EMBO/soda-nel huggingface dataset:
ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport (2018)
Figure 6

Figure 6. Time course by HaloTag pulse-chase of ATZ delivery to endolysosomes.

A, B Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and the luminal ER marker sfGFP-KDEL (green).

C, D Same as A, B, with CNX as ER membranes marker.

E, F Same as A, B, with CLIMP63 as ER membranes marker.

G Quantification of ER markers and HaloTag-ATZ co-localization in time (sfGFP-KDEL n=10, 10, 12, 13, CNX n=8, 10, 9, 8, CLIMP63 n=8, 10, 6, 10 cells for corresponding time points). Mean ± SEM, unpaired two-tailed t-test, **** P<0.0001.

H Quantification of double-positive endolysosomes at 300' (ATZ:sfGFP-KDEL n=14, ATZ:CNX n=8, ATZ:CLIMP63 n=10 cells. One-way ANOVA and Dunnett's multiple comparisons test, ** P<0.01 **** P<0.0001).

Data information: Scale bars 10 μm.
ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport (2018)
Figure 7

Figure 7. STX17 and VAMP8 are required for ATZ delivery within endolysosomes.

A KO efficiency in CRISPR17 MEF.

B Same as A for CRISPR8.

C-E CLSM analysis of ATZ delivery to LAMP1-positive endolysosomes in BafA1-treated CRISPRWT MEF (C); in CRISPR17 MEF(D); in CRISPR8 MEF (E). Scale bars: 10 μm.

F Quantification of C-E (n=10, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, **** P<0.0001.

G-I Distribution of gold-labelled ATZ-HA by IEM in CRISPRWT MEF exposed to BafA1 (G); in CRISPR17 (H); in CRISPR8. (I). ER, endoplasmic reticulum; EV, ER-derived vesicles; EL, endolysosomes. Scale bars: 1 μm.

J GFP reconstitution in ATZ-HA transfected CRISPR17 MEF expressing FAM134B-T11 and GFP1-10-LC3. Scale bar: 10 μm.

K Same as J by CLEM. ATZ-HA staining in red, LAMP1-positive endolysosomes in cyan. FAM134B:LC-induce GFP reconstitution in green. FAM134B in gold is shown. Scale bar: 1 μm.

L ERLAD schematics. 1. Synthesis and CNX-independent polymers formation; 2. CNX-regulated polymers segregation in ER subdomains (ES); 3. FAM134B-dependent (LIR-independent) formation of ER-derived vesicles (EV); 4. FAM134B:LC3II-mediated EV docking to RAB7/LAMP1-positive endolysosomes (EL); 5. STX17/VAMP8-controlled fusion of EV:EL membranes and polymers release in the EL lumen for clearance.

Figure Legends for EV Figures.
Promoter-pervasive transcription causes RNA polymerase II pausing to boost DOG1 expression in response to salt (2023)
Figure 1

Figure 1 - Seed phenotypic and transcriptomic response to salt stress depends on DOG1

A Germination of dog1-3 (knock-out), WT, and dog1-5 (DOG1 upregulation) seeds in media supplemented with NaCl for the indicated number of days after stratification (DAS). Scale bar represents 5 mm.

B Germination rate in 100 mM NaCl 7 days after stratification for seeds of different genotypes. Lines represent the fitted curves with a 95% confidence interval (grey area). ∗ p-value < 0.05 from two-tailed Student's t-test. Data for WT is the same as plotted in Fig 3C.

C shDOG1 expression levels normalized to UBC21 (AT5G25760) in dog1-3 and dog1-5 relative to WT.

D shDOG1 and lgDOG1 expression levels normalized to UBC21 in seeds treated with 100 mM NaCl relative to mock.

E Schematics of the pDOG1-LUC::DOG1 construct with LUC reporter inserted after DOG1 ATG, including complete intergenic regions upstream and downstream of the DOG1 gene (Fedak et al., 2016).

F Luciferase reporter activity in seeds in different concentrations of NaCl or mock. The colour bar shows the luminescence in counts per second on a logarithmic scale.

G Volcano plot shows DEGs as coloured points (DESeq2; |log2fold-change| > log2(1.5), FDR < 0.05). The colour scale shows log2fold-change. DOG1 gene is highlighted in black. The number of downregulated and upregulated genes is provided on the plot.

H, I Top 7 non-redundant Gene Ontology terms for biological process (GO:BP) for down- (H) and upregulated genes (I).

J Mean absolute expression levels from 3'RNA-seq of selected genes known to be involved in salt stress response and/or seed germination. Data from mock (-) and 100 mM NaCl (+).

K Venn diagram shows the overlap between genes affected by salt treatment in WT and dog1-3 (DESeq2; |log2fold-change| > log2(1.5), FDR < 0.05).

L, M Box plots represent the behaviour of genes upregulated (L) and downregulated (M) by salt in WT. Points show genes for which expression is more than 2 times higher or lower in dog1-3 compared to WT. Grey lines connect the same genes. The box plot's central band marks the median, boxes mark the first and third quartiles, and whiskers extend the boxes to the largest value no further than 1.5 times the interquartile range. For the comparison of transcriptomic responses the Wilcoxon rank sum test was applied using each gene (DEG in WT) as a biological replicate. Sample size n = 1136 for both WT and dog1-3 (L) and n = 835 for both WT and dog1-3 (M) ∗∗∗∗ p-value < 0.0001.

Data information: C, D Bars and error bars represent the mean ± SD. Points represent biological replicates. ∗ p-value < 0.05, ∗∗∗ p-value < 0.001 from two-tailed Student's t-test.
Promoter-pervasive transcription causes RNA polymerase II pausing to boost DOG1 expression in response to salt (2023)
Figure 2

Figure 2 - Transcriptional activity of salt-sensing element on DOG1 promoter.

A 3'RNA-seq read coverage on DOG1 locus. Black colour is used for coverage within the scale on the right-hand side (0 to 500), and red is used for coverage within the scale on the left-hand side (0 to 50). Above is a schematic representation of the annotated transcripts from the DOG1 locus and the chromosome coordinates.

B 5'RACE-seq from a primer within the DOG1 promoter reveals a transcription start site (TSS) upstream of the DOG1 gene.

C 3'RACE-seq results show novel transcription termination sites (TTS) along the DOG1 promoter and gene body.

D Schematics of newly annotated PUPPIES isoforms, co-directionally transcribed upstream of DOG1.

E RT-qPCR with primers specific for PUPPIES-uns, PUPPIES-prom, and PUPPIES-fusion isoforms. Expression levels normalized to UBC21 for 100 mM NaCl treated seeds and relative to mock. Bars and error bars represent the mean ± SD. Points represent biological replicates. ∗ p-value < 0.05, ∗∗ p-value < 0.01 from two-tailed Student's t-test.

F Schematics of psDOG1::LUC construct with Luciferase reporter sequence inserted at the end of DOG1 exon 2 (Fedak et al., 2016).

G Luciferase activity in seeds in different concentrations of NaCl or mock. The colour bar shows the luminescence in counts per second on a logarithmic scale.
Promoter-pervasive transcription causes RNA polymerase II pausing to boost DOG1 expression in response to salt (2023)
Figure 3

Figure 3 - PUPPIES-mediated changes in DOG1 gene expression and salt stress response.

A Schematics of DOG1 locus with PUPPIES and DOG1 TSS indicated by black arrows. The T-DNA insertions in puppies-1 and dog1-3 mutants, and CRISPR-Cas9 deletion in puppies-ox are shown.

B RT-qPCR with primers specific for PUPPIES-uns, PUPPIES-prom, PUPPIES-fusion, and shDOG1. Expression levels normalized to UBC21 in seeds treated with 100 mM NaCl in the puppies-1 mutant relative to WT.

C, D Germination time-course in 100 mM NaCl after stratification for puppies-1 (C) and puppies-ox (D) relative to WT. Lines represent fitted curves with a 95% confidence interval (grey area), dots represent data points, ∗ p-value < 0.05 from the two-tailed Student's t-test. (C) Data for WT is the same as plotted in Fig 1B.

E RT-qPCR (same as in B) in puppies-ox relative to WT.

F RT-qPCR fold-change induction of PUPPIES-uns, PUPPIES-prom, PUPPIES-fusion, shDOG1, and lgDOG1 in puppies-ox relative to WT (blue dashed line), in dry (light grey) and imbibed seeds in the presence of 100 mM NaCl (red). Significance from the two-tailed Student's t-test for comparing dry seeds of WT and puppies-ox is represented with light grey asterisks, and salt-imbibed seeds of WT and puppies-ox are represented with red asterisks. Black asterisks represent significance from two-way ANOVA with Tukey's multiple comparisons test for dry seeds vs imbibed seeds. ns p-value > 0.05, ∗ p-value < 0.05, ∗∗ p-value < 0.01, ∗∗∗ p-value < 0.001. Error bars represent the mean ± SD. n = 4 biological replicates.

G RT-qPCR with primers for shDOG1 and lgDOG1 on nascent RNA from seeds imbibed in 100 mM NaCl from puppies-ox relative to WT. Nascent RNA levels were normalized to UBC21. Bars and error bars represent the mean ± SD. Points represent biological replicates. Statistical significance from two-tailed Student's t-test. ∗ p-value < 0.05.

Data information: B, E Bars and error bars represent the mean ± SD. Statistical significance from two-tailed Student's t-test. ∗ p-value < 0.05, ∗∗ p-value < 0.01, ∗∗∗ p-value < 0.001.
Promoter-pervasive transcription causes RNA polymerase II pausing to boost DOG1 expression in response to salt (2023)
Figure 4

Figure 4 - DOG1 transcriptional regulation by PUPPIES revealed by single-molecule RNA FISH

A z-stack max-projection image of smFISH for DOG1 RNA. The "Inferno" colour scale is used for the intensity of fluorescence from Quasar670 fluorophore (DOG1). The blue colour shows fluorescence from DAPI (nuclei staining). Arrowheads point to foci corresponding to transcription sites (TS). The scale bar is 20 μm.

B Distribution of cytoplasmic DOG1 foci per cell in WT. The blue vertical dashed line indicates the average.

C z-stack max-projection images of DOG1 smFISH in seeds imbibed in 100 mM NaCl of WT (left) versus puppies-1 (right). Arrowheads point to foci corresponding to TS. The scale bar is 5 μm.

D Notched box plots showing the cytoplasmic DOG1 foci number per cell in WT and puppies-1. Diamond-shaped points represent each cell, n = 134 cells from WT and n = 182 cells from puppies-1.

E Distribution of intensities of foci corresponding to DOG1 transcription sites in WT and puppies-1. In the x-axis is the fluorescence intensity fold-change of foci classified as TS to the average intensity of nuclear foci. In the y-axis is the number of foci normalized to their maximum value. Vertical dashed lines indicate the average fluorescence fold-change for WT (3.1) and puppies-1 (2.4). n = 135 foci from WT, n = 29 foci from puppies-1.

F Plot showing the frequency of alleles with detected foci corresponding to DOG1 TS for WT and puppies-1. Points and error bars represent the mean ± SD. Diamond-shaped points represent single embryos.

G z-stack max-projection images from seeds imbibed in 100 mM NaCl of WT (left) versus puppies-ox (right). Arrowheads point to foci corresponding to TS. The scale bar is 5 μm.

H Same as (F), for WT and puppies-ox.

I Same as in (E), in WT and puppies-ox. Vertical dashed lines indicate the average fluorescence fold-change for WT (3.1) and puppies-ox (3.9). n = 107 foci from WT and n = 105 foci from puppies-ox.

J Notched box plot, same as (D), in WT and puppies-ox. n = 212 cells from WT, n = 179 cells from puppies-ox.

Data information: D-F, H-J Statistical significance from two-tailed Student's t-test. ∗ p-value < 0.05, ∗∗ p-value < 0.01, ∗∗∗∗ p-value < 0.0001. D and J The box plot's central band marks the median, lower and upper box limits mark the first and third quartiles, whiskers extend the boxes to the largest value no further than 1.5 times the interquartile range, and the notches extend to 1.58 times the interquartile range divided by sqrt(n).
Promoter-pervasive transcription causes RNA polymerase II pausing to boost DOG1 expression in response to salt (2023)
Figure 5

Figure 5 - DOG1 transcription pausing and processivity dependent on PUPPIES.

A-E The profile of Pol II during DOG1 transcription. Plots show the fraction of unique reads from targeted NET-Seq using rolling median (11 nt). Targeted libraries for sequencing of DOG1 locus were obtained by usage of 5 primers across the gene. Pol II dynamics is analyzed separately for each region sequenced in exon 1 (A), exon 2 (B), exon 3 (C), intron 1 (D), and intron 2 (E). The x-axis shows the distance in bp from DOG1. A light red colour fill highlights the regions where Pol II density is higher in puppies-ox. A light blue colour fill highlights the regions where Pol II density is higher in WT. For each plot, coloured tiles represent the fold-change (puppies-ox / WT) on a logarithmic scale. Greyscale tiles represent the result of a statistical test for the difference between genotypes from the two-tailed Student's t-test after Bonferroni correction. Tiles correspond to a 25 bp bin each. Vertical dashed lines connect the exon boundaries on the plots with the schematics of the DOG1 gene with grey boxes representing exons and black lines representing introns.

F, G RT-qPCR measurement of alternative splicing of lgDOG1 alpha and beta isoforms and unspliced intron 1 DOG1 isoforms levels in puppies-ox, and puppies-1 relative to WT, in seeds treated with 100 mM NaCl.

H RT-qPCR quantification of DOG1 intron 1 retention on nascent RNA from seeds imbibed in 100 mM NaCl from puppies-ox and puppies-1 relative to WT. Nascent RNA levels were normalized to UBC21.

Data information: F-H Bar and error bars represent the mean ± SD, and points represent biological replicates. ∗ p-value < 0.05, ∗∗ p-value < 0.01 from two-tailed Student's t-test.
Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling (2020)
Figure 1

Figure 1. Islr is primarily expressed in stromal cells and upregulated in colitis and colorectal cancer.

(A) qRT-PCR analysis for Islr, Chd1 and Vim in total intestinal tissues, intestinal epithelium and lamina propria. n = 4.

(B) Western blotting for Islr and E-cadherin in total intestinal tissues, intestinal epithelium and lamina propria. α-Tubulin was used as a loading control.

(C) qRT-PCR analysis for Islr in CD45+, CD45-Epcam+, CD45-Epcam-CD90+ and CD45-Epcam-CD90- cells sorted from intestinal tissues. n = 3 technical replicates.

(D) In situ hybridization for Islr with RNAscope probe in intestine and colon from 8-week-old mice, showing that Islr is primarily expressed in stromal cells. Negative and positive controls were shown in Appendix Figure S1A. Scale bar: 25 μm.

(E) In situ hybridization for ISLR with RNAscope probe in human mucosa, inflamed mucosa from CD and UC patients. Scale bar: 25 μm.

(F) qRT-PCR analysis showing the dynamic changes of Islr in colonic tissues from mice upon DSS treatment and after DSS removal. n = 4 at each time points.

(G) In situ hybridization for Islr with RNAscope probe in mouse colons without or with 3-day or 5-day DSS treatments. Scale bar: 50 μm.

(H) The cancer genome atlas (TCGA) RNA-seq analysis showing that ISLR is upregulated in human rectal adenocarcinoma relative to normal rectal tissues. The horizontal lines between the box limits represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively. n = 65.

(I) Kaplan-Meier survival curve of 270 CRC cases. P = 0.02 (log-rank test).

(J) ISLR level increased with nodal metastasis in colorectal cancer patients. N0, no regional lymph node metastasis; N1, metastases in 1 to 3 axillary lymph nodes; N2, metastases in 4 to 9 axillary lymph nodes; N3, metastases in 10 or more axillary lymph nodes. The solid horizontal lines represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively.

(K and L) In situ hybridization for ISLR/Islr with RNAscope probes in normal human colon, human malignant adenocarcinoma, metastatic adenocarcinoma (K), and in mouse colon tumors from AOM-DSS model (L). t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm.

Data information: In (A and F), data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t-test).
Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling (2020)
Figure 2

Figure 2. Ets1 activity directly regulated Islr expression.

(A) The schematic diagram showing two potential Ets1 binding sites in the Islr promoter.

(B) qRT-PCR analysis for Ets1 in colonic epithelium and lamina propria. n = 3.

(C) Double immunofluorescence for ETS1 and β-catenin in intestinal biopsies of healthy controls and inflamed mucosa from UC patients. Scale bar: 50 μm.

(D) qRT-PCR for Ets1 in colons with or without 5-day DSS treatment. n = 4.

(E) Double immunofluorescence for Ets1 and β-catenin in normal mouse colons without DSS treatment, mouse colons with 5-day DSS treatments, and AOM-DSS mouse colon tumors. Scale bar: 50 μm.

(F) Pearson correlation analysis on ETS1 and ISLR in colorectal adenocarcinoma TCGA RNA-seq (P < 0.001; R = 0.68).

(G) Western blotting for ISLR and HA in lysates of UC-MSCs transfected with pCMV5-HA or pCMV5-ETS1. α-Tubulin was used as a loading control.

(H) Luciferase activity in lysates of UC-MSCs transfected with luciferase reporter plasmids of pGL3-basic empty vector (basic), wild type ISLR promoter or mutant promoter with mutation of ETS1 binding sites under normal and ETS1 overexpression conditions. n = 3.

(I) Chromatin immunoprecipitation assay was carried out on UC-MSCs cells using antibodies against ETS1 and Histone H3. The antibody against Histone H3 was used as a positive control. IgG was used as a negative control. The enrichment of ETS1 binding to ISLR promoter was quantified using qPCR. n = 3 technical replicates.

(J) Chromatin immunoprecipitation assay was carried out on primary intestinal stromal cells isolated from WT mice without DSS treatment or WT mice after 5-day DSS treatment using antibodies against ETS1 and Histone H3. The antibody against Histone H3 was used as a positive control. IgG was used as a negative control. The enrichment of Ets1 binding to Islr promoter was quantified using qPCR. n = 3 technical replicates.

Data information: In (B, D, H, I and J), data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t-test).
Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling (2020)
Figure 3

Figure 3. Deletion of Islr in stromal cells impaired intestinal regeneration.

(A) Quantification of body weight change in control (n=12) and cKO (n=13) mice after DSS treatment and DSS removal.

(B) Fecal occult blood test in control and cKO mice at indicated timepoints. n=3 at each timepoint.

(C) Histological images of colonic tissues from control (n=10) and cKO (n=10) mice at indicated timepoints after DSS treatment. The Panels with non DSS are identical to Panels in Fig EV1F. Scale bar: 100 μm.

(D) Quantification of the clinical scores in control (n=12) and cKO (n=13) mice at indicated timepoints.

(E) Gross images of colons and quantification of colon length from control (n=6) and cKO (n=6) mice two days after 5-day DSS treatment.

(F) Double immunofluorescence for Ki67 and β-catenin in colons from control (n=4) and cKO (n=4) mice two days after 5-day DSS treatment. Quantification of percentage of Ki67+ epithelial cells. Scale bar: 100 m.

Data information: In (A, D-F), data are presented as mean ± SD. **P < 0.01; ***P < 0.001 (Student's t-test).
Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling (2020)
Figure 4

Figure 4. Deletion of Islr in stromal cells suppressed tumor growth.

(A) Schematics of generating AOM-DSS mouse colon tumor model.

(B) Quantification of body weight changes in control (n=9) and cKO (n=10) mice during tumor development.

(C) Gross images of AOM-DSS tumors in control (n=9) and cKO (n=10) mice.

(D) Quantification of tumor number per mouse and tumor volume in control (n=9) and cKO (n=10) mice.

(E) Histological images of colon tumors from control and cKO mice. Scale bar: 2 mm.

(F and G) Double immunofluorescence for Cyclin D1 and β-catenin (F), as well as Ki67 and β-catenin (G) in colon tumors from control (n=4) and cKO (n=4) mice. Scale bar: 100 μm.

(H) Quantification of percentage of cyclin D1+ and Ki67+ cells in (F and G). n = 4.

(I) The growth curve of HT29 CRC cells cultured in the supernatant with or without ISLR-HA. The supernatant were collected for HT29 CRC cells 24 hours after transfected with pcDNA3.1 empty vector or pcDNA3.1-ISLR-HA vector. n=5 at each timepoint.

Data information: In (B, D, H and I), data are presented as mean ± SD. **P < 0.01; ***P < 0.001 (Student's t-test).
Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling (2020)
Figure 5

Figure 5. Deletion of Islr in stromal cells suppressed epithelial Yap activity.

(A) Heatmap of the altered Hippo related genes in colons from control and cKO mice one day after 5-day DSS treatment. The parameter of the color key indicated the fold changes converted to log2 of signal value normalized.

(B) qRT-PCR analysis validates the altered Hippo-related genes in control and cKO mice. n=4.

(C) Western blotting for pMst1/2, Mst1, pMob1 and Mob1 in colon epithelium from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The quantification of pMst1/2 versus Mst1 and pMob1 versus Mob1 were shown under the corresponding protein bands.

(D) Western blotting for Yap1 in nuclear and cytoplasmic proteins isolated from intestinal epithelial cells from control and cKO mice two days after 5-day DSS treatment. Histone H3, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins. The quantification of Yap1 versus Histone H3 in nuclear proteins was shown under the corresponding band.

(E and F) Double immunofluorescence for Yap1 and β-catenin in the colons from control (n=4) and cKO (n=4) mice two days after DSS removal (E), and in AOM/DSS tumors from control (n=4) and cKO (n=4) mice (F). The percentage of nuclear Yap1+ cells versus epithelial cells was quantified. Scale bar: 50 μm.

(G) Western blotting for Yap1 and pYap1 in colon epithelial cells from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The ratio of Yap1/pYap1 were quantified. n=3.

(H) The growth curve of HCT116 colorectal cancer cells cultured in the supernatant from WT or cKO IMCs, concomitantly transfected with PCDH empty vector or PCDH-YAP1-5SA vector. n = 4 technical replicates.

(I) Pearson correlation analysis of ISLR and CTGF (P < 0.001; R = 0.69), ISLR and FSTL1 (P < 0.001; R = 0.81), as well as ISLR and CYR61 (P < 0.001; R = 0.65) in human colorectal cancer based on TCGA RNA-seq database.

Data information: In (B and E-H), data are presented as mean ± SD. **P < 0.01; ***P < 0.001 (Student's t-test).