EMBO/SourceData huggingface dataset:
Figure legend: "(C, D) Co-localization analysis of neosynthesized alcAp-GFP-SynA, used as a conventional cargo that traffics through the Golgi, with early (SedV) or late (PHospb) markers tagged with mCherry or mRFP, respectively. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization and statistical analysis was performed as in (A, B). Biological/technical replicates: 2/10 for each strain. Statistical analysis showed significant colocalization of SynA with the late Golgi marker (PCC = 0.67±0.04, P&lt;0.0001), but not with early Golgi marker (PCC=0.37±0.06, P&lt;0.0001). An explanation for lack of co-localization of SynA with SedV is given in the text. Scale bars: 2 μm. The cartoon at the bottom depicts the localization of early or late markers as established here in several previous studies"
NCBI gene IDs:
"alcA: 2868277"
Figure legend: "(B) Key endogenous genes controlling Golgi (sedV, geaA, hypB) or post-Golgi trafficking (rabE, ap1σ, claH) were genetically replaced by versions transcribed under the highly repressible thiAp promoter via targeted homologous recombination. In the absence of thiamine from the growth medium (derepressed conditions) the relative proteins are expressed, while upon addition of thiamine at the onset of conidiospore germination (ab initio repression) the expression of these proteins is tightly repressed. Proteins are detected by a standard western blot analysis using either anti-FLAG or anti-GFP antibodies for Golgi and Post-Golgi proteins. Equal loading and protein steady state levels are normalized against the amount of actin, detected with a specific antibody."
NCBI gene IDs:
"thiA: "
Figure legend: "(C) In the absence of thiamine from the growth medium (derepressed conditions) the corresponding strains grow nearly as an isogenic wild-type control, although a delay in growth is observed for thiAp-sedV and less so for thiAp-ap1σ (upper row). In the presence of thiamine to the growth medium most cells do not form colonies, except for thiAp-hypB which forms a compact slow growing colony (lower row)."
NCBI gene IDs:
"ap1σ: 
hypB: 
sedV: 
thiA: "
Figure legend: "(D) Microscopic examination of the corresponding strains under thiamine (repressing conditions) shows that, in most cases, the apical region of germlings is enlarged and growth is arrested. This morphological phenotype, taken as a strong indication of blocked secretion, is more evident in thiAp-sedV and thiAp-rabE but concerns all strains, except for thiAp-hypB. Scale bar: 5 μm."
NCBI gene IDs:
"hypB: 
rabE: 
sedV: 
thiA: "
Figure legend: "(E) Subcellular localization of UapA, after 6-8 hours of initiation of transcription, via its native uapA promoter, while conventional sedV, geaA or hypB transcription is repressed by thiamine ab initio. Scale bars: 2 μm. Notice that UapA-GFP translocates in the PM of germlings in all cases. Results shown are confirmed by quantification (right panel) of UapA-GFP PM/cytosolic intensity ratios for the four strains (for details see Materials and methods). Mean PM/cytosolic intensity ratios for wild-type, thiAp-sedV, thiAp-geaA and thiAp-hypB are 0.53±0.02, 0.52±0.02, 0.52±0.02 and 0.52±0.02, respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). No statistical significance was found between the wild-type and each of the mutant strains. Biological/technical replicates: 3/15 for wild-type, 3/18 for thiAp-sedV, 3/15 for thiAp-geaA and 3/18 for thiAp-hypB."
NCBI gene IDs:
"hypB: 
geaA: 
sedV: 
thiA: 
uapA: 2870384"
Figure legend: "(F) alcAp-UapA-GFP, under derepressing conditions, translocates to the PM of a strain carrying a thermosensitive mutation in SedVts both at the permissive (25oC) and restrictive temperature (42oC). A degree of increased degradation of UapA, caused by exposure to high temperature (42oC), is apparent as cytosolic puncta, which correspond to membrane aggregates and sorting to vacuoles. Scale bar: 5 μm."
NCBI gene IDs:
"SedV: 
alcA: 2868277"
Figure legend: "(G) Unlike UapA, de novo made apical markers, such as ChsB or SynA, lose their polar localization, when the expression of sedV, geaA or hypB is repressed. Scale bars: 2 μm. Quantification: GFP-ChsB PM/cytosolic and alcAp-GFP-SynA PM/cytosolic intensity ratios are plotted to the right upper and lower panel, respectively. For GFP-ChsB, mean PM/cytosolic intensity ratios are: 0.60±0.02 (wild-type), 0.11±0.01 (thiAp-sedV), 0.26±0.02 (thiAp-null) and 0.34±0.01 (thiAp-hypB). For alcAp-GFP-SynA mean PM/cytosolic intensity ratios for wild-type, thiAp-sedV, thiAp-geaA and thiAp-hypB are 0.61±0.03, 0.14±0.02, 0.26±0.03 and 0.39±0.03, respectively. The statistical analysis was performed as in (E). A significant difference (****P&lt;0.0001) of GFP-ChsB fluorescence in the apical PM membrane was found between the wild-type and the three strains lacking the key-Golgi proteins. This is also the case for SynA, as seen in the scatter plot on the lower right panel, where the fluorescence intensity of SynA is diminished (****P&lt;0.0001) when sedV, geaA and hypB are repressed. Biological/technical replicates: 3/15 for each strain."
NCBI gene IDs:
"hypB: 
geaA: 
sedV: 
thiA: 
alcA: 2868277"
Figure legend: "(A) Growth test showing that Sec24 expression is essential for growth as its transcriptional repression by thiamine (+thi) leads to absence of colony formation, despite initial germination The strains shown are isogenic except for the sec24 locus. thiAp-sec24 signifies the strain where the endogenous sec24 promoter was replaced by the thiAp promoter."
NCBI gene IDs:
"sec24: 
thiA: "
Figure legend: "(B) Epifluorescence microscopy analysis of the subcellular localization of UapA-GFP under conditions where sec24 transcription is ab initio derepressed (upper panel) or repressed by thiamine (lower panel). o/n (overnight) means addition of thiamine from the onset of germination. Germination of conidiospores takes place until full repression of Sec24 is achieved (10-12 h). Notice the total lack of PM-associated signal of UapA under conditions of Sec24 repression. Scale bar: 5 μm. Quantification: In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when sec24 is derepressed (-thi) or repressed (+thi). Mean PM/cytosolic intensity ratios are 0.55±0.02 and 0.32±0.02 respectively. To test the significance of differences, an unpaired t-test was performed, which verified the significant difference (****P&lt;0.0001) in the presence of thiamine. Biological/technical replicates: 2/15 for each condition."
NCBI gene IDs:
"sec24: "
Figure legend: "(E) Epifluorescence microscopy analysis of the subcellular localization of UapA-GFP under conditions where sec13 transcription is ab initio derepressed (left panel) or repressed by thiamine (right panel). The inserts in the upper right corner in both panels reflect growth tests showing that Sec13 repression leads to arrest in growth and absence of colony formation. Scale bars: 5 μm. All images reflect practically identical results obtained in several experiments. Quantification: UapA-GFP PM/cytosolic intensity ratios are quantified when sec13 is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.31±0.02, respectively. There is a significant difference on UapA PM fluorescence intensity (****P&lt;0.0001) in the absence of the COPII outer coat protein (statistical analysis as in B). Biological/technical replicates: 2/15 for each condition."
NCBI gene IDs:
"sec13: "
Figure legend: "(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made UapA-GFP, after 6-8 hours of initiation of transcription in strains where the expression of RabE, AP-1σ or ClaH (thiAp-rabE, thiAp-ap1σ or thiAp-claH) has been repressed ab initio (o/n) by addition of thiamine. Notice that when rabE or ap1σ is repressed UapA-GFP still reaches the PM. In contrast, repression of claH abolishes labeling of the PM and leads to the cytosolic puncta and a membranous network. Scale bars: 5 μm. Quantification: UapA-GFP PM/cytosolic intensity ratios are plotted on the right. Mean ratios for wild-type, thiAp-rabE, thiAp-ap1σ and thiAp-claH are 0.55±0.02, 0.53±0.03, 0.54±0.02 and 0.29±0.02 respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). After 6-8h of transcriptional derepression, UapA-GFP fluorescence to the PM does not change when rabE or ap1σ are repressed. On the other hand, in the absence of ClaH, UapA does not reach the PM, as its fluorescence intensity there is statistically lower (****P&lt;0.0001) in comparison to that of the wild-type strain. Biological/technical replicates: 2/15 for each strain."
NCBI gene IDs:
"claH: 
rabE: 
ap1σ: 
thiA: "
Figure legend: "(B) In a similar experiment, the apical polarized localization of de novo made SynA, used as a control of conventional secretion, is shown to be abolished in all three strains where RabE, AP-1σ or ClaH are repressed. Scale bars: 5 μm. mCherry-SynA PM/cytosolic intensity ratios are plotted with values being 0.52±0.02 for wild-type, 0.14±0.02 for thiAp-rabE, 0.24±0.02 for thiAp-ap1σ and 0.16±0.02 for thiAp-claH. The statistical evaluation was performed as in (A). There is a significant difference (****P&lt;0.0001) of mCherry-SynA fluorescence in the apical PM membrane between the wild-type and the three strains lacking the post-Golgi proteins. Biological/technical replicates: 2/15 for each strain."
NCBI gene IDs:
"ap1σ: 
claH: 
rabE: 
thiA: "
Figure legend: "(B) Time course of treatment of strains expressing neosynthesized alcAp-UapA-GFP and mCherry-TubA with the anti-microtubule drug benomyl for 5, 55, or 95 minutes. In all cases benomyl was added at 95 min of UapA derepression, so that total time of UapA-GFP expression was 100, 150 or 190 minutes Benomyl abolished the thread-like appearance microtubules in all samples added, evident by the diffuse cytoplasmic signal of mCherry-TubA. Notice that UapA reaches normally the PM (at 190 min of derepression), similarly to the untreated control (right panel). Scale bar: 5 μm."
NCBI gene IDs:
"alcA: 2868277"
Figure legend: "(C) Subcellular localization of neosynthesized alcAp-GFP-ChsB in the absence (left panel) or presence (≈40 min) of benomyl (middle panel) or latrunculin B (right panel), after ≈2h of derepression. Notice the abolishment of proper polar localization of ChsB at the apical tip in both cases. Scale bars: 5 μm."
NCBI gene IDs:
"alcA: 2868277"
Figure legend: "(A) Subcellular localization of de novo made alcAp-GFP-SsoA in the PM in a single hypha. Notice the ER-like membranous network (white arrows) and several cytosolic puncta labelled before SsoA reaches eventually the PM. Scale bar: 5 μm."
NCBI gene IDs:
"alcA: 2868277"
Figure legend: "(B) Subcellular localization of neosynthesized UapA-GFP (8 hours after transcriptional depression) while ssoA transcription is ab initio repressed. Notice the localization of UapA in the ER-membranous network, often at very close proximity with the PM. Scale bar: 5 μm. In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when ssoA is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.30±0.02 respectively. There is statistically lower fluorescence intensity to the PM (****P&lt;0.0001) when SsoA is absent from the cell, as confirmed by an unpaired t-test. Biological/technical replicates: 2/15 for each condition."
NCBI gene IDs:
"ssoA: "
Figure legend: "(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp-AzgA-GFP and alcAp-FurA-GFP, after 6-8 hours transcriptional derepression in strains where the expression of Sec24, SedV, GeaA, HypB, RabE, AP-1σ and SsoA was repressed ab initio (o/n) by addition of thiamine. Images showing that sorting of AzgA or FurA to the PM does not require early (SedV, GeaA), late (HypB) or post-Golgi (RabE, AP-1σ ) key proteins. On the other hand, COPII coat protein Sec24 and PM t-SNARE SsoA are essential for proper localization of the two transporters to the PM. Scale bar: 5 μm."
NCBI gene IDs:
"alcA: 2868277"
Figure legend: "Keratinocytes were isolated from the skin of a 4-year-old patient with severe-generalised RDEB linked to homozygous insertion-deletion in COL7A1 (Hilal et al, 1993). Cultured RDEB cells (blue line) were serially passaged for more than 4 months, displaying a growth potential similar to non-diseased control cells (YF29) isolated from the foreskin of a newborn (black line). To calculate the percentage of growing colonies, 100 to 1,000 cells were plated into indicator dishes at each passage. Cells were grown for 12 days, fixed and stained with rhodamine B. Colonies were scored as growing or aborted (Barrandon & Green, 1987)."
NCBI gene IDs:
"COL7A1: 1294"
Figure legend: "Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon & Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation.COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm."
NCBI gene IDs:
"COL7A1: 1294"
Figure legend: "Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon & Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Quantitative RT-PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1."
NCBI gene IDs:
"COL7A1: 1294"
Figure legend: "Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon & Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk."
NCBI gene IDs:
"ColVII1: 1308
COL7A1: 1294"
Figure legend: "Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon & Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control."
NCBI gene IDs:
"COL7A1: 1294"
Figure legend: "Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII. Non-COLVII-producing holoclone 54 (black lines) and COLVII-producing holoclone 6 (red lines) were serially transferred once a week until exhaustion (Rochat et al, 1994).Theoretical number of epidermal cells available for characterisation and CEA production from corrected (clone 6) and uncorrected stem cells (clone 54) calculated from the day of cloning. The colony-forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling, the generation number and the total progeny of isolated stem cells. Both corrected and uncorrected epidermal stem cells show high growth potential in vitro."
NCBI gene IDs:
"COLVII: 1308"
Figure legend: "Immunodeficient SCID mice were transplanted with untransduced cultured RDEB keratinocytes (left) or the COLVII-secreting holoclone (clone 6) (right). Punch biopsies were obtained at various times post-transplantation (PT), stained with haematoxylin/eosine (H/E), for human leucocyte antigen-1 (HLA-1) (green) and human COLVII (red). RDEB keratinocytes generated a HLA-1-positive epidermis that adhered poorly to the dermo-epidermal junction (DEJ) (arrow indicates a blister) and absence of COLVII (dotted line delimits the dermis), whereas the corrected keratinocytes produced an epidermis that adhered to the dermis and deposited COLVII (red) at the DEJ. Note the presence of KI67-positive keratinocytes (red) in the basal and suprabasal layers in the corrected epidermis at 385 days post-transplantation, indicating that transplanted cells had self-renewed for more than a year. Scale bar: 50 μm."
NCBI gene IDs:
"COLVII: 1308"
Figure legend: "Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage XII (clone 6). Five specific signals (red) were detected on five different chromosomes. As expected, a COL7A1 probe (red) hybridised to the two endogenous COL7A1 alleles located on the chromosomes 3 as identified by a specific centromeric probe (Cen 3) and to three other chromosomes identified as chromosomes 2, 11 and 22 by means of specific centromeric probes (Cen 2, Cen 11 and Cen 22, respectively). The latter corresponded to proviruses.Determination of proviral integration sites in corrected stem cell. Genomic DNA from clone 6 was submitted to whole-genome sequencing together with PCR and subsequent Sanger sequencing to identify the integration sites to the base-pair level. Mate pairs that span from the viral sequence to a human chromosome were extracted and used to estimate integration regions. The reads were mapped to the hg19 reference sequence. Three integration sites were uncovered: one in chromosome 2, one in chromosome 11 and one in chromosome 22, and targeted genes were identified."
NCBI gene IDs:
"COL7A1: 1294"
Figure legend: "Analysis of the level of expression of targeted genes in corrected stem cells by quantitative RT-PCR. DARS was not significantly changed in clone 6 compared to the cells before transduction. Primers used annealed downstream of the proviral integration site. Error bars represent the standard deviation of three replicates.Identification of sequence abnormalities in the two alleles of the DARS-targeted gene in clone 6 by NGS. Reads were mapped to the hg19 reference sequence. Small insertion-deletions and SNP calling were performed and compared to GWAS databases. The mapping highlighted two indels and one SNP in the DARS genomic sequence. The indels were not associated with disease and the SNP was synonymous (CCU-CCG both codons correspond to proline)."
NCBI gene IDs:
"DARS: 1615"
Figure legend: "To test whether the transduced RDEB keratinocytes had disseminated after the generation of an epidermis onto SCID mice (see Fig4C), the internal organs of the recipient mice (3 mice for clone 6 and 2 mice for clone 22) were harvested 385 days post-transplantation and analysed for COL7A1 proviral sequences. No COL7A1 sequence was detected. A mouse transplanted with a holoclone obtained from a healthy donor (YF29) was used as an internal control. PCR-positive controls were genomic DNA from transduced cells (holoclone 6) and cDNAs isolated from healthy keratinocytes. B-actin was run as a loading control."
NCBI gene IDs:
"B-actin: 
COL7A1: 1294"
Figure legend: "C. Representative  flow cytometric profiles of  thymocytes from 6-week old  Rb∆K4 and control  mice  for CD3-, CD4- and  CD8-positive cell populations. Bottom, average of CD3-, CD4- and  CD8 single- and double-positive cells in Rb∆K4 vs. control  thymocytes showing no significant (n.s.) difference by student's t-test. Bars represent mean ±SD (standard deviation); n=6 biological replicates)."
NCBI gene IDs:
"Rb: 19645"
Figure legend: "D. Short telomeres in Rb∆K4 mice. Images of  splenocyte metaphase spreads hybridized with the telomere repeat probe (pink). Note shorter telomeres in Rb∆K4 splenocytes  (arrows). Bottom, quantitative analysis of 10  splenocyte metaphases from one pair of Rb∆K4 and control littermate; (P&lt;0.001 by two-tailed Mann-Whitney-Wilcoxon test)."
NCBI gene IDs:
"Rb: 19645"
Figure legend: "C. Left, representative  flow cytometric analysis for CD3, CD4 and CD8, showing near normal  T cell  development in 10-day-old Rb∆K7 mice. Right, average of CD3, CD4  and CD8 single- and double-positive cell populations in Rb∆K7 vs control thymocytes  with no significant difference (n.s.) by student's t-test. Results are presented as mean ±SD, n=4 biological replicates)."
NCBI gene IDs:
"Rb: 19645"
Figure legend: "D. Left, images showing apparent normal division of  retinal progenitors in  Rb∆K7 mice. Mice were labeled with BrdU for 2 hrs; retinal sections from indicated genotypes were stained  with  DAPI (blue), Ki67 (green), PH3  (red) or  BrdU (red) to mark nuclei, all dividing cells, mitotic  or S-phase cells, respectively. Right, quantification of  staining showing no significant difference among the genotypes. Bars denote mean ±SD; n=3 biological replicates). Scale bar: 20 μm."
NCBI gene IDs:
"Rb: 19645"
Figure legend: "E. Left, top,  western blot analysis of Rb∆K7 thymocytes with an antibody that recognizes both hyper-phosphorylated (ppRb) and hypo-phosphorylated pRb species. Right, phospho-specific  immunoblots showing absence of phosphorylation at upstream Thr350 and Ser601 or the substituted Ser773Ala site in  Rb∆K7 thymocytes. Right, immuno-precipitation (IP)-Western blot  analysis demonstrating  phosphorylation of  pRb∆K7 on Ser243/Thr246 (top) and Ser605 (bottom). n.s., non-specific band."
NCBI gene IDs:
"Rb: 19645"
Figure legend: "B. Increased resistance of  Rb∆K7 vs. control MEFs to  CDK2 inhibition (P=0.008 by two-tailed unpaired student's t-test). Effect of a pan CDK inhibitor was near significance (P=0.07). Bars represent mean ±SD, n=3 independent MEFs."
NCBI gene IDs:
"Rb: 19645"
Figure legend: "C. Left, representative flow cytometric analysis with  7-AAD  and AnnexinV, showing a significant decrease in apoptosis in  Rb∆K7 MEFs relative to control. Right, significant differences in average percentage of live (7-AAD-:AnnexinV-) vs. apoptotic (7-AAD+:AnnexinV+) cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test; n=3 independent MEFs."
NCBI gene IDs:
"Rb: 19645"
Figure legend: "F-I. Delayed  hair re-growth in eight-month old Rb∆K7 mice two weeks post hair  plucking (F). (G) Abnormal skin histology in Rb∆K7 mice relative to control. FCL, subcutaneous fat cell layer, T,  telogen; A,  anagen. (H-I) Representative  immunostaining for  keratin 14 and PCNA showing reduced expression in  Rb∆K7 hair follicles vs controls. Scale bar, 50 μm."
NCBI gene IDs:
"Rb: 19645"
Figure legend: "J. Hyperglycemia  in Rb∆K7 but not Rb∆K4 mice. Blood glucose in 10-12-month old fasting Rb∆K7 (left, n=11) or 18-month old fasting Rb∆K4 (right, n=15) mice versus control littermates (n=6 and n=8, respectively). Mean ±SD; P values by two-tailed unpaired student's t-test."
NCBI gene IDs:
"Rb: 19645"