR - tubulin . Time in hh : mm is relative to GVBD . Scale bar , 10 µm . ( B , C ) Live - cell imaging of oocytes as in ( A ) was used to quantify the effects of Ran mutant . Means ± SDs . , Mann - Whitney test , oocyte numbers are indicated in brackets .

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(A) The effects of RanT24N, T42A, and RanT24N on MI spindle assembly observed in maximum z-projection images from confocal time-lapse imaging of oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin. Time in hh:mm is relative to GVBD. Scale bar, 10 µm. (B, C) Live-cell imaging of oocytes as in (A) was used to quantify the effects of Ran mutant. Means ± SDs., Mann-Whitney test, oocyte numbers are indicated in brackets.


( D ) MT intensity at MTOCs at the time of GVBD quantified from SiR - tubulin signal that was normalized to mean cytoplasmic SiR - tubulin signal . Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group . Means ± SDs , Kruskal - Wallis , and Dunn ' s tests . 25 control oocytes with 264 MTOCs in total , 10 RanT24N - expression oocytes with 70 MTOCs in total and 19 RanT24N , T42A - expressing oocytes with 96 MTOCs in total were analyzed .

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(D) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group. Means ± SDs, Kruskal-Wallis, and Dunn's tests. 25 control oocytes with 264 MTOCs in total, 10 RanT24N-expression oocytes with 70 MTOCs in total and 19 RanT24N, T42A-expressing oocytes with 96 MTOCs in total were analyzed.


( E ) Time course of the MT intensity in the area of spindle after GVBD . Mean of MT intensity was arbitrarily centered to 1 in the control group . Means ± SDs , mixed - design ANOVA , oocyte numbers are shown in brackets .

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(E) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group. Means ± SDs, mixed-design ANOVA, oocyte numbers are shown in brackets.


( F ) Frequency and severity of MI spindle phenotypes in the oocytes expressing RanT24N or RanT24N , T42A . Oocyte numbers are shown in brackets .

(F) Frequency and severity of MI spindle phenotypes in the oocytes expressing RanT24N or RanT24N, T42A. Oocyte numbers are shown in brackets.


( G , H ) Quantification of the percentage of oocytes ( G ) with chromosome segregation errors in anaphase I and ( H ) with defective MTOC coalescence during metaphase I . Means ± SDs , oocyte numbers are shown in brackets .

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(G, H) Quantification of the percentage of oocytes (G) with chromosome segregation errors in anaphase I and (H) with defective MTOC coalescence during metaphase I. Means ± SDs, oocyte numbers are shown in brackets.


( I ) Detection of MTs and pericentrin - containing MTOCs by IF in control and RanT24N , T42A - expressing oocytes . Arrow indicates unaligned chromosome . ( J ) Quantification of chromosomal spread area in oocytes depicted in ( I ) , Means ± SDs , Mann - Whitney test , oocyte numbers indicated in brackets .

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(I) Detection of MTs and pericentrin-containing MTOCs by IF in control and RanT24N, T42A - expressing oocytes. Arrow indicates unaligned chromosome. (J) Quantification of chromosomal spread area in oocytes depicted in (I), Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets.


( A ) . Maximum intensity z - stack projections of confocal IF images ( DNA stain , indirect IF for tubulin and TPX2 ) in untreated oocytes ( control ) , oocytes expressing RanT24N or injected with importin β ( 71 - 876 ) . The representative of at least 5 experiments is shown .

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(A). Maximum intensity z-stack projections of confocal IF images (DNA stain, indirect IF for tubulin and TPX2) in untreated oocytes (control), oocytes expressing RanT24N or injected with importin β(71-876). The representative of at least 5 experiments is shown.


( B - D ) Maximum intensity z - stack projections of IF images in control and importin β ( 71 - 876 ) - injected cells were used to quantify the spindle vs . whole - cell area fraction ( B ) and the ratios of the average signal in the spindle vs . cytoplasm for the tubulin ( C ) and TPX2 ( D ) staining . Means ± SDs , with t - test in ( B , C ) and Mann - Whitney test in ( D ) , oocyte numbers indicated in brackets .

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(B-D) Maximum intensity z-stack projections of IF images in control and importin β(71-876)-injected cells were used to quantify the spindle vs. whole-cell area fraction (B) and the ratios of the average signal in the spindle vs. cytoplasm for the tubulin (C) and TPX2 (D) staining. Means ± SDs, with t-test in (B, C) and Mann-Whitney test in (D), oocyte numbers indicated in brackets.


( E ) Ratios of the spindle - associated fractions of TPX2 vs . tubulin were calculated in maximum intensity z - stack projections of IF images . Means ± SDs , Mann - Whitney test , oocyte numbers indicated in brackets .

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(E) Ratios of the spindle-associated fractions of TPX2 vs. tubulin were calculated in maximum intensity z-stack projections of IF images. Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets.


( F ) Maximum intensity z - stack projections of IF images were used to quantify the number of ectopic MT asters . Means ± SDs , ANOVA with Kruskal - Wallis and Dunn ' s tests , oocyte numbers indicated in brackets .

(F) Maximum intensity z-stack projections of IF images were used to quantify the number of ectopic MT asters. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests, oocyte numbers indicated in brackets.


( G ) Maximum intensity z - stack projections of confocal IF images stained for DNA , pericentrin , and tubulin ( directly labeled primary antibody ) . A representative example of at least 2 experiments is shown . Scale bar , 20 µm . ( H - I ) The number and size of pericentrin foci were quantified in maximum - intensity z - stack projections of IF images stained as in ( G ) in at least 2 separate experiments . Means ± SDs , ANOVA with Kruskal - Wallis and Dunn ' s tests .

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(G) Maximum intensity z-stack projections of confocal IF images stained for DNA, pericentrin, and tubulin (directly labeled primary antibody). A representative example of at least 2 experiments is shown. Scale bar, 20 µm. (H-I) The number and size of pericentrin foci were quantified in maximum-intensity z-stack projections of IF images stained as in (G) in at least 2 separate experiments. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests.


( a ) Confocal microscopy of synchronized HeLa cells stained for VHR ( green ) and BrdU ( red ) to indicate S - phase entry , and DNA ( DAPI ; blue ) . The cells were synchronized using a double thymidine block and then washed and placed in culture for the indicated times . The cells shown are representative of the majority of cells in each sample .

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(a) Confocal microscopy of synchronized HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI; blue). The cells were synchronized using a double thymidine block and then washed and placed in culture for the indicated times. The cells shown are representative of the majority of cells in each sample.


b ) Anti - VHR , anti - actin and anti - Prc1immunoblots of lysates of the same cells .

b) Anti-VHR, anti-actin and anti-Prc1immunoblots of lysates of the same cells.


( c ) DNA histograms of the same cells at the indicated time points .

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(c) DNA histograms of the same cells at the indicated time points.


( d ) Confocal microscopy of HeLa cells at various stages of mitosis stained for tubulin ( green ) , VHR ( red ) and DNA ( DAPI ; blue ) .

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(d) Confocal microscopy of HeLa cells at various stages of mitosis stained for tubulin (green), VHR (red) and DNA (DAPI; blue).


( e ) Anti - VHR immunoblot of synchronized cells from G1 , S , G2 and M phase treated for 30 min with 50 μg ml − 1 of the protein synthesis inhibitor cycloheximide . Note that only cells in G1 showed a rapid loss of VHR protein . The scale bars represent 5 μm in a and 2 μm in b .

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(e) Anti-VHR immunoblot of synchronized cells from G1, S, G2 and M phase treated for 30 min with 50 μg ml−1 of the protein synthesis inhibitor cycloheximide. Note that only cells in G1 showed a rapid loss of VHR protein. The scale bars represent 5 μm in a and 2 μm in b.


( a ) Anti - VHR and anti - actin immunoblots of lysates of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for the indicated number of days .

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(a) Anti-VHR and anti-actin immunoblots of lysates of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for the indicated number of days.


( b ) Number of cells at the start of the experiment ( 200 , 000 ) and on day 3 for HeLa cells transfected with a control luciferase siRNA ( black bars ) or with VHR siRNA ( white bars ) .

(b) Number of cells at the start of the experiment (200,000) and on day 3 for HeLa cells transfected with a control luciferase siRNA (black bars) or with VHR siRNA (white bars).


( c ) Light microscopy of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days .

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(c) Light microscopy of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days.


( d ) Thymidine incorporation by HeLa cells transfected with a control luciferase siRNA or with VHR siRNA . The data represent the mean ± sd . ( n = 3 ) .

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(d) Thymidine incorporation by HeLa cells transfected with a control luciferase siRNA or with VHR siRNA. The data represent the mean ± sd. (n = 3).


( e ) β - galactosidase staining of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days . Note the increased number of blue - staining granules in many cells . (

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(e) β-galactosidase staining of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days. Note the increased number of blue-staining granules in many cells. (


( f ) Telomerase activity of cells treated with the indicated concentrations of VHR siRNA . Each sample was analysed in triplicate , with RNAse added to a fourth sample .

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(f) Telomerase activity of cells treated with the indicated concentrations of VHR siRNA. Each sample was analysed in triplicate, with RNAse added to a fourth sample.


( g ) Anti - VHR immunoblot of the same cells as in f . ( h ) Anti - actin control immunoblot of the same cells as in f . The scale bars represent 10 μm in c and 25 μm in e .

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(g) Anti-VHR immunoblot of the same cells as in f. (h) Anti-actin control immunoblot of the same cells as in f. The scale bars represent 10 μm in c and 25 μm in e.


( a ) Cell - cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4 - 5 days . The cells were stained with propidium iodide for DNA content and analysed by FACS after the indicated number of days in culture .

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(a) Cell-cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4-5 days. The cells were stained with propidium iodide for DNA content and analysed by FACS after the indicated number of days in culture.


( b ) Confocal microscopy of the same HeLa cells stained for VHR ( green ) and BrdU ( red ) to indicate S - phase entry , and DNA ( DAPI , blue ) . Control siRNA - treated cells and cells treated with VHR are shown . The bottom panels represent merges of the 3 images

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(b) Confocal microscopy of the same HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI, blue). Control siRNA-treated cells and cells treated with VHR are shown. The bottom panels represent merges of the 3 images above


( c ) Quantification of the BrdU positive cells in b . The indicated numbers of cells were counted and the data are given as percentage of BrdU positive cells of this total number .

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(c) Quantification of the BrdU positive cells in b. The indicated numbers of cells were counted and the data are given as percentage of BrdU positive cells of this total number.


d ) Quantification of cells staining for phosphoH3 . The indicated number of cells were counted and the data are given as percentage of phosphoH3 - positive cells of this total number .

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d) Quantification of cells staining for phosphoH3. The indicated number of cells were counted and the data are given as percentage of phosphoH3-positive cells of this total number.


( e ) Confocal microscopy of control or VHR siRNA - transfected HeLa cells stained for VHR ( green ) and phospho H3 ( red ) to indicate M - phase entry , and DNA ( DAPI , blue ) . The bottom panels represent merges of the images above .

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(e) Confocal microscopy of control or VHR siRNA-transfected HeLa cells stained for VHR (green) and phospho H3 (red) to indicate M-phase entry, and DNA (DAPI, blue). The bottom panels represent merges of the images above.


( f ) Immunoblots of total lysates of control siRNA transfected cells and VHR siRNA cells with the indicated antibodies . Note that VHR was downregulated by approximately 80 % , wheras actin levels were unchanged .

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(f) Immunoblots of total lysates of control siRNA transfected cells and VHR siRNA cells with the indicated antibodies. Note that VHR was downregulated by approximately 80%, wheras actin levels were unchanged.


( g ) Immunoblot with anti - p21Cip1 - WAF1 of total lysates of cells transfected with control siRNA ( lanes 1 - 2 ) , two different VHR siRNAs cells ( lanes 3 - 6 ) or another control siRNA ( lanes 7 - 8 ) . The scale bars represent 10 μm in b and e .

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(g) Immunoblot with anti-p21Cip1-WAF1 of total lysates of cells transfected with control siRNA (lanes 1-2), two different VHR siRNAs cells (lanes 3-6) or another control siRNA (lanes 7-8). The scale bars represent 10 μm in b and e.


( a ) Anti - VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b - e . The cells were stimulated with 10 % serum for 0 , 1 , 5 , 15 , 30 and 60 min , as indicated at the bottom of the figure . Equal amounts of total protein were loaded into each lane . ( b - e ) Immunoblots of the same lysates with the indicated antibodies for assement of MAP kinase activation .

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(a) Anti-VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b-e. The cells were stimulated with 10% serum for 0, 1, 5, 15, 30 and 60 min, as indicated at the bottom of the figure. Equal amounts of total protein were loaded into each lane. (b-e) Immunoblots of the same lysates with the indicated antibodies for assement of MAP kinase activation.


( a ) Proliferation of cells transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek , Jnk or Mek plus Jnk siRNAs . The data are calculated as percentage of control ( control siRNA alone ) and represent the mean ± s . d . from three independent experiments . The starting cell numbers were 150 , 000 or 200 , 000 and the control cells had increased by 5 . 3 , 5 . 0 and 13 - fold in the three experiments .

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(a) Proliferation of cells transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek, Jnk or Mek plus Jnk siRNAs. The data are calculated as percentage of control (control siRNA alone) and represent the mean ± s.d. from three independent experiments. The starting cell numbers were 150,000 or 200,000 and the control cells had increased by 5.3, 5.0 and 13-fold in the three experiments.


( b ) Immunoblots of the same cells with the indicated antibodies .

(b) Immunoblots of the same cells with the indicated antibodies.


( A and B ) One hundred human keratinocytes ( YF29 , passage V ) were cultured in duplicate for twelve days in individual Thermo Scientific Heracell 150 incubators with the temperature set at 35oC , 36oC , 37oC , or 38oC . Temperature of each incubator was monitored with independent thermo probes In parallel , the temperature of the culture medium was recorded every ten seconds using immerged RFID temperature sensors Sharp spikes resulted from wireless communication artifacts . ( B ) Cells were fixed and stained with 1 % Rhodamine B . Temperature did not affect colony - forming efficiency ( CFE ) but impacted colony size ; colonies were smaller when temperatures were below 37oC and larger at 38oC . Asterisks indicate thermo probes for monitoring the temperature .

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

(A and B) One hundred human keratinocytes (YF29, passage V) were cultured in duplicate for twelve days in individual Thermo Scientific Heracell 150 incubators with the temperature set at 35oC, 36oC, 37oC, or 38oC. Temperature of each incubator was monitored with independent thermo probes In parallel, the temperature of the culture medium was recorded every ten seconds using immerged RFID temperature sensors Sharp spikes resulted from wireless communication artifacts. (B) Cells were fixed and stained with 1% Rhodamine B. Temperature did not affect colony-forming efficiency (CFE) but impacted colony size; colonies were smaller when temperatures were below 37oC and larger at 38oC. Asterisks indicate thermo probes for monitoring the temperature.


( C and D ) Human keratinocytes ( YF29 ) were cultured in mass for seven days at different temperatures before the cultures were dissociated . ( C ) Colony size was quantified using an image processing software ( ImageJ ) . ( D ) The number and the size of the cells were determined using a TaliTM cytometer . Temperature impacted both cell number and size . Data information : In C and D , data are presented as the mean ± SD ( n = 4 biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Two - tailed Student ' s t - test ) . The data shown were confirmed in two technical replicates .

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(C and D) Human keratinocytes (YF29) were cultured in mass for seven days at different temperatures before the cultures were dissociated. (C) Colony size was quantified using an image processing software (ImageJ). (D) The number and the size of the cells were determined using a TaliTM cytometer. Temperature impacted both cell number and size. Data information: In C and D, data are presented as the mean ± SD (n = 4 biological replicates). * p < 0.05, ** p < 0.01 (Two-tailed Student's t-test). The data shown were confirmed in two technical replicates.


( A ) The transcripts of eight thermoTRP channels were quantified by qPCR in two different strains of human diploid keratinocytes ( YF29 and OR - CA ) . Data are presented as the mean ± SEM ( n = 3 biological replicates ) .

(A) The transcripts of eight thermoTRP channels were quantified by qPCR in two different strains of human diploid keratinocytes (YF29 and OR-CA). Data are presented as the mean ± SEM (n = 3 biological replicates).


( F and G ) YF29 cells were cultured for seven days at 37oC in a Nuaire 8700E culture incubator . Cells were then maintained at 37oC or switched to an adjacent incubator with temperature set at 32oC . Temperature was monitored with independent thermo probes in the presence or absence of rapamycin ( 100 nM ) , or a cold agonist ( menthol - 500 μM ) , a warm agonist ( 2 - APB - 200 μM ) and the vehicle ( control - ethanol 70 % ) for 30 and 60 minutes . ( F ) mTORC1 kinase activity decreases in the presence of rapamycin and when the temperature is switched from 37oC to 32oC . ( G ) mTORC1 kinase activity decreases in the presence of menthol and increases in presence of 2 - APB , indicating that ThermoTRP and mTOR signaling are connected . Rapamycin ( Rapa ) , menthol , and 2 - APB were added in the keratinocyte cultures 1 h before starting the temperature changes .

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(F and G) YF29 cells were cultured for seven days at 37oC in a Nuaire 8700E culture incubator. Cells were then maintained at 37oC or switched to an adjacent incubator with temperature set at 32oC. Temperature was monitored with independent thermo probes in the presence or absence of rapamycin (100 nM), or a cold agonist (menthol - 500 μM), a warm agonist (2-APB - 200 μM) and the vehicle (control - ethanol 70%) for 30 and 60 minutes. (F) mTORC1 kinase activity decreases in the presence of rapamycin and when the temperature is switched from 37oC to 32oC. (G) mTORC1 kinase activity decreases in the presence of menthol and increases in presence of 2-APB, indicating that ThermoTRP and mTOR signaling are connected. Rapamycin (Rapa), menthol, and 2-APB were added in the keratinocyte cultures 1 h before starting the temperature changes.


( A ) Colony - forming efficiency ( CFE ) of keratinocytes was unaltered when keratinocytes were cultured at 35oC or in the presence of 100 nM rapamycin . However , the mean area of colonies was smaller when compared to cells cultured at 37oC . ( B ) Culture at 35oC and treatment with rapamycin decreased keratinocyte proliferation as shown by the lower total number of cells after 7 days of culture compared with in the control condition . ( C ) Culture at 35oC and treatment with rapamycin also reduced cell growth as shown by a smaller mean diameter of keratinocytes compared with that in the control condition after 7 days of culture . Data information : In B and C , data are presented as the mean ± SD ( n = 4 biological replicates ) . * p < 0 . 05 ( Two - tailed Student ' s t - test ) . The data shown were confirmed in two technical replicates .

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(A) Colony-forming efficiency (CFE) of keratinocytes was unaltered when keratinocytes were cultured at 35oC or in the presence of 100 nM rapamycin. However, the mean area of colonies was smaller when compared to cells cultured at 37oC. (B) Culture at 35oC and treatment with rapamycin decreased keratinocyte proliferation as shown by the lower total number of cells after 7 days of culture compared with in the control condition. (C) Culture at 35oC and treatment with rapamycin also reduced cell growth as shown by a smaller mean diameter of keratinocytes compared with that in the control condition after 7 days of culture. Data information: In B and C, data are presented as the mean ± SD (n = 4 biological replicates). * p < 0.05 (Two-tailed Student's t-test). The data shown were confirmed in two technical replicates.


( D ) Long - term exposure to temperatures below 37oC decreased mTORC1 kinase activity .

(D) Long-term exposure to temperatures below 37oC decreased mTORC1 kinase activity.


( B ) Proteins were extracted from different YF29 subcellular components with ProteoExtractTM Subcellular Proteome Extraction kit ( Calbiochem ) . The purity of each subcellular fraction was confirmed using specific markers , GAPDH for the cytosolic fraction , calnexin ( CANX ) for the membrane and organelles fraction , keratin 5 ( KRT5 ) for the cytoskeletal fraction and HDAC2 for the nuclear fraction . mTOR , p - mTOR , RICTOR and RAPTOR were identified both in the membranes / organelles and nuclear fractions by western blotting . RICTOR and RAPTOR were also identified in the cytosolic fraction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proteins were extracted from different YF29 subcellular components with ProteoExtractTM Subcellular Proteome Extraction kit (Calbiochem). The purity of each subcellular fraction was confirmed using specific markers, GAPDH for the cytosolic fraction, calnexin (CANX) for the membrane and organelles fraction, keratin 5 (KRT5) for the cytoskeletal fraction and HDAC2 for the nuclear fraction. mTOR, p-mTOR, RICTOR and RAPTOR were identified both in the membranes/organelles and nuclear fractions by western blotting. RICTOR and RAPTOR were also identified in the cytosolic fraction.


( D ) CHO cells engineered to transiently express SNAP - mTOR , SNAP - RAPTOR , and SNAP - RICTOR ( Gautier et al . , 2008 ) were incubated with the nuclear export inhibitor leptomycin B ( LMB ) ( 6 . 4 ng / mL ) for six hours at 37 ° C . Cells were labeled with TMR - Star ( 1 μM ) for 30 min , washed and then imaged . This experiment demonstrated that mTOR , RAPTOR , and RICTOR accumulated in the nucleus after LMB treatment . Scale bars , 10μm .

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(D) CHO cells engineered to transiently express SNAP-mTOR, SNAP-RAPTOR, and SNAP-RICTOR (Gautier et al., 2008) were incubated with the nuclear export inhibitor leptomycin B (LMB) (6.4 ng/mL) for six hours at 37°C. Cells were labeled with TMR-Star (1 μM) for 30 min, washed and then imaged. This experiment demonstrated that mTOR, RAPTOR, and RICTOR accumulated in the nucleus after LMB treatment. Scale bars, 10μm.


A genome - wide expression screen was performed in parallel on normal human epidermal keratinocytes ( YF29 ) cultured for 10 days at 32oC , 35oC , 36oC , 37oC , 38oC and at 37oC in the presence of rapamycin ( 100 nM ) . ( C ) Quantitative PCR analysis of epidermal differentiation - associated genes . Data are presented as the mean ± SD ( n = 3 biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - way ANOVA with Dunnett ' s multiple comparisons test vs 37oC ) .

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A genome-wide expression screen was performed in parallel on normal human epidermal keratinocytes (YF29) cultured for 10 days at 32oC, 35oC, 36oC, 37oC, 38oC and at 37oC in the presence of rapamycin (100 nM). (C) Quantitative PCR analysis of epidermal differentiation-associated genes. Data are presented as the mean ± SD (n = 3 biological replicates). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA with Dunnett's multiple comparisons test vs 37oC).


Comprehensive miRNA expression was performed in parallel on normal human epidermal keratinocytes ( YF29 ) cultured for 10 days at 32oC , 35oC , 36oC , 37oC , 38oC and at 37oC in the presence of rapamycin ( 100 nM ) . ( B ) The MA plot showing all miRNAs without low expression or log2FC = 0 . miRNAs indicated in pink : upregulated ( ≥ 1 log2 ( FC ) ) , blue : downregulated ( ≤ - 1 log2 ( FC ) ) , grey : others ( - 1 < log2 ( FC ) < 1 ) and black : miRNAs related to epidermal differentiation . log2 ( FC ) values are calculated using the edgeR package .

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

Comprehensive miRNA expression was performed in parallel on normal human epidermal keratinocytes (YF29) cultured for 10 days at 32oC, 35oC, 36oC, 37oC, 38oC and at 37oC in the presence of rapamycin (100 nM). (B) The MA plot showing all miRNAs without low expression or log2FC = 0. miRNAs indicated in pink: upregulated (≥ 1 log2(FC)), blue: downregulated (≤ -1 log2(FC)), grey: others (-1 < log2(FC) < 1) and black: miRNAs related to epidermal differentiation. log2(FC) values are calculated using the edgeR package.


( B ) Cultured human keratinocytes ( YF29 passage III ) treated with rapamycin maintained a basal phenotype as shown by the expression of several basal ( KRT14 , BNC1 ) and terminal differentiation markers ( SPINK5 , IVL , KRT1 , KRT10 ) using quantitative PCR ( left panel ) and Western blotting ( right panel ) . Data are presented as the mean ( n = 2 biological replicates ) .

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(B) Cultured human keratinocytes (YF29 passage III) treated with rapamycin maintained a basal phenotype as shown by the expression of several basal (KRT14, BNC1) and terminal differentiation markers (SPINK5, IVL, KRT1, KRT10) using quantitative PCR (left panel) and Western blotting (right panel). Data are presented as the mean (n = 2 biological replicates).


( E ) Rapamycin - treated keratinocytes ( YF29 passage IV ) can fully regenerate an epidermis when transplanted onto SCID mice ; grafts were harvested after 71 days and immunostained with an HLA - 1 antibody to identify the human cells ( white dotted line indicates the boundary between mouse and human epidermis ) . Scale bar , 100 μm .

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(E) Rapamycin-treated keratinocytes (YF29 passage IV) can fully regenerate an epidermis when transplanted onto SCID mice; grafts were harvested after 71 days and immunostained with an HLA-1 antibody to identify the human cells (white dotted line indicates the boundary between mouse and human epidermis). Scale bar, 100 μm.


B Hops − / − mouse genotyping and protein expression . Genotyping by PCR using genomic DNA showed a 242bp band for wild type ( Hops + / + ) allele and a 331bp band for mutant ( Hops − / − ) allele . NTC is Null Template Control ( left panel ) . Genotyping by real time PCR confirmed the absence of Hops mRNA in Hops − / − mice ( middle panel ) . Protein extracts from Hops + / + and Hops − / − MEFs were analysed by Western Blot using anti - HOPS antibody and anti - α - tubulin antibody as loading control ( right panel ) . Data information : in B data are presented as mean ± SD .

O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Hops−/− mouse genotyping and protein expression. Genotyping by PCR using genomic DNA showed a 242bp band for wild type (Hops+/+) allele and a 331bp band for mutant (Hops−/−) allele. NTC is Null Template Control (left panel). Genotyping by real time PCR confirmed the absence of Hops mRNA in Hops−/− mice (middle panel). Protein extracts from Hops+/+ and Hops−/− MEFs were analysed by Western Blot using anti-HOPS antibody and anti-α-tubulin antibody as loading control (right panel). Data information: in B data are presented as mean ±SD.


C Offspring distribution . Different HOPS genotypes at weaning age ( approximately postnatal day 21 ) from crosses between homozygous knockout mice males and heterozygous females ( n = 1525 pups ) . Data information : In C the χ2 test was performed to determine significant differences between the expected and observed ratios of Hops − / − to Hops + / − mice . P < 0 . 001 , by two tailed Student ' s t - test .

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C Offspring distribution. Different HOPS genotypes at weaning age (approximately postnatal day 21) from crosses between homozygous knockout mice males and heterozygous females (n=1525 pups). Data information: In C the χ2 test was performed to determine significant differences between the expected and observed ratios of Hops−/− to Hops+/− mice. P < 0.001, by two tailed Student's t-test.


A Reduced caspase - 3 activation in Hops − / − mice upon apoptotic stimulus . Hops + / + and Hops − / − mice were treated with etoposide for the indicated times and thymus , spleen , testis and liver were harvested and Tissue - Tek ® O . C . T . embedded . Sections were immunostained with anti - cleaved caspase - 3 antibody followed by appropriate secondary antibody labelling ( green ) . The graphs display the relative positive cells number . Scale bars , 10µm . Data information : all the experiments were performed three times in three different mice per condition and a representative example is shown . In A data are presented as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test .

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A Reduced caspase-3 activation in Hops−/− mice upon apoptotic stimulus. Hops+/+ and Hops−/− mice were treated with etoposide for the indicated times and thymus, spleen, testis and liver were harvested and Tissue-Tek® O.C.T. embedded. Sections were immunostained with anti-cleaved caspase-3 antibody followed by appropriate secondary antibody labelling (green). The graphs display the relative positive cells number. Scale bars, 10µm. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In A data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


B HOPS affects p53 increase after stress in vivo . Whole - cell homogenates from organs , were immediately prepared and immunoblotted with antibodies to detect p53 and GAPDH . The p53 levels were semi - quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 1 . Data information : all the experiments were performed three times in three different mice per condition and a representative example is shown . In B data are presented as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test .

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B HOPS affects p53 increase after stress in vivo. Whole-cell homogenates from organs, were immediately prepared and immunoblotted with antibodies to detect p53 and GAPDH. The p53 levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 1. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In B data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


A Thymocytes from Hops + / + and Hops − / − mice were treated with dexamethasone ( Dex ) and the apoptotic cells were measured by flow cytometry . The flow cytometry analysis plots are shown on the left and the data represented as a histogram on the right . Data information : the experiments were performed three times in primary cells from three different mice per condition . In A data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test .

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A Thymocytes from Hops+/+ and Hops−/− mice were treated with dexamethasone (Dex) and the apoptotic cells were measured by flow cytometry. The flow cytometry analysis plots are shown on the left and the data represented as a histogram on the right. Data information: the experiments were performed three times in primary cells from three different mice per condition. In A data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


B HOPS was pivotal for apoptosis induction . Hops + / + and Hops − / − MEFs were subjected to etoposide ( Eto ) and apoptosis was analysed by flow cytometry ( left ) and showed as a graph ( right ) . Data information : the experiments were performed three times in primary cells from three different mice per condition . The experiments in MEFs were performed three times . Representative panels are shown . In data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test .

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B HOPS was pivotal for apoptosis induction. Hops+/+ and Hops−/− MEFs were subjected to etoposide (Eto) and apoptosis was analysed by flow cytometry (left) and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


C HOPS overexpression in MEFs promotes apoptosis . Hops + / + and Hops − / − MEFs were transiently transfected with an empty vector ( pEGFP - N1 ) or with a Hops encoding vector ( pEGFP - N1 - Hops ) . The apoptotic cells were measured by flow cytometry ( left ) on the GFP positive and negative populations and showed as a graph ( right ) . Data information : the experiments were performed three times in primary cells from three different mice per condition . The experiments in MEFs were performed three times . Representative panels are shown . In C data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test .

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C HOPS overexpression in MEFs promotes apoptosis. Hops+/+ and Hops−/− MEFs were transiently transfected with an empty vector (pEGFP-N1) or with a Hops encoding vector (pEGFP-N1-Hops). The apoptotic cells were measured by flow cytometry (left) on the GFP positive and negative populations and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In C data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


D HOPS affects p53 levels following stress in vitro . Hops + / + and Hops − / − MEFs were treated with etoposide and harvested at the indicated time points . The p53 protein levels were semi - quantified using α - tubulin as loading control and relative p53 levels at 0 µM dosage were assumed as 1 . Data information : the experiments were performed three times in primary cells from three different mice per condition . The experiments in MEFs were performed three times . Representative panels are shown .

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

D HOPS affects p53 levels following stress in vitro. Hops+/+ and Hops−/− MEFs were treated with etoposide and harvested at the indicated time points. The p53 protein levels were semi-quantified using α-tubulin as loading control and relative p53 levels at 0 µM dosage were assumed as 1. Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown.


A HOPS loss and p53 half - life . Hops + / + and Hops − / − MEFs untransfected or transfected with HOPS , were analysed by immunoblotting following cycloheximide treatment for the indicated time ( left panel ) . The p53 protein levels were semi - quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 100 % ( right panel ) . Data information : all the experiments in MEFs were performed three times . Representative panels are shown . In A data are presented as mean ± SD .

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

A HOPS loss and p53 half-life. Hops+/+ and Hops−/− MEFs untransfected or transfected with HOPS, were analysed by immunoblotting following cycloheximide treatment for the indicated time (left panel). The p53 protein levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 100% (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In A data are presented as mean ±SD.


B p19Arf - independent HOPS control of p53 half - life . p19Arf − / − MEFs untransfected ( CTRL ) , transfected with empty vector ( pSGV ) or HOPS were treated with cycloheximide for the indicated times and subjected to immunoblot analysis with anti - p53 , anti - HOPS and anti - GAPDH antibodies ( left panel ) . The p53 protein levels were semi - quantified as above ( right panel ) . Data information : all the experiments in MEFs were performed three times . Representative panels are shown . In B data are presented as mean ± SD .

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B p19Arf-independent HOPS control of p53 half-life. p19Arf−/− MEFs untransfected (CTRL), transfected with empty vector (pSGV) or HOPS were treated with cycloheximide for the indicated times and subjected to immunoblot analysis with anti-p53, anti-HOPS and anti-GAPDH antibodies (left panel). The p53 protein levels were semi-quantified as above (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In B data are presented as mean ±SD.


C HOPS and p53 ubiquitination . p53 , HDM2 , His - tagged ubiquitin were co - expressed in H1299 cells with HOPS or HOPS - G176A or HOPS - K126A mutants and treated with MG132 . p53 ubiquitination rate was evaluated by elution using Ni2 + - NTA His - bind resin followed by immunoblotting with anti - p53 and the indicated antibodies .

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C HOPS and p53 ubiquitination. p53, HDM2, His-tagged ubiquitin were co-expressed in H1299 cells with HOPS or HOPS-G176A or HOPS-K126A mutants and treated with MG132. p53 ubiquitination rate was evaluated by elution using Ni2+-NTA His-bind resin followed by immunoblotting with anti-p53 and the indicated antibodies.


D HOPS depletion and HOPS - G176A mutant affect p53 ubiquitination . Hops + / + and Hops − / − MEFs untransfected and Hops − / − MEFs transfected with HOPS or HOPS - G176A mutant were co - expressed with p53 , HDM2 and His - tagged ubiquitin . Cells were treated with MG132 and the ubiquitinated p53 fraction was recovered as above . Immunodetection of ubiquitinated p53 was performed using anti - p53 antibody and the samples were tested with the indicated antibodies . Data information : all the experiments in MEFs were performed three times . Representative panels are shown .

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D HOPS depletion and HOPS-G176A mutant affect p53 ubiquitination. Hops+/+ and Hops−/− MEFs untransfected and Hops−/− MEFs transfected with HOPS or HOPS-G176A mutant were co-expressed with p53, HDM2 and His-tagged ubiquitin. Cells were treated with MG132 and the ubiquitinated p53 fraction was recovered as above. Immunodetection of ubiquitinated p53 was performed using anti-p53 antibody and the samples were tested with the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.


E HOPS - G176A mutant ability to stabilize p53 . Hops − / − MEFs were transfected with HOPS - G176A , subjected to cycloheximide treatment for the indicated times and analysed by immunoblotting using the indicated antibodies . Data information : all the experiments in MEFs were performed three times . Representative panels are shown .

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E HOPS-G176A mutant ability to stabilize p53. Hops−/− MEFs were transfected with HOPS-G176A, subjected to cycloheximide treatment for the indicated times and analysed by immunoblotting using the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.


A Protein binding in RKO cells . PLA showed HOPS / p53 interaction with or without etoposide treatment at the indicated time . Red spots showed a single interaction . DNA counterstaining was performed with DAPI . α - Tubulin - FITC antibody was used to identify the cellular edge . Background control was performed according to the manufacturer ' s instruction . Scale bars , 10µm . The graph represents the binding increase . Non treated cells binding was assumed as 1 . Data information : all the experiments were performed three times , and representative examples are shown . In A data are presented as mean ± SD .

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A Protein binding in RKO cells. PLA showed HOPS/p53 interaction with or without etoposide treatment at the indicated time. Red spots showed a single interaction. DNA counterstaining was performed with DAPI. α-Tubulin-FITC antibody was used to identify the cellular edge. Background control was performed according to the manufacturer's instruction. Scale bars, 10µm. The graph represents the binding increase. Non treated cells binding was assumed as 1. Data information: all the experiments were performed three times, and representative examples are shown. In A data are presented as mean ±SD.


B Biochemical analysis of nuclear - cytoplasmic protein distribution . RKO cells were treated with etoposide , collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions . Aliquots relative to the same cell number per fraction were immunoblotted using anti - HOPS and anti - p53 antibodies . GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively .

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B Biochemical analysis of nuclear-cytoplasmic protein distribution. RKO cells were treated with etoposide, collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions. Aliquots relative to the same cell number per fraction were immunoblotted using anti-HOPS and anti-p53 antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively.


C HOPS and p53 binding in cytoplasm following apoptosis induction . RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions . WCL and cytoplasmic fractions were immunoprecipitated with anti - HOPS antibody and subsequently immunoblotted using anti - p53 antibody .

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C HOPS and p53 binding in cytoplasm following apoptosis induction. RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions. WCL and cytoplasmic fractions were immunoprecipitated with anti-HOPS antibody and subsequently immunoblotted using anti-p53 antibody.


A HOPS localization at mitochondria . RKO cells were stained by MitoTracker ™ ( red ) , immediately fixed and stained to visualize HOPS ( green ) . Nuclei were DAPI stained ( blue ) . Samples were analysed with fluorescent microscope and representative images were shown . Scale bars , 10µm .

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A HOPS localization at mitochondria. RKO cells were stained by MitoTracker™ (red), immediately fixed and stained to visualize HOPS (green). Nuclei were DAPI stained (blue). Samples were analysed with fluorescent microscope and representative images were shown. Scale bars, 10µm.


B RKO cells were lysed and processed for mitochondria isolation . WCL , cytoplasm and mitochondria were analysed using anti - HOPS antibody . GAPDH , Lamin B1 and Tom20 are purity controls of mitochondrial fraction .

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B RKO cells were lysed and processed for mitochondria isolation. WCL, cytoplasm and mitochondria were analysed using anti-HOPS antibody. GAPDH, Lamin B1 and Tom20 are purity controls of mitochondrial fraction.


C HOPS and p53 translocation to mitochondria . RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53 , HOPS and the reported extraction purity controls .

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C HOPS and p53 translocation to mitochondria. RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53, HOPS and the reported extraction purity controls.


D HOPS / p53 binding at mitochondria . RKO cells were treated with etoposide and subjected to mitochondrial purification , and subsequent immunoprecipitation using anti - HOPS antibody . Samples were immunoblotted to detect p53 , HOPS and the reported controls .

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D HOPS/p53 binding at mitochondria. RKO cells were treated with etoposide and subjected to mitochondrial purification, and subsequent immunoprecipitation using anti-HOPS antibody. Samples were immunoblotted to detect p53, HOPS and the reported controls.


E HOPS and p53 mitochondrial translocation in vivo . Hops + / + and Hops − / − mice were treated with etoposide and thymus , spleen and testis were harvested . Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53 . GAPDH and Tom20 are purity controls . p53 mitochondrial protein levels were semi - quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1 . Data information : assays involving animals were performed three times in three different mice per condition . In E data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two tailed Student ' s t - test .

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E HOPS and p53 mitochondrial translocation in vivo. Hops+/+ and Hops−/− mice were treated with etoposide and thymus, spleen and testis were harvested. Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53. GAPDH and Tom20 are purity controls. p53 mitochondrial protein levels were semi-quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1. Data information: assays involving animals were performed three times in three different mice per condition. In E data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.


F p53 - dependent mitochondrial membrane potential ( Δψm ) induction in MEFs . Hops + / + and Hops − / − MEFs were treated with etoposide and then incubated with JC - 1 vital dye . The images were collected by fluorescent microscopy and representative images are shown ( left panel ) . Scale bars , 10µm . Fluorescence was analysed by TECAN plate reader and plotted in the graph ( right panel ) . Data information : assays involving animals were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times . In F data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two tailed Student ' s t - test .

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F p53-dependent mitochondrial membrane potential (Δψm) induction in MEFs. Hops+/+ and Hops−/− MEFs were treated with etoposide and then incubated with JC-1 vital dye. The images were collected by fluorescent microscopy and representative images are shown (left panel). Scale bars, 10µm. Fluorescence was analysed by TECAN plate reader and plotted in the graph (right panel). Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times. In F data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.


G p53 MOMP induction in HOPS deficiency . Mitochondria from Hops + / + and Hops − / − MEFs , were purified and incubated with p53 recombinant protein . Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting . Samples were tested to detect p53 as control and Tom20 as mitochondria structural marker . Data information : assays involving animals were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times .

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G p53 MOMP induction in HOPS deficiency. Mitochondria from Hops+/+ and Hops−/− MEFs, were purified and incubated with p53 recombinant protein. Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting. Samples were tested to detect p53 as control and Tom20 as mitochondria structural marker. Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times.


A HOPS expression and p53 nuclear accumulation . HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 ( green ) . Nuclei were stained with DAPI ( blue ) . Samples were analysed with fluorescent microscope and merged images are shown ( right panel ) . Scale bars , 10µm . Cells were scored for p53 localization . The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells ( left panel ) . Data information : all the experiments detailed above were performed three times , and representative panels are shown . In A data are presented as mean ± SD .

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A HOPS expression and p53 nuclear accumulation. HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 (green). Nuclei were stained with DAPI (blue). Samples were analysed with fluorescent microscope and merged images are shown (right panel). Scale bars, 10µm. Cells were scored for p53 localization. The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells (left panel). Data information: all the experiments detailed above were performed three times, and representative panels are shown. In A data are presented as mean ±SD.


B Biochemical analysis of HOPS intracellular localization . RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component . Aliquots relative to the same cell number per fraction were immunoblotted using anti - p53 and anti - HOPS antibodies . GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively . Data information : all the experiments detailed above were performed three times , and representative panels are shown .

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B Biochemical analysis of HOPS intracellular localization. RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component. Aliquots relative to the same cell number per fraction were immunoblotted using anti-p53 and anti-HOPS antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


HOPS and p53 binding to importin α . Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti - p53 and analysed by immunoblotting using anti - importin α antibody . Arrowhead indicates the importin α specific signal . Data information : all the experiments detailed above were performed three times , and representative panels are shown .

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HOPS and p53 binding to importin α. Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti-p53 and analysed by immunoblotting using anti-importin α antibody. Arrowhead indicates the importin α specific signal. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


D PCAF - mediated p53 acetylation . p53 and PCAF Flag - tagged were co - expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide . p53 acetylation levels were evaluated by immunoprecipitation with anti - p53 antibody followed by immunoblotting with anti - acetylated - Lysine ( AcK ) , anti - p53 , anti - Flag , anti - HOPS and anti - α - tubulin antibodies . Data information : all the experiments detailed above were performed three times , and representative panels are shown .

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D PCAF-mediated p53 acetylation. p53 and PCAF Flag-tagged were co-expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide. p53 acetylation levels were evaluated by immunoprecipitation with anti-p53 antibody followed by immunoblotting with anti-acetylated-Lysine (AcK), anti-p53, anti-Flag, anti-HOPS and anti-α-tubulin antibodies. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


( A - B ) Live third instar discs incubated in DiBAC . DiBAC fluorescence is observed in the wing imaginal disc in both apical ( A - A ' ) , and basal ( B - B ' ) optical sections . Increased fluorescence is observed in the center of the pouch ( white arrow , B ' ) , and at the dorsoventral ( D - V ) compartment boundary in the anterior compartment ( yellow arrow , B ' , and inset , D ) .

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(A-B) Live third instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A-A'), and basal (B-B') optical sections. Increased fluorescence is observed in the center of the pouch (white arrow, B'), and at the dorsoventral (D-V) compartment boundary in the anterior compartment (yellow arrow, B', and inset, D).


( C - E ) Comparison of DiBAC with the voltage - insensitive dye FM4 - 64 . Incubation of live discs in FM4 - 64 ( E ) shows more uniform fluorescence when compared to DiBAC ( D ) . Quantitative comparison of fluorescence in the two white boxes in each panel is shown in ( C ) . N = 7 discs for each treatment , data compared using an unpaired t test , * * indicates p < 0 . 001 , error bars are standard deviations . The region boxed in yellow in ( D ) is shown at higher magnification to show DiBAC fluorescence at the D - V compartment boundary .

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(C-E) Comparison of DiBAC with the voltage-insensitive dye FM4-64. Incubation of live discs in FM4-64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N=7 discs for each treatment, data compared using an unpaired t test, ** indicates p<0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D-V compartment boundary.