EMBO/soda-nel huggingface dataset:
Lipid induced lysosomal damage after demyelination corrupts microglia protective function in Lysosomal Storage Disorders (2018)
Figure 5

Figure 5. Lysosomal exocytosis of CathB in ASMko conditions mediates neuronal death.

(A) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt BMDMs. Staining of Ponceau-S is shown as loading control.

(B) Mean ± SEM fold increase in CathB levels in the culture media from wt and ASMko BMDMs (n = 5 independent cultures, Student´s t-test).

(C) Immunofluorescence staining against LAMP-1 (yellow) and Iba-1 (red) in Cb of ASMko and wt mice. Scale bar, 50 μm, zoomed images 20 μm.

(D) Immunofluorescence staining against LAMP-1 in non-permeabilised BMDMs from ASMko and wt mice. Scale bar, 50 μm.

(E) Mean ± SEM of LAMP-1 levels on the surface of ASMko and wt BMDMs (n = 4 independent cultures, >40 cells per culture, Student´s t-test).

(F) Mean ± SEM microvesicle and exosome numbers in the culture media from ASMko and wt BMDMs (n = 6 independent cultures, Student´s t-test).

(G) Western blot of CathB and Flotillin1 levels in the secreted exosome fractions from ASMko and wt BMDMs.

(H) Western blot of CathB levels in the exosome depleted culture media from wt and ASMko BMDMs.

(I) Mean ± SEM MTT absorbance reflecting cell viability in primary neurons from wt mice treated with conditioned media from ASMko and wt BMDMs in the presence or absence of the CathB inhibitor Ca074 (n = 4 independent cultures Two-way Anova, Bonferroni post hoc).

(J) Immunofluorescence staining against Iba-1 (green) and CathB (red) in lobes of the Cb sensitive or resistant to Purkinje cell death. Asterisks indicate Purkinje cells in the resistant lobes. Scale bar, 50 μm.

(K) Mean ± SEM percentage of CathB positive microglia in resistant and sensitive Cb lobules of ASMko mice (n = 5 mice, χ2 test).

(L) Western blot of Cath B and β-Actin levels in cerebellar extracts of wt and ASMko mice treated or not with PLX.

(M) Mean ± SEM of CathB levels normalized by β-Actin in cerebellar extracts of wt and ASMko mice treated or not with PLX (n= 6 mice Two-way Anova, Bonferroni post hoc).

(N) Diagram showing the experimental procedure to obtain media from cerebellar organotypic cultures dissected from the brain of wt and ASMko mice orally treated or not with PLX.

(O) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt cerebellar organotypic cultures from mice fed with vehicle (veh) or PLX (PLX). Below, Western blots of organotypic culture lysates against Iba-1, to confirm microglia depletion, and against GAPDH as loading control.

(P) Mean ± SEM increase in pro-CathB (left) and CathB (right) levels in the culture media from cerebellar organotypic cultures from wt and ASMko mice treated or not with PLX (n = 3 mice per group Two-way Anova, Bonferroni post hoc).

Data information: P*<0.05; P**<0.005; P***<0.001.
Lipid induced lysosomal damage after demyelination corrupts microglia protective function in Lysosomal Storage Disorders (2018)
Figure 6

Figure 6. CathB inhibition prevents disease progression in ASMko mice.

(A) Mean ± SEM body weight of ASMko and wt mice treated or not with Ca074Me at the indicated time during treatment (n = 7 mice per group).

(B, C) Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time spent in the rotating rod on each of four trials (B) or as mean ± SEM time of the four trials (C) (n = 7 mice per group Two-way Anova, Bonferroni post hoc).

(D) Immunofluorescence staining against the PC marker Calbindin in the posterior lobules of the Cb in ASMko and wt mice treated or not with Ca074Me for 1 month. DAPI staining shows cell nuclei. Scale bar, 200 μm.

(E) Mean ± SEM number of Calbindin positive cells in the posterior zone (lobules IX-X) of the Cb from the different mouse groups (n= 7 mice per group Two-way Anova, Bonferroni post hoc).

Data information: P*<0.05; P**<0.005.
Lipid induced lysosomal damage after demyelination corrupts microglia protective function in Lysosomal Storage Disorders (2018)
Figure 7

Figure 7. Alterations in NPC1-deficient microglia.

(A) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 2 month-old Npc1nmf164 and wt mice. DAPI staining shows cell nuclei. Scale bars, 30 μm.

(B) 3D rendered images obtained with Imaris Surface software from high magnification z-stack images of immunofluorescence staining against Iba-1 showing representative microglial morphology in Cx, Hip and Cb of Npc1nmf164 and wt mice. Scale bars, 10 μm.

(C) Mean ± SEM number (upper panel) and area (lower panel) of Iba-1 positive cells in different brain regions from Npc1nmf164 and wt mice (n = 5 mice per group, >30 cells per mouse, Student´s t-test).

(D) Immunofluorescence staining against Iba-1 in the Cb of a NPC affected and a control child. Scale bars, 50 μm. Graph shows mean ± SEM area of Iba-1 positive cells (n = 30 cells, Student´s t-test).

(E) Immunofluorescence staining against MBP in Cb of Npc1nmf164 and wt mice. Scale bar, 50 μm. Graph shows mean ± SEM number of MBP aggregates per area (n = 5 mice per group, Student´s t-test).

(F) High magnification images and their 3D rendered replicates from cerebellar Npc1nmf164 microglia stained against Iba-1 and MBP containing myelin debris (arrowheads). Scale bars, 10 μm (up), 2 μm (down). The orthogonal projection of the cells in the right panels confirms the presence of myelin debris inside the cell and underneath the plasma membrane. DAPI staining shows cell nuclei. Scale bars, 10 μm.

(G) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and Npc1nmf164 mice (n = 5 mice per group).

(H) Immunofluorescence staining against F4/80 and CathB in Cb of Npc1nmf164 mice. Scale bar, 30 μm. The table shows mean ± SEM percentage of microglia presenting CathB staining in Npc1nmf164 and wt mice. Below, magnified images of the selected areas. Scale bar, 20 μm.

(I) Immunofluorescence staining against Iba-1 and CathB in Cb of a NPC patient. DAPI shows cell nuclei. Scale bar, 50 μm. A merged magnified image of the selected area is shown in the right down panel, scale bar 10 μm. Table shows mean ± SEM percentage of microglia presenting CathB staining in NPC and control children.

Data information: P*<0.05; P**<0.005; P***<0.001.
Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3 (2021)
Figure 1

Figure 1. GOLPH3 interacts with the cytosolic tails of selected GSL synthetic enzymes

A. Pull-down experiments using as a bait the biotinylated peptides mimicking the N-terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot (LxxR motif in red, biotin at the C-terminus). Immunoblotting shows affinity-captured GOLPH3 (endogenous and recombinant).

B. Control or GOLPH3-KD HeLa cells expressing luminal SI-GFP or the chimeras of sucrose-isomaltase containing the N-terminal cytosolic tails of LCS, Gb3S, GM3S and GD3S were fixed and labelled for surface localized GFP (red). Scale bar, 20 μm.

C. Pull-down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail of LCS WT and LCS mutants (mut. 1, 2 and 3) is shown on the top of the blot (mutations are in blue, LxxR motif in red, biotin is at the C-terminus).

D. HeLa cells expressing luminal SI-GFP chimeras of sucrose-isomaltase (SI-GFP, green) containing the WT and mutant N-terminal cytosolic tails of LCS, were fixed and labelled for surface localized GFP (red). Cells expressing LCS-SI-RxR and LCS-SI-LxxR chimeras shown PM staining. Scale bar, 20 μm, (left). PM (surface) fluorescence intensity (in Table S10 are listed the software used in this study) of each chimera is normalised on the LCS-SI-GFP surface intensity (data are means ± SEM derived from two biological replicates; n is indicated in the graph, ***p <0.001 [Student's t-test]) (right).

E. Quantitative flow cytometry-based analysis of the experiment reported in D; n is indicated in the graph (left). Percentage of cells with plasma membrane staining (surface) is reported for each chimera (right).

F. GOLPH3-KD or OE HeLa cells were processed for ShTxB (upper panels) or ChTxB (lower panels) staining followed by flow cytometry analysis. Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition.
Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3 (2021)
Figure 2

Figure 2. GOLPH3 drives the intra-Golgi recycling of LCS

A. HeLa cells mock-treated or GOLPH3-KD were transiently transfected with LCS_GFP_RUSH (for simplicity, referred in the text as LCS-GR) and subjected to the synchronisation protocol described in Materials and Methods. Cells were then subjected to IF at each indicated time point: LCS-GR (green), cis Golgi marker GM130 (red), and TGN marker TGN46 (blue). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm. LCS-GR intra-Golgi trafficking in mock or GOLPH3-KD conditions it has been quantified (right graph) by computational coalescence of line scans, and was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1). The n indicated in the graph refers to the number of Golgi stacks analysed in each condition.

B. Mock and GOLPH3-KD HeLa cells co-expressing LCS-GR and VSV-G were subjected to the co-synchronisation protocol described in Materials and Methods. Cells were then subjected to IF: LCS-GR or VSV-G (green), GM130 (blue), and TGN46 (red). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm.

C. Quantification of the experiment in (B) was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1); n indicated in the graph refers to the number of Golgi stacks analysed in each condition, individual data points and means ± SEM are shown, ***p < 0.001 (Student's t test); ns, not significant.

D. HeLa cells were transfected with LCS-GR, subjected to the synchronisation protocol (see Materials and Methods), fixed and processed for cryoimmunolabeling with an anti-GFP antibody (15 nm gold particles) and anti-GM130 antibody (10 nm gold particles, black arrowheads) peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. Scale bar, 150 nm.

E, F. Quantification of the distribution of LCS-GR as measured by Frequency Distribution Analysis (E) and linear density (LD, in F) (data are means± SEM, number of Golgi stacks is n=15 at 0 min, n=14 at 10 min, n=17 at 20 min. (E) The red arrows indicate the fraction of LCS-GR gold particles (%) in vesicles at each indicated time-point.

G. HeLa cells expressing LCS-GR mock or GOLPH3-KD were treated and stained as in D. Black arrowheads indicate GM130; peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. LCS-GR in the TGN is marked by blue arrowheads. Scale bar, 150 nm.

H. Quantification of LCS-GR distribution (linear density, LD) across the stack and in peri-Golgi vesicles (data are means ± SEM, number of Golgi stacks is n=17 at 20 min for the CTRL, n=13 at 20 min for the GOLPH3 KD; *p <0.05, **p < 0.01; ***p <0.001 [Student's t-test]).
Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3 (2021)
Figure 3

Figure 3. GOLPH3 regulates the lysosomal degradation of its clients

A. PHFs GOLPH3 OE (left), or KD (right), were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), TGN46 or DAPI (blue). Dashed lines indicate cells overexpressing or interfered for GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi; data are means ± SEM derived from two biological replicates, n=42 in CTRL, n=49 in GOLPH3 KD and n=27 in GOLPH3 OE; ***p <0.001 [Student's t-test]).

B. GOLPH3-OE HeLa cells were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), and TGN46 (blue). Dashed lines indicate cells overexpressing GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi (data are means ± SEM derived from two biological replicates, n=21 in CTRL, n=11 in GOLPH3 OE; ***p <0.001 [Student's t-test]).

C. GOLPH3 OE or KD HeLa cells expressing LCS-HA were fixed and processed for IF: HA (green). Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.

D. Assessment of LCS-GR synchronized transport in GOLPH3 OE or KD HeLa cells. Micrographs show cells at the indicated trafficking time points. Scale bar, 20 µm.

E. Representative WB of cells treated as in (D) (n = 3).

F. Plots of the densitometric quantification of the amount of LCS-GR (Golgi mature band) over time, starting from 2 hr chase (data are means of at least 3 experiments ± SD).

G. GOLPH3 OE or KD HeLa cells expressing LCS-HA were treated with bafilomycin A1 (BafA1, 10 nM) for 16 hr, fixed, and processed for IF labelling: LCS (green), LAMP1 (red), and DAPI (blue). Arrowheads indicate LCS and LAMP1 co-localisation. Scale bar, 20 µm.

H. WB of cells treated as in (G). Densitometric quantification of the blot is plotted in the top panel (data are means of at least 3 experiments ± SD).

I. GOLPH3 OE or KD HeLa cells expressing the indicated HA-tagged glycoenzymes were fixed and processed for IF. Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.

J. Number of glycoenzymes-HA positive spots per cell in GOLPH3-KD cells (left panel) and percentage of cells with glycoenzymes-HA in the ER in GOLPH3-OE cells (right panel) are plotted (n is indicated in the graph; data are means ± SEM; ***p <0.001 [Student's t-test]).
Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3 (2021)
Figure 4

Figure 4. GOLPH3 controls GSL synthesis

A. Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [3H]-sphingosine pulse (2 hr) and chase (indicated times), lipid extraction and HPTLC separation and radioactive counting (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]).

B. Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (bottom) (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.

C. Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI-MS after lipid extraction. Profiles are representative of 3 independent experiments. Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID: ST001674.

D. Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig. 1F (data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001 [Student's t-test]).

E. Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (bottom). The dotted black box highlights the effects on C16:0 Cer (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.
Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3 (2021)
Figure 5

Figure 5. GOLPH3 impacts growth signalling through LCS

A. Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS-PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway. Data are representative of at least 3 independent experiments.

B. Clonogenic assay of HeLa cells in adherence. 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days. Afterward, colonies were fixed and stained as detailed in Materials and Methods. The graph on right reports the number of colonies per well in each indicated condition (data are means ± SEM of 3 independent experiments; *p <0.05, **p <0.01 [Student's t-test]).

C. Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 (left upper graph) or LCS (left bottom graph) encoding plasmids. Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS (right upper graph). Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD (right bottom graph). Colonies were stained and quantified as described in Materials and Methods; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001, n.s. = not significant [Student's t-test]).

D. Protein lysates from parental (left), GCS-KO (middle), and LCS-KO (right) HeLa cells, CTRL or GOLPH3-OE were processed for SDS-PAGE and WB with the indicated antibodies. Data are representative of at least 3 independent experiments.

E. Soft agar colony formation assay of parental and LCS-KO HeLa cells expressing LCS-HA or GOLPH3. Colony number was quantified; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; ***p <0.001 [Student's t-test]).

F. Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS (data are means ± SEM of at least 3 independent experiments; ***p <0.001 [Student's t-test]).
Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3 (2021)
Figure 6

Figure 6. GOLPH3 and LCS protein levels correlate in NSCLC tissue samples

A, B, C. Surgical specimens were collected from 72 non-small cells lung cancer (NSCLC) patients. In (A) age at surgery and sex distribution of the patients are reported. Data are depicted as a box plot. The central band represents the mean ± quartiles, the whiskers mark the range. (B) Copy number (CN) variations in GOLPH3 gene were assessed in cancer tissue section by DNA FISH as described in Materials and Methods and are expressed as a pie chart. (C) Cancer tissue sections were processed for IHC using antibodies against endogenous GOLPH3 and LCS. The IHC intensity of the staining for each section and for both the tested proteins was classified according to the criteria described in Materials and Methods. A contingency table for the GOLPH3 and LCS staining is reported in Table EV1.

D. Representative images of NSCLC sections stained for GOLPH3 and LCS. Scale bar, 100 µm.

Expanded View Figure legends
Organismal Benefits of Transcription Speed Control at Gene Boundaries (2020)
Figure 1

Figure 1 Altering transcription activity of RNAPII by targeted mutagenesis in NRPB2.

Schematic drawing of S. cerevisiae RNAPII transcription active center. Trigger loop is shown in blue. TL-interacting Rpb2 domain is shown in beige. Proline 1018 (P1018, green) and gating tyrosine 769 (Y769, red) are highlighted. The schematic drawing is based on PDB: 2e2h [15].

Protein sequence alignment of RNAPII Rpb2 Y769 and P1018 regions in S. cerevisiae and A. thaliana. P979S and Y732F are the yeast equivalent point mutations in Arabidopsis. The color scheme indicates conservation from variable (blue) to conserved (red).

Detection of NRPB2WT-FLAG, NRPB2P979S-FLAG and NRPB2Y732F-FLAG protein by western blotting in NRPB2WT-FLAG Col-0, NRPB2P979S-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 plants. Untagged NRPB2 (Col-0) was used as a negative control. Histone H3 was used as an internal control and total protein level detected by stain-free blot was used as a loading control. Quantification was done by normalizing to the loading control and anti-H3 blot based on 3 independent replicates.

Transmission rate of nrpb2-2 allele in nrpb2-2 +/- line (n=197) and nrpb2- +/- lines combined with homozygous NRPB2P979S-FLAG +/+ (n=280), NRPB2Y732F-FLAG +/+ (n=240) and NRPB2WT-FLAG +/+ (n=210), respectively. Fisher´s exact test was used as a statistic test, three asterisks denote p<0.001 between samples and n.s. stands for not significant.

Image of homozygous mutant nrpb2-2 fully complemented by NRPB2WT-FLAG (top, NRPB2WT +/+ nrpb2-2 -/-) and partially complemented by NRPB2Y732F-FLAG (bottom, NRPB2WT +/+ nrpb2-2 -/-). Plants were grown for 4 weeks in soil. Scale bars represent 1 cm.
Organismal Benefits of Transcription Speed Control at Gene Boundaries (2020)
Figure 2

Figure 2 NRPB2Y732F accelerates RNAPII transcription in vivo.

Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo. In brief, Arabidopsis seedlings of NRPB2WT-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days. Flagellin peptides (flagellin 22) were added into media and treated samples were collected in a 0 minute (no treatment), 2 minutes and 4 minutes time course. The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes. See Methods for technical details.

Nascent RNA profile of AT5G41750. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represent 0.5 kb.

Nascent RNA profile of AT4G19520. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represents 0.5 kb.