Fig 2: ZMPSTE24 colocalises with IFITM proteins and interacts via a C-terminal interaction with IFITM3. (A) ZMPSTE24 is localised to early and late endosomal compartments. ZMPSTE24-HA was transiently expressed in A549 cells which were then probed with antibodies against HA, early endosomal (EEA1) or late endosomal (Rab9) markers, or LAMP1. Nuclei were counterstained with DAPI. Panels are of representative images. Pearson’s correlation coefficient of ZMPSTE24 with the early and late endosomal compartment markers was calculated by measuring cells of interest using ImageJ software. (B) A549 cells transiently expressing HA-tagged IFITM proteins 1, 2, and 3 were probed with antibodies against endogenous ZMPSTE24 and HA and imaged by confocal microscopy. Panels are of representative images. Pearson’s correlation coefficient of ZMPSTE24 with individual IFITM proteins was calculated by measuring cells of interest using ImageJ software. (C) For the endogenous IP, A549 cells were pre-treated with IFN prior to cell lysis. For the co-IP, IFN pre-treated A549 cells were mock-transfected or transfected with ZMPSTE24-FLAG. Cell lysates were immunoprecipitated with anti-IFITM2/3 monoclonal antibody prior to resolving by SDS-PAGE and analysis by western blotting for endogenous ZMPSTE24 or ZMPSTE24-FLAG along with IFITMs 1, 2, and 3. (D) Schematic illustration of NanoBiT construct expression. IFITM3 or ZMPSTE24 were fused to large (LgBiT) or small (SmBiT) subunits of NanoLuc luciferase at N or C terminals and co-transfected in pairs (hatched boxes) into HEK293T cells. (E) Luminescence produced by interaction of co-expressed NanoBiT pairs was measured in live cells and analysed relative to a control consisting of the target-LgBiT co-transfected with a HaloTag-SmBiT control. LgBiT-PRKACA and SmBiT-PRKAR2A were co-transfected as a positive control. Green arrow indicates interaction between ZMPSTE24-C-SmBiT and IFITM3-C-LgBiT. Data are shown as mean ± SE (standard error) of n = 3 independent experiments. Significance is indicated as p-values *** p < 0.0001, ** p < 0.001.

Fig 3: FASN expression negatively associates with TFEB activity.A Heatmaps of the correlation between FASN and TFEB target genes extracted from the TCGA-AML cohort analyzed by the UCSD xena platform and from the blood spot data bank (Spearman, p values in Supplementary Table 1 and 2). B mRNA sequencing data of NB4 cells treated with ATRA. Relative expression of FASN, TFEB and TFEB transcriptional targets involved in lysosomal function and biogenesis are shown. C–H NB4 control (SHC002) and FASN knockdown (shFASN_1, _2) cells were treated for 1–3 days with ATRA. C Immunofluorescence microscopy of endogenous TFEB (red) and a-tubulin (green). IgG staining was used as negative control. Nuclei were stained with DAPI (blue). D Immunofluorescence microscopy of endogenous LAMP1 (red). Nuclei were stained with DAPI (blue). Scale: 10 µm (E) LAMP1 punctae quantification of cells shown in (D). Scale: 10 µm (F–H) Acridine Orange staining. F Histogram representation of the ratio between RED and GREEN of NB4 control (SHC002) cells treated as described in 6 C. G Representative histogram of NB4 control (SHC002) and FASN knockdown (shFASN_1, _2) cells treated as in 6 C. H Overton percentage positive quantification of the RED/GREEN ratio of NB4 control (SHC002) and FASN knockdown (shFASN_1, _2) cells treated with ATRA at indicated times. I Evaluation of BECN1, GABARAP, STK4 and WDR45 mRNA transcripts was done by qPCR. Values were normalized to the HMBS housekeeping gene. Results shown are from at least two biological duplicates.

Fig 4: FASN localizes at the lysosome to increase mTOR activity.A NB4 cells were treated as in 2D and fixed and stained for endogenous FASN (red) and LAMP1 (green) protein. The colocalization analysis was performed using ImageJ software. Scale: 10 µm. Results shown are from at least two biological duplicates. B–D NB4 control (SHC002) and FASN knockdown (shFASN_1, _2) cells were treated for 1–3 days with ATRA. B Immunofluorescence microscopy of endogenous mTOR (green) and LAMP1 (red). The colocalization analysis was performed using ImageJ software. Scale: 10 µm. C Total protein was extracted and subjected to immunoblotting using anti-FASN, anti-pmTOR(Ser2448), anti-pTFEB(Ser211), anti-TFEB, anti-pAKT(Ser473) anti-PTEN and anti-p4E-BP1 (Thr37/46) antibodies. D Relative protein expressions of two independent experiments were normalized to total protein or the respective non-phosphorylated protein and quantified using ImageJ software (NIH, Bethesda, MD, USA).

Fig 5: Tissue degradation and signal loss vs. preserved tissue and signal with refixation. (A) FoxP3 staining (brown) after 5 min retrieval in high pressure cooker in citric buffer pH 6, followed by KLRG1 (red) after proteinase K pretreatment and (B) vice versa (proteinase K followed by high pressure cooking) lead to strong tissue degradation. (C–F) show examples with refixation between the retrievals. (C) Protocols from (B) with interposed refixation step lead to preservation of tissue structure with maintained signals (KLRG1 red, FoxP3 brown). (D,E) Examples of triple staining with up to two retrievals: (D) human tonsil, CD8 (red, 2 min high pressure cooking) followed by Bcl6 (brown, 5 min high pressure cooking) together with PD1 (blue, cocktail with Bcl6); (E) human lymph node, IgD (red, without retrieval), followed by Bcl6/PD1 (brown and blue, respectively, after 5 min high pressure cooking). (F) immunofluorescence in human tonsil: LAMP1 (488 green, after 15 min TRIS pH 9 retrieval), ADA2 (555 red, after 20 min TRIS pH 6.1 retrieval), nuclear counterstain with DAPI (blue fluorescence).