Figure 5 ( A ) 3 . 8 Å resolution mFo - DFc omit map density ( green , calculated with sharpened B - factors and contoured at 2 . 0  ) reveals the presence of ADP and a short segment of RNA bound to FRH . ( B ) 3 . 8 Å resolution 2mFo - DFc calculated without ADP or RNA contributing to the model . Green electron density shown for ADP and RNA ( 0 . 6     orange density shown for surrounding protein ( 0 . 6   . ( C ) Comparison of Mtr4 and FRHADP binding ( FRH in cyan with ADP , RNA , and F179 in orange bonds ; Mtr4 in green with ADP , RNA , and F148 in yellow bonds ) . ( c ) Unwound DNA from Hel308 ( pdb 2PR6 ; magenta , DNA in orange bonds ) overlaid in FRH with RNA and ADP ( FRH in cyan with yellow RNA and cyan ADP in stick ) after superposition of the respective RecA domains . ( d ) Hel308 DNA helicase DNA ( pdb 2PR6 ; backbone in orange , bases in gray bonds ) after aligning the RecA domains of Hel308 with those of FRH ( cyan , RNA in yellow ) ; see also Appendix Fig S4 . The superposition indicates that the KOW module is positioned to interact with double stranded RNA that is being unwound by the RecA and DHSCT domains .

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 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 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 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

Figure 5(A) 3.8 Å resolution mFo-DFc omit map density (green, calculated with sharpened B-factors and contoured at 2.0) reveals the presence of ADP and a short segment of RNA bound to FRH.(B) 3.8 Å resolution 2mFo-DFc calculated without ADP or RNA contributing to the model. Green electron density shown for ADP and RNA (0.6orange density shown for surrounding protein (0.6.(C) Comparison of Mtr4 and FRHADP binding (FRH in cyan with ADP, RNA, and F179 in orange bonds; Mtr4 in green with ADP, RNA, and F148 in yellow bonds). (c) Unwound DNA from Hel308 (pdb 2PR6; magenta, DNA in orange bonds) overlaid in FRH with RNA and ADP (FRH in cyan with yellow RNA and cyan ADP in stick) after superposition of the respective RecA domains.(d) Hel308 DNA helicase DNA (pdb 2PR6; backbone in orange, bases in gray bonds) after aligning the RecA domains of Hel308 with those of FRH (cyan, RNA in yellow); see also Appendix Fig S4. The superposition indicates that the KOW module is positioned to interact with double stranded RNA that is being unwound by the RecA and DHSCT domains.


Figure 6 ( A ) Key residues for FRH function shown on the large - cell structure : K208 and S324 compose the ATP binding site , E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis ; P871 resides in the elbow region of the arch domain . K811 , R806 , R712 and K766 contribute to a positively charged surface on the KOW domain . R806H disrupts the clock . Q131 , G132 and V142 in the N - terminus provide interactions to the helicase RecA domains ( FRH domains are colored according to Fig1 , ADP and RNA are in orange bonds ) . ( B ) Same as ( A ) after rotation about a horizontal axis . ( C ) Close - up of the ADP - binding pocket and N - terminal interaction for the large - cell structure ( cyan ribbons ) and the small - cell structure ( blue ribbons ) . ( D ) Electrostatic potential surface of FRH ( blue positive , red negative ) shown with the same orientation as in ( B ) . ( E ) Close - up of the KOW domain showing the large number of basic ( aqua sticks ) and acidic side chains ( yellow sticks ) on the surface that also includes the mutational sites R806 , R712 , K811 and K766 ( grey sticks )

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 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 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 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

Figure 6(A) Key residues for FRH function shown on the large-cell structure: K208 and S324 compose the ATP binding site, E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis; P871 resides in the elbow region of the arch domain. K811, R806, R712 and K766 contribute to a positively charged surface on the KOW domain. R806H disrupts the clock. Q131, G132 and V142 in the N-terminus provide interactions to the helicase RecA domains (FRH domains are colored according to Fig1, ADP and RNA are in orange bonds). (B) Same as (A) after rotation about a horizontal axis.(C) Close-up of the ADP-binding pocket and N-terminal interaction for the large-cell structure (cyan ribbons) and the small-cell structure (blue ribbons).(D) Electrostatic potential surface of FRH (blue positive, red negative) shown with the same orientation as in (B).(E) Close-up of the KOW domain showing the large number of basic (aqua sticks) and acidic side chains (yellow sticks) on the surface that also includes the mutational sites R806, R712, K811 and K766 (grey sticks)


Figure 7 ( A ) Race tube assays with FRH mutants . Every circadian cycle N . crassa lays down aerial hyphae that appear as fluffy yellow / orange conidial bands . The distance between the bands reflects the clock period . The strains were grown in constant darkness and growth fronts were labeled every 24 h ( black vertical lines ) , rhythmicity on right ( τ = period in hours , σ = standard deviation , n = number of race tubes , ARR , arrhythmic ) . The race tubes shown here are representative samples from three replicate tubes . ( B ) Pull - down assays evaluating interactions among FRH , FRQ , WC - 1 and WC - 2 . Ratio of FRQ , WC - 1 and WC - 2 binding to the V5 tagged FRH mutants as compared to the inter - strain variations in FRH levels , error bars represent standard deviation for the assay run in triplicate . * P < 0 . 05 ( unpaired two - tailed t - test with Gaussian distribution assumption ) . Representative immunoblots are shown in Appendix Fig S5 .

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 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

Figure 7(A) Race tube assays with FRH mutants. Every circadian cycle N. crassa lays down aerial hyphae that appear as fluffy yellow/orange conidial bands. The distance between the bands reflects the clock period. The strains were grown in constant darkness and growth fronts were labeled every 24 h (black vertical lines), rhythmicity on right (τ= period in hours, σ = standard deviation, n = number of race tubes, ARR, arrhythmic). The race tubes shown here are representative samples from three replicate tubes.(B) Pull-down assays evaluating interactions among FRH, FRQ, WC-1 and WC-2. Ratio of FRQ, WC-1 and WC-2 binding to the V5 tagged FRH mutants as compared to the inter-strain variations in FRH levels, error bars represent standard deviation for the assay run in triplicate. *P < 0.05 (unpaired two-tailed t-test with Gaussian distribution assumption). Representative immunoblots are shown in Appendix Fig S5.


Figure 1B sgRNA frequencies ( log2 of fold change ) in PD - L1low cells unstimulated ( left ) or IFN - γ stimulated ( right ) . Enriched sgRNAs ( dashed line ) are highlighted ; hit genes ( black , red ) and controls ( green , yellow or purple ) are shown and labeled with target gene names . C sgRNA frequencies ( log2 of fold change ) in viability screen . sgRNAs targeting controls are highlighted ; essential ( orange ) , non - essential ( blue ) . Genes attributed to PD - L1 modulation are shown in different colors ( red , yellow and purple ) .

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 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

Figure 1B sgRNA frequencies (log2 of fold change) in PD-L1low cells unstimulated (left) or IFN-γ stimulated (right). Enriched sgRNAs (dashed line) are highlighted; hit genes (black, red) and controls (green, yellow or purple) are shown and labeled with target gene names.C sgRNA frequencies (log2 of fold change) in viability screen. sgRNAs targeting controls are highlighted; essential (orange), non-essential (blue). Genes attributed to PD-L1 modulation are shown in different colors (red, yellow and purple).


Figure 2A PD - L1 cell surface presentation ( percentage ± SD ) in RKO mock control ( black ) or monoclonal MLLT6 knockout cells ( grey ) ( Student ' s t - test ; * * p < 0 . 01 ; n = 3 biological replicates ; degrees of freedom ( df ) = 2 ) . B MLLT6 BAC mediated rescue in U2OS cells ( left ) showing percentage ± SD of PD - L1 cell surface presentation in mock control ( black ) , MLLT6 polyclonal knockout ( light grey ) and MLLT6 knockout + BAC cells ( dark grey ) ( Student ' s t - test ; * p < 0 . 05 ; n = 3 biological replicates ; df = 2 ) . Representative flow cytometry plots ( right ) of PD - L1 cell surface presentation after staining with anti - PD - L1 antibody ( top right , bottom left , bottom right ) and isotype control ( top left ) . Percentages of cells in the PD - L1 + gate as indicated . C Total cellular protein levels of PD - L1 ( top ) and GAPDH ( bottom ) in monoclonal RKO MLLT6 knockout , mock control or MLLT6 knockout + BAC cells visualized by immunoblotting . Numbers indicate relative band intensities of PD - L1 protein normalized to GAPDH . D PD - L1 transcript levels in U2OS mock ( black ) and polyclonal MLLT6 knockout lines ( grey ) treated with and without IFN - γ normalized to expression in mock U2OS cells without IFN - γ treatment ( ANOVA ; * * p < 0 . 01 ; n = 4 biological replicates ; df = 12 ) . E Representative flow cytometry plots of PD - L1 cell surface presentation in the presence and absence of IFN - γ in mock , polyclonal MLLT6 knockout and PD - L1 knockout cells . Percentages of cells in the PD - L1 + gate after staining with anti - PD - L1 antibody or isotype control as indicated . F Total cellular protein levels of PD - L1 ( top ) and GAPDH ( bottom ) in U2OS polyclonal MLLT6 knockout or mock control cells with and without IFN - γ stimulation . Numbers indicate relative band intensities of PD - L1 protein normalized to GAPDH .

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

Figure 2A PD-L1 cell surface presentation (percentage ± SD) in RKO mock control (black) or monoclonal MLLT6 knockout cells (grey) (Student's t-test; **p < 0.01; n=3 biological replicates; degrees of freedom (df)=2). B MLLT6 BAC mediated rescue in U2OS cells (left) showing percentage ± SD of PD-L1 cell surface presentation in mock control (black), MLLT6 polyclonal knockout (light grey) and MLLT6 knockout + BAC cells (dark grey) (Student's t-test; *p < 0.05; n=3 biological replicates; df=2). Representative flow cytometry plots (right) of PD-L1 cell surface presentation after staining with anti-PD-L1 antibody (top right, bottom left, bottom right) and isotype control (top left). Percentages of cells in the PD-L1+ gate as indicated.  C Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in monoclonal RKO MLLT6 knockout, mock control or MLLT6 knockout + BAC cells visualized by immunoblotting. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.  D PD-L1 transcript levels in U2OS mock (black) and polyclonal MLLT6 knockout lines (grey) treated with and without IFN-γ normalized to expression in mock U2OS cells without IFN-γ treatment (ANOVA; **p < 0.01; n=4 biological replicates; df=12).  E Representative flow cytometry plots of PD-L1 cell surface presentation in the presence and absence of IFN-γ in mock, polyclonal MLLT6 knockout and PD-L1 knockout cells. Percentages of cells in the PD-L1+ gate after staining with anti-PD-L1 antibody or isotype control as indicated.  F Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in U2OS polyclonal MLLT6 knockout or mock control cells with and without IFN-γ stimulation. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.


Figure 3 B Relative cell numbers ( ± SD ; n = 3 ; biological replicates ) of U2OS mock ( black ) or polyclonal MLLT6 knockout ( grey ) cells treated with varying amounts of EpCAM - CD3 BiTE and CTLs ( Student ' s t - test ; * * p < 0 . 01 ; degrees of freedom ( df ) = 4 ) . C Kaplan Meier survival plot ( left ) showing changes in survival ( percent ) of mock ( grey ) and MLLT6 knockout ( red ) cells treated with CTLs and EpCAM - CD3 BiTE over a course of 9 . 5 hours . Representative images ( right ) of time - lapse fluorescence microscopy following U2OS mock ( eGFP tagged , green ) and MLLT6 knockout ( mCherry tagged , red ) cells immediately ( top left and bottom left ) after adding CTLs and EpCAM - CD3 BiTE and 9 . 5 hours later ( top right and bottom right ) ( scale bars = 100 μm ) . D Relative cell numbers ( left ) ( ± SD ; n = 3 ; biological replicates ) of mock ( black ) , PD - L1 ( dark grey ) or MLLT6 ( light grey ) knockout cells treated with BiTE or bi - specific antibodies and CTLs as indicated ( Student ' s t - test ; * * p < 0 . 01 ; df = 4 ) . Representative images ( right ) from bright field microscopy of polyclonal U2OS mock ( top ) , PD - L1 ( middle ) or MLLT6 ( bottom ) knockout cells treated without or with EpCAM - CD3 BiTE or bispecific antibodies . E Relative normalized cell numbers ( ± SD ; n = 3 ; biological replicates ) of U2OS mock ( left ) , MLLT6 ( middle ) or PD - L1 ( right ) knockout cells treated with EpCAM - CD3 BiTE and CTLs in the presence ( grey ) or absence ( black ) of IFN - γ ( Student ' s t - test ; * * p < 0 . 01 ; df = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B Relative cell numbers (± SD; n=3; biological replicates) of U2OS mock (black) or polyclonal MLLT6 knockout (grey) cells treated with varying amounts of EpCAM-CD3 BiTE and CTLs (Student's t-test; **p < 0.01; degrees of freedom (df)=4).  C Kaplan Meier survival plot (left) showing changes in survival (percent) of mock (grey) and MLLT6 knockout (red) cells treated with CTLs and EpCAM-CD3 BiTE over a course of 9.5 hours. Representative images (right) of time-lapse fluorescence microscopy following U2OS mock (eGFP tagged, green) and MLLT6 knockout (mCherry tagged, red) cells immediately (top left and bottom left) after adding CTLs and EpCAM-CD3 BiTE and 9.5 hours later (top right and bottom right) (scale bars = 100 μm).  D Relative cell numbers (left) (± SD; n=3; biological replicates) of mock (black), PD-L1 (dark grey) or MLLT6 (light grey) knockout cells treated with BiTE or bi-specific antibodies and CTLs as indicated (Student's t-test; **p < 0.01; df=4). Representative images (right) from bright field microscopy of polyclonal U2OS mock (top), PD-L1 (middle) or MLLT6 (bottom) knockout cells treated without or with EpCAM-CD3 BiTE or bispecific antibodies.  E Relative normalized cell numbers (± SD; n=3; biological replicates) of U2OS mock (left), MLLT6 (middle) or PD-L1 (right) knockout cells treated with EpCAM-CD3 BiTE and CTLs in the presence (grey) or absence (black) of IFN-γ (Student's t-test; **p < 0.01; df=4 ).


Figure 4A Scatter plots of pairwise comparisons of transcript expression in U2OS cells . Plots describe log2 fold change in transcript expression in mock and polyclonal MLLT6 knockout cells treated with and without IFN - γ as indicated . Horizontal axis shows log2 fold change in transcript expression and vertical axis represents statistical significance ( log10 of p - value , n = 3 ) with candidates nominated for further validation ( red ) . B Correlation plots describing log2 fold change of transcripts in RNA sequencing ( x axis ) and Nanostring ( y axis ) measurements in mock cells + / - IFN - γ ( left ) and MLLT6 knockout cells with IFN - γ ( right ) . Transcripts previously described to have a role in tumor immunity ( red ) and PD - L1 ( blue ) are represented on the plots as indicated . C Changes ( log2 fold ) in transcript expression ( left ) of select immune - related genes in U2OS mock or MLLT6 knockout cells treated with or without IFN - γ and total protein levels measured by immunoblotting ( right ) . Numbers indicate band intensities normalized to GAPDH .

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 O 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

Figure 4A Scatter plots of pairwise comparisons of transcript expression in U2OS cells. Plots describe log2 fold change in transcript expression in mock and polyclonal MLLT6 knockout cells treated with and without IFN-γ as indicated. Horizontal axis shows log2 fold change in transcript expression and vertical axis represents statistical significance (log10 of p-value, n=3) with candidates nominated for further validation (red).B Correlation plots describing log2 fold change of transcripts in RNA sequencing (x axis) and Nanostring (y axis) measurements in mock cells +/- IFN-γ (left) and MLLT6 knockout cells with IFN-γ (right). Transcripts previously described to have a role in tumor immunity (red) and PD-L1 (blue) are represented on the plots as indicated.C Changes (log2 fold) in transcript expression (left) of select immune-related genes in U2OS mock or MLLT6 knockout cells treated with or without IFN-γ and total protein levels measured by immunoblotting (right). Numbers indicate band intensities normalized to GAPDH.


Figure 5A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN - γ . Numbers indicate band intensities normalized to GAPDH . B Immunoblot showing total protein levels of STAT1 isoforms ( α and β ) , IDO1 , GBP5 and CD74 in wild type U2OS cells transfected with esiRNAs targeting renilla luciferase ( Rluc , negative control ) , total STAT1 , STAT1α and STAT1β treated with and without IFN - γ .

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 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 5A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN-γ. Numbers indicate band intensities normalized to GAPDH.B Immunoblot showing total protein levels of STAT1 isoforms (α and β), IDO1, GBP5 and CD74 in wild type U2OS cells transfected with esiRNAs targeting renilla luciferase (Rluc, negative control), total STAT1, STAT1α and STAT1β treated with and without IFN-γ.


Figure 1 ( E ) Representative photos of iWAT , eWAT and BAT from Ftoflox / flox and FtoAKO mice fed a HFD . Scale bar , 1 cm . ( F ) Representative H & E staining of iWAT , eWAT and BAT from Ftoflox / flox and FtoAKO mice fed a HFD . Scale bar , 200 μm . ( ( I - K ) Serum levels of glucose ( I ) , triglyceride ( J ) and free fatty acid ( FFA ) ( K ) in Ftoflox / flox and FtoAKO mice fed a HFD followed by fasting . Data information : The data were presented as the mean ± SD ( n = 6 ) . Statistical analyses were performed using Student t ‐ test . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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 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

Figure 1(E) Representative photos of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 1 cm. (F) Representative H&E staining of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 200 μm. ( (I-K) Serum levels of glucose (I), triglyceride (J) and free fatty acid (FFA) (K) in Ftoflox/flox and FtoAKO mice fed a HFD followed by fasting. Data information: The data were presented as the mean ± SD (n = 6). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


Figure 2 ( A ) O2 consumption of 16 - week - old Ftoflox / flox and FtoAKO mice fed a HFD . White and gray areas in the graphs indicate day and night , respectively . ( B ) CO2 generation of 16 - week - old Ftoflox / flox and FtoAKO mice fed a HFD . White and gray areas in the graphs indicate day and night , respectively . ( Data information : The data were presented as the mean ± SD of n = 6 mice per group or triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 . ( F ) qPCR analysis of genes involved in thermogenesis , mitochondrial biogenesis and adipogenesis in iWAT of Ftoflox / flox and FtoAKO mice . ( G ) Western blot analysis of the protein levels of UCP1 , PPARGC1A and PPARG in iWAT of Ftoflox / flox and FtoAKO mice . ( I ) Immunofluorescence detection of UCP1 in iWAT from Ftoflox / flox and FtoAKO mice following 6 days cold exposure ( 4 ° C ) . Scale bar , 20 μm . ( M ) Western blot analysis of UCP1 , PPARGC1A and PPARG protein levels in differentiated beige adipocytes described in ( J ) . ( N ) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in ( J ) . Scale bar , 20 μm .

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 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 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 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 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 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

Figure 2(A) O2 consumption of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively. (B) CO2 generation of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively. ( Data information: The data were presented as the mean ± SD of n = 6 mice per group or triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗∗p < 0.01, ∗∗∗p < 0.001.(F) qPCR analysis of genes involved in thermogenesis, mitochondrial biogenesis and adipogenesis in iWAT of Ftoflox/flox and FtoAKO mice.(G) Western blot analysis of the protein levels of UCP1, PPARGC1A and PPARG in iWAT of Ftoflox/flox and FtoAKO mice.(I) Immunofluorescence detection of UCP1 in iWAT from Ftoflox/flox and FtoAKO mice following 6 days cold exposure (4°C). Scale bar, 20 μm.(M) Western blot analysis of UCP1, PPARGC1A and PPARG protein levels in differentiated beige adipocytes described in (J).(N) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (J). Scale bar, 20 μm.


Figure 3 ( G ) Western blot analysis of the protein levels of FTO , HIF1A , UCP1 , PPARGC1A and PPARG in differentiated beige adipocytes described in ( F ) .

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

Figure 3(G) Western blot analysis of the protein levels of FTO, HIF1A, UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (F).


Figure 4 ( C ) The O2 consumption rate ( OCR ) in differentiated beige adipocytes from 3T3 - L1 cells overexpressing Vec or HIF1A plasmid . The average basal and maximal respiration rates are shown in right panel . Data information : The data were presented as the mean ± SD of triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 . ( G ) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in ( C ) . ( H ) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in ( D ) . ( Data information : The data were presented as the mean ± SD of triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 . ( I ) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in ( D ) . Scale bar , 20 μm .

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 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 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

Figure 4(C) The O2 consumption rate (OCR) in differentiated beige adipocytes from 3T3-L1 cells overexpressing Vec or HIF1A plasmid. The average basal and maximal respiration rates are shown in right panel. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.(G) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (C). (H) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (D). ( Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.(I) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (D). Scale bar, 20 μm.


Figure 5 ( C ) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl and siFto 3T3 - L1 cells . Blue boxes represent exons , and blue lines represent introns . The m6A peaks were in the dashed red box . ( D ) Methylated RNA immunoprecipitation ( MeRIP ) - qPCR analysis of m6A levels of Hif1a mRNA in siCtrl and siFto 3T3 - L1 cells . Data information : The data were presented as the mean ± SD of triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 . ( H ) Western blot analysis of the protein levels of HIF1A , UCP1 , PPARGC1A , PPARG and GFP in 3T3 - L1 cells infected with lentiviruses pCDHpuro expressing Vec , WT - or MUT - HIF1A .

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 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

Figure 5(C) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl and siFto 3T3-L1 cells. Blue boxes represent exons, and blue lines represent introns. The m6A peaks were in the dashed red box.(D) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m6A levels of Hif1a mRNA in siCtrl and siFto 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.(H) Western blot analysis of the protein levels of HIF1A, UCP1, PPARGC1A, PPARG and GFP in 3T3-L1 cells infected with lentiviruses pCDHpuro expressing Vec, WT- or MUT-HIF1A.


Figure 6 ( A ) Western blot analysis of the protein levels of YTHDF1 , YTHDF2 and HIF1A in siCtrl , siYthdf1 and siYthdf2 3T3 - L1 cells . ( G ) Polysome profiling of 3T3 - L1 cells overexpressing Vec or YTHDC2 plasmid . ( H ) qPCR analysis of mRNA levels of Hif1a in 40 - 80S or polysome fraction in 3T3 - L1 cells overexpressing Vec or YTHDC2 plasmid . ( I ) Polysome profiling of siCtrl and siYthdc2 3T3 - L1 cells . ( J ) qPCR analysis of mRNA levels of Hif1a in 40 - 80S or polysome fraction in siCtrl and siYthdc2 3T3 - L1 cells . Data information : The data were presented as the mean ± SD of triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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 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) Western blot analysis of the protein levels of YTHDF1, YTHDF2 and HIF1A in siCtrl, siYthdf1 and siYthdf2 3T3-L1 cells.(G) Polysome profiling of 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (H) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (I) Polysome profiling of siCtrl and siYthdc2 3T3-L1 cells. (J) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in siCtrl and siYthdc2 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.


Figure 5 ( B ) Levels of PIN2 phosphopeptide in our experiments . Bars represent the mean plus standard error of replicates ( 2 biological replicates for nitrate treatments at 20 min ( Col - 0 roots ) and 3 biological replicates for all other experimental conditions ) . Each independent biological replicate consisted of a pool of 4 . 500 roots collected from Arabidopsis plants grown independently under the same experimental conditions . The asterisk indicates statistically significant differences in phosphoproteomic analysis ( multiple t - test comparison without testing corrections , assuming same variance , p < 0 . 05 ) . ( C ) Detection of phosphorylation PIN2 by Phos - tag Western blotting . Arabidopsis plants ( Col - 0 ) were growth in ammonium as only nitrogen source , and treated with 5 mM KNO3 or 5mM KCl as control . Total protein from roots were analyzed in SDS PAGE using Phos - tag to detect changes in phosphorylation status . Immunoblotting was performed with PIN2 antibody . Total proteins isolated from eir1 . 1 roots were used as a negative control . White and red asterisks indicate a slow - or fast - mobility band corresponding to a more or less phosphorylated PIN2 , respectively . ( D ) Western blot against PIN2 protein comparing nitrate treated ( KNO3 ) and control ( KCl ) condition in Arabidopsis roots for all genotypes ( eir1 - 1 mutant background was complemented with PIN2 : : PIN2wt - GFP ( PIN2wt ) , PIN2 : : PIN2S439D - GFP ( PIN2S439D phospho - mimic point mutation ) or PIN2 : : PIN2S439A - GFP ( PIN2S439A , phospho - null point mutation ) .

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

Figure 5(B) Levels of PIN2 phosphopeptide in our experiments. Bars represent the mean plus standard error of replicates (2 biological replicates for nitrate treatments at 20 min (Col-0 roots) and 3 biological replicates for all other experimental conditions). Each independent biological replicate consisted of a pool of 4.500 roots collected from Arabidopsis plants grown independently under the same experimental conditions. The asterisk indicates statistically significant differences in phosphoproteomic analysis (multiple t-test comparison without testing corrections, assuming same variance, p < 0.05).(C) Detection of phosphorylation PIN2 by Phos-tag Western blotting. Arabidopsis plants (Col-0) were growth in ammonium as only nitrogen source, and treated with 5 mM KNO3 or 5mM KCl as control. Total protein from roots were analyzed in SDS PAGE using Phos-tag to detect changes in phosphorylation status. Immunoblotting was performed with PIN2 antibody. Total proteins isolated from eir1.1 roots were used as a negative control. White and red asterisks indicate a slow- or fast-mobility band corresponding to a more or less phosphorylated PIN2, respectively.(D) Western blot against PIN2 protein comparing nitrate treated (KNO3) and control (KCl) condition in Arabidopsis roots for all genotypes (eir1-1 mutant background was complemented with PIN2::PIN2wt-GFP (PIN2wt), PIN2::PIN2S439D-GFP (PIN2S439D phospho-mimic point mutation) or PIN2::PIN2S439A-GFP (PIN2S439A, phospho-null point mutation).


Figure 6 ( A ) Primary root length of Col - 0 wild type plants or eir1 - 1 mutant plants was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 10 - 15 roots each replicate ) . The box plot shows the data within the interquartile range ( 25th and 75th percentiles ) and a solid black line represents the median . Whiskers show maximum and minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicates statistically significant difference between analyzed by unpaired , two - tailed and assuming equal variance t - test ( * * p < 0 . 01 ) . ( B ) Number of initiating and emerging lateral roots of Col - 0 or eir1 - 1 mutant plants were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 10 - 15 roots each replicate ) . The box plot shows the data within the interquartile range ( 25th and 75th percentiles ) and a solid black line represents the median . Whiskers show maximum and minimum values no further than 1 . 5x IQR ( interquartile range ) . Asterisk indicates statistically significant difference between analyzed by unpaired , two - tailed and assuming equal variance t - test ( * * p < 0 . 01 ) .

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


Figure 7 ( A ) Primary root length of the different genotypes was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 8 - 10 roots each replicate ) . The box plot shows the data within the interquartile range ( 25th and 75th percentiles ) and a solid black line represents the median . Whiskers show maximum and minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicate statistically significant difference between means analyzed by unpaired , two - tailed and assuming equal variance t - test ( * p < 0 . 05 , * * p < 0 . 01 ) . ( B ) Number of initiating and emerging lateral roots for all genotypes were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 8 - 10 roots each replicate ) . The box plot shows the data within the interquartile range ( 25th and 75th percentiles ) and a solid black line represents the median . Whiskers show maximum and minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicate statistically significant difference between means analyzed by unpaired , two - tailed and assuming equal variance t - test ( * p < 0 . 05 , * * p < 0 . 01 ) .

O O 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 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

Figure 7(A) Primary root length of the different genotypes was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).(B) Number of initiating and emerging lateral roots for all genotypes were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).


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

O O 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

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


Figure 1Left graph reports the tumor volume in NSG mice injected with control ( shCTR ) and antimiR - 9 FaDu cells followed for up to 35 days ( n = 5 mice / group ) . Data represent the mean ( ± SD ) and unpaired t - test was used to verify the statistical significance at each time point . On the right , typical images of explanted tumors formed by control ( shCTR ) and antimiR - 9 FaDu cells at necropsy . Typical images of Hematoxylin & Eosin ( H & E - upper images ) and Ki67 expression ( Ki67 - bottom images ) evaluated by immunohistochemistry ( IHC ) in tumors explanted from mice treated as in H . 4X and 20X magnification are shown . Right graph reports the percentage of Ki67 positive cells . Each dot represents one tumor . Data are expressed as mean ( ± SD ) and unpaired t - test was used to verify the statistical significance .

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 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

Figure 1Left graph reports the tumor volume in NSG mice injected with control (shCTR) and antimiR-9 FaDu cells followed for up to 35 days (n=5 mice/group). Data represent the mean (±SD) and unpaired t-test was used to verify the statistical significance at each time point. On the right, typical images of explanted tumors formed by control (shCTR) and antimiR-9 FaDu cells at necropsy.Typical images of Hematoxylin&Eosin (H&E - upper images) and Ki67 expression (Ki67 - bottom images) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in H. 4X and 20X magnification are shown. Right graph reports the percentage of Ki67 positive cells. Each dot represents one tumor. Data are expressed as mean (±SD) and unpaired t-test was used to verify the statistical significance.


Figure 2qRT - PCR analyses of normalized miR - 9 expression , expressed as fold over the untreated condition , in control ( shCTR ) and antimiR - 9 FaDu cells treated with Cetuximab ( CTX ) for up to four hours . Data represent the mean ( ± SD ) of three independent experiments performed in duplicate and two - way ANOVA test was used to verify the statistical significance . Graph reporting the cell viability of control ( shCTR ) and antimiR - 9 FaDu cells treated with increasing concentration of Gefitinib as indicated and evaluated using the MTS assay . Data represent the mean ( ± SD ) of two independent experiments performed in sextuplicate , and unpaired t - test was used to verify the statistical significance per each dose .

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

Figure 2qRT-PCR analyses of normalized miR-9 expression, expressed as fold over the untreated condition, in control (shCTR) and antimiR-9 FaDu cells treated with Cetuximab (CTX) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA test was used to verify the statistical significance.Graph reporting the cell viability of control (shCTR) and antimiR-9 FaDu cells treated with increasing concentration of Gefitinib as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and unpaired t-test was used to verify the statistical significance per each dose.


Figure 3qRT - PCR analyses of Sp1 expression in control ( shCTR ) and antimiR - 9 FaDu cells transiently transduced with PGK - miR - 9 or control vector as indicated . Data represent the mean ( ± SD ) of three independent experiments performed in duplicate and two - way ANOVA with Sidak ' s multiple comparison test was used to verify the statistical significance . qRT - PCR analyses of Sp1 expression in CAL27 cells transfected with pcDNA miR - 9 or control vector . Data represent the mean ( ± SD ) of three independent experiments performed in duplicate and unpaired t - test was used to verify the statistical significance . Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of Gefitinib ( GEFI - left panel ) or Cetuximab ( CTX - right panel ) as indicated and evaluated using the MTS assay . Data represent the mean ( ± SD ) of two independent experiments performed in sextuplicate , and two - way ANOVA with Sidak ' s multiple comparison test was used to verify the statistical significance . Graph reporting the normalized Luciferase activity of Sp1 promoter fragments in control ( shCTR ) or antimiR - 9 FaDu cells . Data represent the mean ( ± SD ) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance .

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 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 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

Figure 3qRT-PCR analyses of Sp1 expression in control (shCTR) and antimiR-9 FaDu cells transiently transduced with PGK-miR-9 or control vector as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance. qRT-PCR analyses of Sp1 expression in CAL27 cells transfected with pcDNA miR-9 or control vector. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t-test was used to verify the statistical significance.Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of Gefitinib (GEFI - left panel) or Cetuximab (CTX - right panel) as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.Graph reporting the normalized Luciferase activity of Sp1 promoter fragments in control (shCTR) or antimiR-9 FaDu cells. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance.


Figure 4On the left , WB analyses of KLF5 and Sp1 expression in control ( shCTR ) and antimiR - 9 FaDu . Histone H3 expression was used as loading control . On the right , protein quantification analyses of KLF5 and SP1 expression normalized on H3 loading control . Data represent the mean ( ± SD ) of three biological replicates and unpaired t test was used to verify the statistical significance . Upper panel shows WB analyses of KLF5 and Sp1 expression in FaDu cells . Actin expression was used as loading control . Lower graph shows the SP1 protein quantification normalized over actin expression . Data represents the mean ( ± SD ) of three independent experiments and unpaired t test was used to verify the statistical significance .

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

Figure 4On the left, WB analyses of KLF5 and Sp1 expression in control (shCTR) and antimiR-9 FaDu. Histone H3 expression was used as loading control. On the right, protein quantification analyses of KLF5 and SP1 expression normalized on H3 loading control. Data represent the mean (±SD) of three biological replicates and unpaired t test was used to verify the statistical significance.Upper panel shows WB analyses of KLF5 and Sp1 expression in FaDu cells. Actin expression was used as loading control. Lower graph shows the SP1 protein quantification normalized over actin expression. Data represents the mean (±SD) of three independent experiments and unpaired t test was used to verify the statistical significance.


Figure 5Left , WB analyses of the indicated protein expression in control ( shCTR ) and antimiR - 9 FaDu cells not irradiated ( NIR ) or treated with 5Gy IR and allowed to repair for the indicated hours ( h ) . Tubulin was used as loading control . On the right , protein quantification analyses of KLF5 and Sp1 expression normalized on tubulin loading control . On the right , graphs report the quantification of the indicated proteins normalized on tubulin expression . Data are expressed as mean ( ± SD ) of three independent experiments and unpaired t test was used to calculate the statistical significance at time point . Left panel reports typical immunofluorescence images of control ( shCTR ) or antimiR - 9 FaDu cells , not irradiated ( NIR ) and analyzed 1 , 4 or 8 hours after 2Gy IR ( γH2AX green , pS10 - H3 red , nuclei in blue ) . Graphs report the percentage of γH2AX ( middle ) and pS10 - H3 ( right ) positive cells . Data represent the mean ( ± SD ) of three independent experiments in which at least 10 randomly selected fields were evaluated . Unpaired t test was used to calculate the statistical significance at each time point .

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 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

Figure 5Left, WB analyses of the indicated protein expression in control (shCTR) and antimiR-9 FaDu cells not irradiated (NIR) or treated with 5Gy IR and allowed to repair for the indicated hours (h). Tubulin was used as loading control. On the right, protein quantification analyses of KLF5 and Sp1 expression normalized on tubulin loading control. On the right, graphs report the quantification of the indicated proteins normalized on tubulin expression. Data are expressed as mean (±SD) of three independent experiments and unpaired t test was used to calculate the statistical significance at time point.Left panel reports typical immunofluorescence images of control (shCTR) or antimiR-9 FaDu cells, not irradiated (NIR) and analyzed 1, 4 or 8 hours after 2Gy IR (γH2AX green, pS10-H3 red, nuclei in blue). Graphs report the percentage of γH2AX (middle) and pS10-H3 (right) positive cells. Data represent the mean (±SD) of three independent experiments in which at least 10 randomly selected fields were evaluated. Unpaired t test was used to calculate the statistical significance at each time point.


Figure 6Graph reports the tumor volume of tumors described in A ( n = 10 mice / group ) . Data represent the mean ( ± SD ) and two - way ANOVA was used to verify the statistical significance . Typical images of Hematoxylin & Eosin ( H & E ) ( C ) and Ki67 expression ( Ki67 ) ( D ) evaluated by immunohistochemistry ( IHC ) in tumors explanted from mice treated as in A / B . Kaplan - Mayer curve evaluating the overall survival of HNSCC patients treated with RT + Cetuximab ( CTX ) combination included in the TCGA dataset , segregated on the expression of miR - 9 in the primary tumor ( Low expression < 75819 reads n = 8 ; High expression ≥ 75819 reads n = 23 ) . Number of evaluated samples ( n ) and p value are reported in the graph . Statistical significance was calculated with long - rank test . Kaplan - Mayer curve evaluating the progression free survival of HNSCC patients treated with RT + Cetuximab ( CTX ) combination at the CRO - Aviano National Cancer Institute and at the University Cattolica segregated based on miR - 9 expression in primary tumors , defined as the expression in above ( high expression n = 18 ) or below ( low expression n = 17 ) the median expression , as defined by ddPCR . Hazard Ratio ( HR ) and statistical significance were calculated with long - rank ( Mentel - Cox ) test and are reported in the graph .

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 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 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

Figure 6Graph reports the tumor volume of tumors described in A (n=10 mice/group). Data represent the mean (±SD) and two-way ANOVA was used to verify the statistical significance.Typical images of Hematoxylin&Eosin (H&E) (C) and Ki67 expression (Ki67) (D) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in A/B.Kaplan-Mayer curve evaluating the overall survival of HNSCC patients treated with RT+Cetuximab (CTX) combination included in the TCGA dataset, segregated on the expression of miR-9 in the primary tumor (Low expression <75819 reads n=8; High expression ≥75819 reads n=23). Number of evaluated samples (n) and p value are reported in the graph. Statistical significance was calculated with long-rank test.Kaplan-Mayer curve evaluating the progression free survival of HNSCC patients treated with RT+Cetuximab (CTX) combination at the CRO-Aviano National Cancer Institute and at the University Cattolica segregated based on miR-9 expression in primary tumors, defined as the expression in above (high expression n=18) or below (low expression n=17) the median expression, as defined by ddPCR. Hazard Ratio (HR) and statistical significance were calculated with long-rank (Mentel-Cox) test and are reported in the graph.


Figure 1B . Normalized fitness functions of E . coli BW25113 in the absence of tet ( B ) and their corresponding model fits in the presence of varying concentrations of antibiotics TET , DOX , and MCN in the media ( [ S ] M ) . Normalized quadruplicate data is shown for each antibiotic .

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

Figure 1B. Normalized fitness functions of E. coli BW25113 in the absence of tet(B) and their corresponding model fits in the presence of varying concentrations of antibiotics TET, DOX, and MCN in the media ([S]M). Normalized quadruplicate data is shown for each antibiotic.


Figure 2B . Normalized fitness functions of cells expressing wild type tet ( B ) and their corresponding model fits in the presence of TET , DOX and MCN . Triplicate data is shown for each

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

Figure 2B. Normalized fitness functions of cells expressing wild type tet(B) and their corresponding model fits in the presence of TET, DOX and MCN. Triplicate data is shown for each antibiotic


Figure 3B . Model fits for E . coli BW25113 strains expressing a chromosomal copy of tet ( B ) WT and seven variants in TET , DOX , and MCN . Our model fit triplicate fitness function data for all strains ( Figure S2 ) . At low drug concentrations for all antibiotics , tet ( B ) G366C exhibits much slower growth rates than wild - type . Compared to wild - type , strains expressing tet ( B ) L9F exhibit similar growth rates at low and high drug concentrations but have faster growth rates at intermediate drug concentrations . C . Fitness landscapes with mutants overlaid for TET = 35 µg / mL , DOX = 10 µg / mL , and MCN = 2 . 5 µg / mL corresponding to the dashed lines in 3B .

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 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

Figure 3B. Model fits for E. coli BW25113 strains expressing a chromosomal copy of tet(B)WT and seven variants in TET, DOX, and MCN. Our model fit triplicate fitness function data for all strains (Figure S2). At low drug concentrations for all antibiotics, tet(B)G366C exhibits much slower growth rates than wild-type. Compared to wild-type, strains expressing tet(B)L9F exhibit similar growth rates at low and high drug concentrations but have faster growth rates at intermediate drug concentrations.C. Fitness landscapes with mutants overlaid for TET=35 µg/mL, DOX=10 µg/mL, and MCN=2.5 µg/mL corresponding to the dashed lines in 3B.


Figure 4E . coli BW25113 strains expressing a chromosomal copy of tet ( B ) WT or variant were grown in 2 % arabinose supplemented LB in the absence of drug and harvested at mid - exponential phase . Membrane samples were prepared from three individual colonies grown independently ( Figures S5 and S6 ) . Compared to wild - type , strains expressing tet ( B ) L9F consistently have higher in vivo protein levels where as strains expressing tet ( B ) V339C and tet ( B ) G366C have lower in vivo protein levels as detected by Western Blot analysis . All four variants located in the interdomain loop exhibited in vivo protein levels similar to wild - type .

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

Figure 4E. coli BW25113 strains expressing a chromosomal copy of tet(B)WT or variant were grown in 2% arabinose supplemented LB in the absence of drug and harvested at mid-exponential phase. Membrane samples were prepared from three individual colonies grown independently (Figures S5 and S6). Compared to wild-type, strains expressing tet(B)L9F consistently have higher in vivo protein levels where as strains expressing tet(B)V339C and tet(B)G366C have lower in vivo protein levels as detected by Western Blot analysis. All four variants located in the interdomain loop exhibited in vivo protein levels similar to wild-type.


Figure 5A . The spread of κ and γ fits for 1000 randomly selected parameters A and B from a set gamma distribution shows a linear correlation between κ and γ . B . The calculated mean and error of γ / κ for the sampled parameter fittings shown in Figure S2 . The large error seen for TetBV339C and TetBG366C in MCN reveals that we cannot make the first order approximation within $ \ frac { \ gamma } { \ kappa } \ \ approx k _ { 1 } \ frac { { 2P } _ { \ text { tot } } } { D _ { 2 } } $ .

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 O O O O 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 5A. The spread of κ and γ fits for 1000 randomly selected parameters A and B from a set gamma distribution shows a linear correlation between κ and γ.B. The calculated mean and error of γ/κ for the sampled parameter fittings shown in Figure S2. The large error seen for TetBV339C and TetBG366C in MCN reveals that we cannot make the first order approximation within $\frac{\gamma}{\kappa}\ \approx k_{1}\frac{{2P}_{\text{tot}}}{D_{2}}$.


Figure 6A . Changes in drug binding affinity Δk1 and pumping efficiency Δr for TetB variants relative to wild type in TET , DOX and MCN . Error bars are comprised from a propagation of errors from A and B global parameter modeling and in vivo protein level determination .

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

Figure 6A. Changes in drug binding affinity Δk1 and pumping efficiency Δr for TetB variants relative to wild type in TET, DOX and MCN. Error bars are comprised from a propagation of errors from A and B global parameter modeling and in vivo protein level determination.


Figure 7A . Triplicate normalized fitness functions ( * ) for E . coli BW25113 strains harboring plasmid copies of tet ( B ) WT , tet ( B ) K183E , A290V , T326I ( variant 6 ) and tet ( B ) F151I , F179L , T326A ( variant 7 ) in TET and their corresponding least squares model fits ( solid line ) where both variants display decreased fitness in TET compared to WT .

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 7A. Triplicate normalized fitness functions (*) for E. coli BW25113 strains harboring plasmid copies of tet(B)WT, tet(B)K183E,A290V,T326I (variant 6) and tet(B)F151I,F179L,T326A (variant 7) in TET and their corresponding least squares model fits (solid line) where both variants display decreased fitness in TET compared to WT.


Figure 1D - F . Volcano plots showing RNA - seq data in mouse islets ( 20 % vs 5 % O2 for 24 hours ; n = 3 mice / group ; D ) , human islets ( 20 % [ n = 2 biological replicates from donor 1 sample ] vs 2 % [ n = 3 biological replicates from donor 1 sample ] O2 for 24 hours ; E ) , and MIN6 cells ( 20 % vs 5 % O2 for 6 hours ; n = 3 biological replicates ; F ) . Atf3 , Bhlhe40 , and other reported hypoxia - inducible transcriptional repressor genes ( Cavadas et al . , 2017 ) are shown ( red , significantly upregulated genes ) .

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

Figure 1D-F. Volcano plots showing RNA-seq data in mouse islets (20% vs 5% O2 for 24 hours; n = 3 mice/group; D), human islets (20% [n = 2 biological replicates from donor 1 sample] vs 2% [n = 3 biological replicates from donor 1 sample] O2 for 24 hours; E), and MIN6 cells (20% vs 5% O2 for 6 hours; n = 3 biological replicates; F). Atf3, Bhlhe40, and other reported hypoxia-inducible transcriptional repressor genes (Cavadas et al., 2017) are shown (red, significantly upregulated genes).


Figure 2Western blots of BHLHE40 expression across tissues ( A ) , BHLHE40 expression in MIN6 cells cultured under 20 % or 5 % O2 for the indicated time ( B ) and the quantification of B ( n = 3 biological replicates ) Data information : Glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) or β - actin was used as a loading control . HPT , hypothalamusD - F . The effect of oxidative stress , endoplasmic reticulum stress , and energy stress on BHLHE40 expression . qRT - PCR analysis of Bhlhe40 in MIN6 cells incubated with 10μM H2O2 ( n = 3 biological replicates ; D ) , 2μM thapsigargin ( Thap ) or 5 μg / ml tunicamycin ( Tun ) ( n = 7 biological replicates ; E ) , or 2mM metformin ( Met ) for 24 hours ( n = 3 biological replicates ; F ) . Data information : Data are mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . H - K . BHLHE40 expression in islets from diabetic mice . BHLHE40 expression was analyzed in ob / ob mouse islets by qRT - PCR ( n = 4 biological replicates ; H ) and Western blotting ( I ) and in db / db mouse islets by Western blotting ( J ) . Subcellular localization of BHLHE40 in ob / ob mice islets by immunohistochemical analysis ( K ) . Data information : Data are mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . β - actin was used as a loading control . Scale bar , 100 μm .

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 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 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 2Western blots of BHLHE40 expression across tissues (A), BHLHE40 expression in MIN6 cells cultured under 20% or 5% O2 for the indicated time (B) and the quantification of B (n = 3 biological replicates) Data information: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin was used as a loading control. HPT, hypothalamusD-F. The effect of oxidative stress, endoplasmic reticulum stress, and energy stress on BHLHE40 expression. qRT-PCR analysis of Bhlhe40 in MIN6 cells incubated with 10μM H2O2 (n = 3 biological replicates; D), 2μM thapsigargin (Thap) or 5 μg/ml tunicamycin (Tun) (n = 7 biological replicates; E), or 2mM metformin (Met) for 24 hours (n = 3 biological replicates; F). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.H-K. BHLHE40 expression in islets from diabetic mice. BHLHE40 expression was analyzed in ob/ob mouse islets by qRT-PCR (n = 4 biological replicates; H) and Western blotting (I) and in db/db mouse islets by Western blotting (J). Subcellular localization of BHLHE40 in ob/ob mice islets by immunohistochemical analysis (K). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. β-actin was used as a loading control. Scale bar, 100 μm.


Figure 3A . Glucose - stimulated insulin secretion in MIN6 cells expressing short hairpin RNA against a non - targeting Ctrl or Bhlhe40 knockdown ( B40 KD ) were cultured under 20 % or 5 % O2 for 24 hours ( n = 8 biological replicates ) . B . Glucose - stimulated insulin secretion in MIN6 cells infected with retroviruses generated with pMx - Ctrl ( Ctrl ) or pMx - Bhlhe40 ( B40 OE ; n = 4 biological replicates ) . Data information : Data are mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . Ctrl , control ; n . s . , not significant . D . KCl - stimulated insulin secretion in Ctrl and B40 OE MIN6 cells ( n = 3 biological replicates ) . Data information : Data are mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . Ctrl , control ; n . s . , not significant . G . Cellular ATP content in Ctrl and B40 KD MIN6 cells cultured under 20 % or 5 % O2 for 24 hours ( n = 3 biological replicates ) . Data information : Data are mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . Ctrl , control ; n . s . , not significant . I - K . Mitochondrial DNA ( mtDNA ) content ( n = 4 biological replicates ; I ) , mitochondrial mass ( n = 4 biological replicates ; J ) , and mitochondrial membrane potential ( n = 4 biological replicates ; K ) in Ctrl and B40 KD MIN6 cells cultured under 20 % or 5 % O2 for 24 hours . Data information : Data are mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . Ctrl , control ; n . s . , not significant .

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 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 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 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

Figure 3A. Glucose-stimulated insulin secretion in MIN6 cells expressing short hairpin RNA against a non-targeting Ctrl or Bhlhe40 knockdown (B40 KD) were cultured under 20% or 5% O2 for 24 hours (n = 8 biological replicates). B. Glucose-stimulated insulin secretion in MIN6 cells infected with retroviruses generated with pMx-Ctrl (Ctrl) or pMx-Bhlhe40 (B40 OE; n = 4 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.D. KCl-stimulated insulin secretion in Ctrl and B40 OE MIN6 cells (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.G. Cellular ATP content in Ctrl and B40 KD MIN6 cells cultured under 20% or 5% O2 for 24 hours (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.I-K. Mitochondrial DNA (mtDNA) content (n = 4 biological replicates; I), mitochondrial mass (n = 4 biological replicates; J), and mitochondrial membrane potential (n = 4 biological replicates; K) in Ctrl and B40 KD MIN6 cells cultured under 20% or 5% O2 for 24 hours. Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.


Figure 4B , Expression of DEGs shown in A was confirmed by qRT - PCR in Ctrl and B40 OE MIN6 cells ( n = 3 biological replicates ; B ) Data information : Data are mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . D . qRT - PCR of MAFA target genes in Ctrl and B40 KD MIN6 cells cultured under 20 % or 5 % O2 for 24 hours ( n = 3 biological replicates ) . Data information : Data are mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . G . Western blot of oxidative phosphorylation complex proteins and TOM20 protein in Ctrl and B40 KD MIN6 cells cultured under 20 % or 5 % O2 for 24 hours , and quantification of the band intensities ( n = 3 biological replicates ) . Data information : Data are mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . β - actin was used as a loading control . Ctrl , control .

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 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

Figure 4B, Expression of DEGs shown in A was confirmed by qRT-PCR in Ctrl and B40 OE MIN6 cells (n = 3 biological replicates; B) Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired two-tailed Student's t test.D. qRT-PCR of MAFA target genes in Ctrl and B40 KD MIN6 cells cultured under 20% or 5% O2 for 24 hours (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired two-tailed Student's t test.G. Western blot of oxidative phosphorylation complex proteins and TOM20 protein in Ctrl and B40 KD MIN6 cells cultured under 20% or 5% O2 for 24 hours, and quantification of the band intensities (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired two-tailed Student's t test. β-actin was used as a loading control. Ctrl, control.


Figure 5C . Luciferase reporter assay was performed with MIN6 cells transfected with BHLHE40 expression plasmids , pRL - SV40 plasmid , and pGL3 - Mafa plasmids ( wildtype [ black ] and E - box mutated [ red ] sites ; n = 4 biological replicates ) . D . Luciferase reporter activity in MIN6 cells cultured under 20 % or 5 % O2 for 24 hours ( n = 3 biological replicates ) . Data information : Data are mean ± SEM ; * p < 0 . 05 * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . Ctrl , control ; n . s . , not significant . H - J . Proteins sampled from Ctrl and B40 OE MIN6 cells with FLAG - Pdx1 expression ( H ) or from MIN6 cells with FLAG - Pdx1 expression pre - cultured under 20 % or 5 % O2 for 24 hours ( I ) were immuno - precipitated by IgG or anti - FLAG antibody , after which qRT - PCR was performed for the indicated regions ( n = 9 biological replicates for H and n = 6 biological replicates for I ) . FLAG ( PDX1 ) expression in I was confirmed by Western blotting ( J ) . Data information : Data are mean ± SEM ; * p < 0 . 05 * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . Ctrl , control ; n . s . , not significant .

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 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

Figure 5C. Luciferase reporter assay was performed with MIN6 cells transfected with BHLHE40 expression plasmids, pRL-SV40 plasmid, and pGL3-Mafa plasmids (wildtype [black] and E-box mutated [red] sites; n = 4 biological replicates). D. Luciferase reporter activity in MIN6 cells cultured under 20% or 5% O2 for 24 hours (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.H-J. Proteins sampled from Ctrl and B40 OE MIN6 cells with FLAG-Pdx1 expression (H) or from MIN6 cells with FLAG-Pdx1 expression pre-cultured under 20% or 5% O2 for 24 hours (I) were immuno-precipitated by IgG or anti-FLAG antibody, after which qRT-PCR was performed for the indicated regions (n = 9 biological replicates for H and n = 6 biological replicates for I). FLAG (PDX1) expression in I was confirmed by Western blotting (J). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.


Figure 6C , D . Glucose tolerance test of Ctrl : ob / ob and βB40KO : ob / ob mice ( n = 9 mice and n = 8 mice , respectively ; 6 weeks old ) ( C ) and AUC ( D ) . Data information : Data are mean ± SEM ; * p < 0 . 05 * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . E . Glucose - stimulated insulin secretion in Ctrl : ob / ob and βB40KO : ob / ob mice ( n = 9 mice and n = 6 mice , respectively ; 8 weeks old ) . F . Glucose - stimulated insulin secretion in isolated islets from Ctrl and βB40KO mice after culture under 5 % O2 for 24 hours ( n = 8 biological replicates ) . Data information : Data are mean ± SEM ; * p < 0 . 05 * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . I , J . Representative images of pancreatic islets stained for insulin and glucagon in Ctrl : ob / ob and βB40KO : ob / ob mice ( 12 weeks old ) ( I ) . The ratios of total islet area to whole pancreas area ( % ) are shown ( n = 3 biological replicates ; J ) . Data information : Data are mean ± SEM ; * p < 0 . 05 * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . Scale bar , 10 μm . K , L . Representative images of pancreatic islets stained for insulin , MAFA , and BHLHE40 in Ctrl : ob / ob and βB40KO : ob / ob mice ( 12 weeks old ) ( K ) . Fluorescence intensities of nuclear and cytosolic MAFA in K were quantified ( n = 30 cells ; L ) . Data information : Data are mean ± SEM ; * p < 0 . 05 * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test . Scale bar , 10 μm . M . qRT - PCR of Mafa and its target genes in Ctrl : ob / ob and βB40KO : ob / ob mice ( n = 4 mice ) . Data information : Data are mean ± SEM ; * p < 0 . 05 * * p < 0 . 01 , and * * * p < 0 . 001 by unpaired two - tailed Student ' s t test .

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

Figure 6C, D. Glucose tolerance test of Ctrl:ob/ob and βB40KO:ob/ob mice (n = 9 mice and n = 8 mice, respectively; 6 weeks old) (C) and AUC (D). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.E. Glucose-stimulated insulin secretion in Ctrl:ob/ob and βB40KO:ob/ob mice (n = 9 mice and n = 6 mice, respectively; 8 weeks old). F. Glucose-stimulated insulin secretion in isolated islets from Ctrl and βB40KO mice after culture under 5% O2 for 24 hours (n = 8 biological replicates). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.I, J. Representative images of pancreatic islets stained for insulin and glucagon in Ctrl:ob/ob and βB40KO:ob/ob mice (12 weeks old) (I). The ratios of total islet area to whole pancreas area (%) are shown (n = 3 biological replicates; J). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Scale bar, 10 μm.K, L. Representative images of pancreatic islets stained for insulin, MAFA, and BHLHE40 in Ctrl:ob/ob and βB40KO:ob/ob mice (12 weeks old) (K). Fluorescence intensities of nuclear and cytosolic MAFA in K were quantified (n = 30 cells; L). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Scale bar, 10 μm.M. qRT-PCR of Mafa and its target genes in Ctrl:ob/ob and βB40KO:ob/ob mice (n = 4 mice). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.


Figure 1F : Full - length GST - tagged wild type ( wt ) and RG mutant p14ARF protein , GST alone as well as GST - PRMT1 were purified from bacteria , as visualized by SDS - PAGE and Coomassie Blue staining ( left and middle panel ) , with asterisks indicating the corresponding PRMT1 and p14ARF protein bands . GST and GST - p14ARF proteins ( wt , RG ) were subjected to in vitro - MT assays in the presence of GST - PRMT1 as described in ( B ) . Methylation activities were detected subsequent to protein blotting by autoradiography ( upper right panel ) . The asterisk highlights the methylated p14ARF wt protein , whereas the arrowhead indicates p14ARF - unrelated background signals ( likely deriving from PRMT1 automethylation ) . Amounts of p14ARF proteins were detected by immunostaining of the autoradiographed blot using α - p14ARF antibody ( lower right panel ) . Size markers ( in kDa ) are shown on the left .

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

Figure 1F: Full-length GST-tagged wild type (wt) and RG mutant p14ARF protein, GST alone as well as GST-PRMT1 were purified from bacteria, as visualized by SDS-PAGE and Coomassie Blue staining (left and middle panel), with asterisks indicating the corresponding PRMT1 and p14ARF protein bands. GST and GST-p14ARF proteins (wt, RG) were subjected to in vitro-MT assays in the presence of GST-PRMT1 as described in (B). Methylation activities were detected subsequent to protein blotting by autoradiography (upper right panel). The asterisk highlights the methylated p14ARF wt protein, whereas the arrowhead indicates p14ARF-unrelated background signals (likely deriving from PRMT1 automethylation). Amounts of p14ARF proteins were detected by immunostaining of the autoradiographed blot using α-p14ARF antibody (lower right panel). Size markers (in kDa) are shown on the left.


Figure 2A , B : HeLa cells were transfected with empty vector ( e . v . , control ) or Myc - tagged wild type PRMT1 - containing plasmid . Immunofluorescence ( IF ) staining was performed using α - p14ARF ( red , endogenous p14ARF ) , α - Myc ( green , exogenous PRMT1 ) antibodies , DAPI ( blue , nuclei / DNA ) and Phalloidin ( grey , cytoplasm / F - actin ) . In ( A ) , representative IF results are shown , with asterisks indicating cells with exclusive nucleolar and arrowheads indicating cells with predominantly nucleo - / cytoplasmic p14ARF localization . The Merge displays the combination of p14ARF and PRMT1 staining . Scale bars : 15 μm . The quantification of exclusive nucleolar p14ARF was performed by cell counting ( percentage of cells ) and is shown for three independent experiments in ( B ) ( mean ± SD ) . C , D : U2OS cells were transfected with EGFP - tagged p14ARF alone or in combination with Myc - tagged wild type PRMT1 - containing plasmids . p14ARF - EGFP ( green ) , DAPI ( blue , nuclei / DNA ) and PRMT1 ( red , using α - Myc antibody ) were visualized by fluorescence microscopy , for which representative results are shown in ( C ) , with the lower images displaying a magnification as indicated in the upper images by the rectangles . Scale bars : 10 μm . The subcellular distribution of p14ARF - EGFP ( exclusively nucleolar or not - exclusively nucleolar but predominantly nucleo - / cytoplasmic ) was quantified in p14ARF - positive and p14ARF / Myc - PRMT1 - double positive cells by cell counting ( percentage of cells ) for three independent experiments in ( D ) ( mean ± SD , * * * : p - value ≤ 0 . 001 using Welch ´ s t - test ) . HeLa cells were transfected with the indicated siRNAs ( two control / non - targeting siRNAs and three PRMT1 - specific siRNAs ) . The cellular p14ARF distribution was determined by immunofluorescence staining ( IF ) using α - p14ARF antibody . Representative IF images for the siControl _ 1 and siPRMT1 _ 1 condition are shown in ( G ) ( endogenous p14ARF in red and the merge additionally with DAPI in blue for nuclei / DNA ) . Scale bars : 15 μm .

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 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 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

Figure 2A, B: HeLa cells were transfected with empty vector (e.v., control) or Myc-tagged wild type PRMT1-containing plasmid. Immunofluorescence (IF) staining was performed using α-p14ARF (red, endogenous p14ARF), α-Myc (green, exogenous PRMT1) antibodies, DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin). In (A), representative IF results are shown, with asterisks indicating cells with exclusive nucleolar and arrowheads indicating cells with predominantly nucleo-/cytoplasmic p14ARF localization. The Merge displays the combination of p14ARF and PRMT1 staining. Scale bars: 15 μm. The quantification of exclusive nucleolar p14ARF was performed by cell counting (percentage of cells) and is shown for three independent experiments in (B) (mean ± SD).C, D: U2OS cells were transfected with EGFP-tagged p14ARF alone or in combination with Myc-tagged wild type PRMT1-containing plasmids. p14ARF-EGFP (green), DAPI (blue, nuclei/DNA) and PRMT1 (red, using α-Myc antibody) were visualized by fluorescence microscopy, for which representative results are shown in (C), with the lower images displaying a magnification as indicated in the upper images by the rectangles. Scale bars: 10 μm. The subcellular distribution of p14ARF-EGFP (exclusively nucleolar or not-exclusively nucleolar but predominantly nucleo-/cytoplasmic) was quantified in p14ARF-positive and p14ARF/Myc-PRMT1-double positive cells by cell counting (percentage of cells) for three independent experiments in (D) (mean ± SD, ***: p-value ≤ 0.001 using Welch´s t-test).HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and three PRMT1-specific siRNAs). The cellular p14ARF distribution was determined by immunofluorescence staining (IF) using α-p14ARF antibody. Representative IF images for the siControl_1 and siPRMT1_1 condition are shown in (G) (endogenous p14ARF in red and the merge additionally with DAPI in blue for nuclei/DNA). Scale bars: 15 μm.


Figure 3A , U2OS cells were transfected with EGFP - tagged wild type ( wt ) or mutant ( RG , RF , RK ) p14ARF - containing plasmids . p14ARF - EGFP ( green ) , endogenous NPM as a nucleolar marker ( red , using α - NPM antibody ) , DAPI ( blue , nuclei / DNA ) and Phalloidin ( grey , cytoplasm / F - actin ) were visualized by fluorescence microscopy , for which representative results are shown in ( A ) . Scale bars : 10 μm . D : HeLa cells were transfected with empty vector ( e . v . , control ) or Myc - tagged wild type PRMT1 - containing plasmid . Immunoprecipitation ( IP ) of endogenous p14ARF or NPM was performed from cell lysates using the corresponding antibodies or IgG as negative control . IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies . Staining results of the IP reactions derive from the same blot and exposure times with the white lines indicating where tracks were cut .

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 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

Figure 3A, U2OS cells were transfected with EGFP-tagged wild type (wt) or mutant (RG, RF, RK) p14ARF-containing plasmids. p14ARF-EGFP (green), endogenous NPM as a nucleolar marker (red, using α-NPM antibody), DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin) were visualized by fluorescence microscopy, for which representative results are shown in (A). Scale bars: 10 μm.D: HeLa cells were transfected with empty vector (e.v., control) or Myc-tagged wild type PRMT1-containing plasmid. Immunoprecipitation (IP) of endogenous p14ARF or NPM was performed from cell lysates using the corresponding antibodies or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Staining results of the IP reactions derive from the same blot and exposure times with the white lines indicating where tracks were cut.


Figure 4C : HeLa cells were transfected with the indicated siRNAs ( five control / non - targeting siRNAs and four PRMT1 - specific siRNAs ) . PRMT1 depletion and endogenous p14ARF protein levels were analyzed by immunoblotting using α - PRMT1 , α - p14ARF and α - β - TUBULIN ( loading control ) antibodies . The p14ARF signals were densitometrically quantified and normalized to the respective β - TUBULIN signal , as specified by the numbers below the blot , with the siControl _ 2 condition set to 1 . PRMT1 - proficient ( - Dox ) as well as PRMT1 - depleted cells ( + Dox ) were further treated with the protein synthesis inhibitor cycloheximide ( CHX ) and harvested after 0 , 0 . 5 , 1 , 2 , 3 , 4 , and 6 hours . Cell lysates were subjected to immunoblotting using α - p14ARF and α - β - TUBULIN ( loading control ) antibodies and conventional ECL detection ( F ) . For precise quantification of the p14ARF protein stability in the - / + Dox conditions during the CHX time course , immunoblotting was performed with independent cell lysates and primary antibodies , as in ( F ) , which were then detected using fluorescence dye - coupled secondary antibodies and the LI - COR Odyssey system . The quantitative p14ARF signals were normalized to the corresponding quantitative β - TUBULIN signals and displayed in ( G ) . The CHX - untreated samples ( 0 hour ) were set to 100 % for each condition .

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 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

Figure 4C: HeLa cells were transfected with the indicated siRNAs (five control/non-targeting siRNAs and four PRMT1-specific siRNAs). PRMT1 depletion and endogenous p14ARF protein levels were analyzed by immunoblotting using α-PRMT1, α-p14ARF and α-β-TUBULIN (loading control) antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blot, with the siControl_2 condition set to 1.PRMT1-proficient (-Dox) as well as PRMT1-depleted cells (+ Dox) were further treated with the protein synthesis inhibitor cycloheximide (CHX) and harvested after 0, 0.5, 1, 2, 3, 4, and 6 hours. Cell lysates were subjected to immunoblotting using α-p14ARF and α-β-TUBULIN (loading control) antibodies and conventional ECL detection (F). For precise quantification of the p14ARF protein stability in the -/+ Dox conditions during the CHX time course, immunoblotting was performed with independent cell lysates and primary antibodies, as in (F), which were then detected using fluorescence dye-coupled secondary antibodies and the LI-COR Odyssey system. The quantitative p14ARF signals were normalized to the corresponding quantitative β-TUBULIN signals and displayed in (G). The CHX-untreated samples (0 hour) were set to 100% for each condition.


Figure 5HeLa cells were transfected with the indicated siRNAs ( two control / non - targeting siRNAs and two PRMT1 - specific siRNAs ) and irradiated at 150 J / cm2 UVC or not irradiated . After 24 hours , the cellular distribution of endogenous p14ARF was determined by immunofluorescence ( IF ) staining using α - p14ARF antibody ( red , endogenous p14ARF ) and in the merge additionally DAPI ( blue , nuclei / DNA ) . Representative IF results are shown for two conditions ( siControl _ 4 and siPRMT1 _ 1 ) in ( A ) , with asterisks indicating exclusively nucleolar and arrowheads indicating not - exclusively nucleolar but additionally or predominantly nucleo - / cytoplasmic p14ARF localization . Scale bars : 15 μm . The subcellular distribution of p14ARF , as determined by IF , was quantified by cell counting ( percentage of cells ) for all conditions ( B ) .

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

Figure 5HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and two PRMT1-specific siRNAs) and irradiated at 150 J/cm2 UVC or not irradiated. After 24 hours, the cellular distribution of endogenous p14ARF was determined by immunofluorescence (IF) staining using α-p14ARF antibody (red, endogenous p14ARF) and in the merge additionally DAPI (blue, nuclei/DNA). Representative IF results are shown for two conditions (siControl_4 and siPRMT1_1) in (A), with asterisks indicating exclusively nucleolar and arrowheads indicating not-exclusively nucleolar but additionally or predominantly nucleo-/cytoplasmic p14ARF localization. Scale bars: 15 μm. The subcellular distribution of p14ARF, as determined by IF, was quantified by cell counting (percentage of cells) for all conditions (B).