EMBO/soda-nel huggingface dataset:
3,4-Dimethoxychalcone induces autophagy through activation of transcription factors TFE3 and TFEB (2019)
Figure 5

Figure 5 3,4-DC induced TFEB and TFE3-depdendent autophagy (A-F) U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) (A-C), or U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells (D-F) were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot (A,D). GFP-LC3 dots were quantified as indicator of autophagy (C,F). Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown in (B,E). Scale bar equals 10 µm. (G-H) U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. Following, cells were collected and SDS-PAGE and immunoblots were performed as described before. TFEB, TFE3, LC3, p62, and GAPDH protein levels were measured with specific antibodies (G). GFP-LC3 dots were quantified as indicator of autophagy (H). Data are means ± SD (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 versus Ctr, respectively). (I) U2OS WT, knockout for TFEB (TFEB-/-) or TFE3 (TFE3-/-), double knockout (DKO) were treated with 3,4-DC for 24 h and stained with LysoTracker Red for 30 min. Thereafter, the red (positive) dots were measured. Data are means ± SD (* = p < 0.05; ** = p < 0.01; *** = p < 0.001). (J) Relative mRNA expression levels of key autophagy genes such as Lamp1, Lc3b and p62 were detected in controls and 3,4-DC treated U2OS cells. Data is depicted as a heatmap showing means of at least three independent experiments (*** = p < 0.001 versus WT/Ctr and # = p < 0.05, ## = p < 0.01, as compared to WT treated with 3,4-DC). Samples for immunoblots in A and B were run together on one gel and in G were run in parallel gels, then blotted, cut into horizontal stripes and probed separately.
3,4-Dimethoxychalcone induces autophagy through activation of transcription factors TFE3 and TFEB (2019)
Figure 6

Figure 6. 3,4-DC induces autophagy in vivo. (A-D) Female C57/BL6 animals were treated with 3,4-DC for 24 h. Following, liver and heart tissue was excised and cells were subjected to subcellular fractionation. Thereafter proteins were separated by SDS PAGE and immunoblots were performed to detect the nuclear translocation of TFEB and TFE3 (A, C). GAPDH and H3 were used as controls for cytosolic and nuclear fractions, respectively. Band intensities of nuclear TFEB, TFE3 and H3 were measured and ratios of nuclear TFs versus H3 (TFsnuc/H3) were calculated in (B, D). Data are means ± SEM of at least three mice (*** = p < 0.001 versus Ctr) (E, F, I, J) GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in liver (E, F) and heart (I, J) tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu). (G, H, K, L) C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following liver and heart tissue was excised and subjected to SDS PAGE and immunoblot (G, K). GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated in (H, L). Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01). Samples for immunoblots were run together on the same gel (G,K) or on parallel gels (A, C), then blotted, cut into horizontal stripes, and probed separately.
3,4-Dimethoxychalcone induces autophagy through activation of transcription factors TFE3 and TFEB (2019)
Figure 7

Figure 7. 3,4-DC confers autophagy-dependent cardio-protection from ischemic injury. (A) Twelve weeks-old wild-type and cardiac specific Atg7 knockout (Atg7cKO) mice were injected with corn oil (vehicle control, Ctr) or 3,4-DC 24 h before surgery and subjected to 3 h prolonged ischemia. Representative images of left ventricular (LV) myocardial sections after alcian blue and triphenyltetrazolium chloride (TTC) staining are depicted. Scale bar equals 1 mm. (B,C) The size of the infarction area (indicated by pale color) per area at risk (AAR) (B) and the global AAR per LV (C) were measured (mean value ±SEM, **p<0.01, #p<0.05 vs WT/Ctr).
3,4-Dimethoxychalcone induces autophagy through activation of transcription factors TFE3 and TFEB (2019)
Figure 8

Figure 8. 3,4-DC improves the efficacy of anticancer chemotherapy. A. Induction of autophagy in murine MCA205 fibrosarcomas. Cells were treated with 3,4 DC alone or in combination with chloroquine, and were harvested 6 hours later for immunoblot detection of LC3 lipidation. B. Schematic overview on the in vivo treatment of MCA205 fibrosarcomas with mitoxantrone (MTX) and 3,4-DC, alone or in combination. (C-E). Cytofluorometric characterization of tumor infiltrating lymphocytes (TIL), in particular FOXP3+ regulatory T cells (Treg), CD8+ cytotoxic T lymphocytes and the ratio of CD8+ T cells over Treg. (F-I). Growth kinetic of MCA205 fibrosarcomas that were either wild type (F,G) or Atg5KD (H-I) and were evolving in immunocompetent C57Bl/6 mice (F,I) or immunodeficient nu/nu mice (G), treated as indicated in B. (J-L) Immunocompetent C57Bl/6 mice were subcutaneously inoculated with TFEB/TFE3 double knockdown MCA205 cells or its scramble control cells (K). When tumors became palpable, mice were treated as indicated in (B). Tumor growth curves from mice subjected to 3,4-DC administration alone or in combination with MTX are shown (J,L). Asterisks indicate significant effect of MTX with respect to untreated controls (mean value ±SEM, *p<0.05, **p<0.01, ***p<0.001), while hash symbols refer to the comparison of the effects of MTX plus 3,4-DC to MTX alone (#p<0.05, ##p<0.01, ###p<0.001)(n=6-14). Samples for immunoblots were run on one gel (A, H) or several parallel gels (K), then blotted, cut into horizontal stripes, and probed separately.

Expanded view legends
UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development (2019)
Figure 1

Figure 1. UV-B inhibits the growth of lateral roots in a UVR8-dependent manner.

A-C. Phenotypic analysis. Wild-type, uvr8, and rup1 rup2 seedlings were grown in 1/2 MS with or without UV-B (1 W/m2) for 2 weeks. Images are shown in (A); scale bars = 5 mm. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (each plant) (C) of the indicated genotypes were measured. SDs (n > 8 independent seedlings) are indicated. Letters "a" to "d" indicate statistically significant differences for the indicated values, as determined by a one-way analysis of variance (ANOVA), followed by a Tukey's least significant difference (LSD) test (P < 0.05). There was statistically significant difference between values marked with different letters (for example "a" and "b"), while there was no statistically significant difference between values marked with the same letters (for example "a" and "a").

D. Distribution of lateral root primordia stages. Seedlings of the indicated genotypes were grown in 1/2 MS treatment with or without UV-B (1 W/m2) for 2 weeks. SDs (n > 8) are indicated. "***" indicates statistically significant differences (P < 0.001), as determined by Student's t-test.

E and F. GUS staining of seedlings expressing DR5p::GUS transgene in the WT, uvr8 and rup1 rup2 background. Seedlings were grown in 1/2 MS with or without UV-B for 2 weeks. Images of lateral root primordia (E) and lateral roots (F) are shown. Scale bars = 100 and 50 μm, respectively.
UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development (2019)
Figure 2

Figure 2. UV-B inhibits auxin responses through UVR8.

A and B. Phenotypic analysis. Seedlings of the indicated genotypes were grown in 1/2 MS with the addition of a series of concentrations of NAA under continuous white light or white plus UV-B (1 W/m2) light for 2 weeks. The lateral root density (number of lateral roots/length of primary root) (A) and average length of lateral roots (B) of the indicated genotypes were measured. SDs (n > 8) are indicated. "*" indicates statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001), as determined by Student's t-test.

C-E. UV-B inhibits the response of Arabidopsis seedlings to exogenously added NAA in a UVR8-dependent manner, since the response to NAA was not repressed in uvr8. Seedlings of the indicated genotypes were grown in LD (16-h light/ 8-h dark) conditions for 5 days, then transplanted to new plates containing 0.4 μM NAA and kept in continuous white light or white light plus UV-B (1 W/m2) for 7 days. Images are shown in (C); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (D) and average length of lateral roots (E) of the indicated genotypes were measured. SDs (n > 8) are indicated. Letters "a" to "c" indicate statistically significant differences for the indicated values, as determined by a one-way analysis of variance (ANOVA), followed by a Tukey's least significant difference (LSD) test (P < 0.05). There was statistically significant difference between values marked with different letters (for example "a" and "b"), while there was no statistically significant difference between values marked with the same letters (for example "a" and "a").
UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development (2019)
Figure 3

Figure 3. Monomeric UVR8 inhibits auxin responses in a tissue-autonomic manner.

A-C. Phenotypic analysis. WT, GR-UVR8R338A/uvr8 and GR-UVR8W285F/uvr8 seedlings were grown in LD (16-h light/ 8-h dark) for 5 days, then transplanted to new medium with 0.4 μM NAA and with or without 20 μM DEX and kept in white light plus UV-B (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of indicated genotypes were measured. SDs (n > 8) are indicated. Letters "a" and "b" indicate statistically significant differences for the indicated values, as determined by a one-way analysis of variance (ANOVA), followed by a Tukey's least significant difference (LSD) test (P < 0.05). There was statistically significant difference between values marked with different letters (for example "a" and "b"), while there was no statistically significant difference between values marked with the same letters (for example "a" and "a").

D-F. UVR8 inhibited auxin responses under UV-B in a tissue-autonomous way. WT and uvr8 seedlings grown in LD for 5 days were used for reciprocal grafting. Seedlings were kept in LD for 7 days after grafting, then transplanted to new medium containing 0.4 μM NAA and kept in UV-B light (1 W/m2) for 10 days. Images are shown in (D); scale bar = 2 mm. The lateral root density (number of lateral roots/length of primary root) (E) and average length of lateral roots (F) of indicated genotypes were measured. SDs (n > 8) are indicated. Letters "a" and "b" indicate statistically significant differences for the indicated values, as determined by a one-way analysis of variance (ANOVA), followed by a Tukey's least significant difference (LSD) test (P < 0.05). There was statistically significant difference between values marked with different letters (for example "a" and "b"), while there was no statistically significant difference between values marked with the same letters (for example "a" and "a").
UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development (2019)
Figure 4

Figure 4. UV-B represses the expression of auxin signaling genes.

A and B. Transcriptome analysis of gene expression regulated by auxin, UV-B, and UVR8. (A) Heat map of UV-B-, UVR8-, and auxin-regulated genes. The parameter measured by color key shows the Log-fold change. (B) Auxin signaling genes are up-regulated by auxin treatment but down-regulated by UV-B in a UVR8-dependent manner. Three biological replicates were analyzed and final Log fold-change is shown.

Quantitative RT-PCR results showing that UV-B represses the expression of auxin signaling genes. Six-day-old WT seedlings grown in continuous white light were transferred to UV-B light (2 W/m2) for the indicated period. Error bars are SD of three biological replicates.

D and E. Quantitative RT-PCR analysis of auxin signaling gene expression in wild-type (Col-0), uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 5 days were kept in white light (D) or transferred to white light plus UV-B (E) for 1 day with the addition of 1 μM IAA. Error bars are SD of three biological replicates.

F and G. qPCR analysis of auxin signaling gene expression in roots of WT, uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 12 days were kept in white light (F) or transferred to white light plus UV-B (G) for 1 day with the addition of 0.4 μM NAA. Error bars are SD of three biological replicates.
UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development (2019)
Figure 5

Figure 5. UVR8 physically interacts with MYB73/MYB77 in a UV-B-dependent manner.

A and B. In vitro pull-down assay showing that UV-B treatment promotes the interaction between MYB73 (A) or MYB77 (B) and UVR8. The bound proteins were eluted and analyzed by probing immunoblots with an anti-His antibody.

C. BiFC assay showing that UVR8 interacts with MYB73/MYB77 in white light condition. N. benthamiana was co-transformed with MYB73-nYFP/MYB77-nYFP and cCFP, nYFP, and UVR8-cCFP, or MYB73-nYFP/MYB77-nYFP and UVR8-cCFP. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bar = 20 μm.

D-G. Co-IP assays showing that MYB73 and MYB77 interact with UVR8 in a UV-B-dependent manner in whole seedlings (D, F) and roots (E, G). Input: immunoblots showing the levels of UVR8, MYB73-TAP, and MYB77-TAP in the total protein extract. UVR8 IP: the IP products precipitated by the UVR8 antibody. Total proteins (Input) or IP product were probed, on immunoblots, with an anti-UVR8 or anti-Myc antibody, which detects the TAP tag.
UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development (2019)
Figure 6

Figure 6. MYB73/MYB77 acts downstream of UVR8 in controlling the auxin response.

A-C. Phenotypic analysis. Seedlings of the indicated genotypes were grown in LD conditions (16-h light/ 8-h dark) for 5 days, and then transplanted to new medium containing 0.4 μM NAA and kept in continuous white light or white plus UV-B light (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. Lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of the indicated genotypes were measured. SDs (n > 8) are indicated. Letters "a" to "c" indicate statistically significant differences for the indicated values, as determined by a one-way analysis of variance (ANOVA), followed by a Tukey's least significant difference (LSD) test (P < 0.05). There was statistically significant difference between values marked with different letters (for example "a" and "b"), while there was no statistically significant difference between values marked with the same letters (for example "a" and "a").

D and E. Quantitative RT-PCR analysis of auxin signaling gene expression in roots of wild type (Col-0), uvr8, myb73 myb77, and myb73 myb77 uvr8. (D) Results under white light and (E) under white plus UV-B light. Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV-B for 1 day with the addition of 0.4 μM NAA, and roots were collected to analyze auxin signaling gene expression. Error bars represent SD of three biological replicates.
UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development (2019)
Figure 7

Figure 7. UVR8 inhibits the DNA-binding activity of MYB73/MYB77.

A. Electrophoretic mobility-shift assay (EMSA) showing that MYB73/MYB77 binds to the MBSI motif in vitro. Cold probe was added as a competitor.

B. EMSA showing that MYB73/MYB77 binds to the promoter of IAA19. Cold IAA19p or IAA19mp (MBSI motif mutated) was added as a competitor.

C and D. EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB73(C) and MYB77(D).

E. ChIP-qPCR analysis showed that UV-B inhibits the DNA-binding activity of MYB73. ChIP-qPCR assays were performed using transgenic seedlings expressing 35S::MYB73-TAP, treated with or without UV-B for 5 h before harvesting samples. Chromatin fragments (~500 bp) were immunoprecipitated by anti-Myc antibody, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/Input ratios are shown with SDs (n=3).

F. ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).

G and H. ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in roots (G) in a UVR8-dependent manner (H). The IP/Input ratios are shown with SDs (n=3).

I. ChIP-qPCR showing that UV-B inhibits MYB77 from binding to the indicated promoters in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).
TMEM41B is a novel regulator of autophagy and lipid mobilization (2018)
Figure 1

Figure 1 - Sensitized CRISPR screens identify TMEM41B as a novel regulator of autophagy.

A Schematic representation of pooled CRISPR screening workflow. H4 cells stably expressing Cas9 were transduced with a lentiviral sgRNA mini-pool library, selected for stable integration, expanded and treated for 24 h with the mTOR inhibitor AZD8055 (activated autophagy) or DMSO as vehicle control (basal autophagy). Endogenous p62 or NDP52 were visualized by immunostaining and cells separated by FACS into populations with high or low signal. Abundance of sgRNAs was quantified in cell populations by next generation sequencing.

B, C Relative abundance of sgRNAs in cell populations with high versus low signal were visualized as log2 fold chances for basal as well as activated autophagy conditions for (B) p62 or (C) NDP52. Entire data is reported in Dataset EV1.

D-F Validation of TMEM41B. H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or a non-targeting (NT) control, treated with 500 nM AZD8055, 50nM Bafilomycin A1 (BafA1) or DMSO vehicle control for 24 h, and analyzed by immunoblotting. (E, F) p62 and NDP52 band intensities are depicted as total levels relative to vehicle control in H4 Cas9 NT cells (E), or as flux by calculating the ratio in BafA1-treated cells versus vehicle control (F). Data are presented as mean ± SD (n=6 independent experiments) with paired t-test values.

G LAMP1 and p62 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at p62-filled and empty LAMP1-positive organelles. Scale bar: 5 µm.

H LAMP1-positive organelles were manually quantified for containing a p62-filled or empty lumen. A total of 50-60 LAMP1-positive organelles were analyzed in each experiment. Data are presented as mean ± SD (n=3 independent experiments) with paired t-test values.

I HeLa cells were transduced with Cas9 and TMEM41B or NT sgRNAs followed by infection with luciferase-expressing S. typhimurium. Luciferase readings were taken up to 8.5 h post-infection. Results from one representative experiment are presented as mean ± SD (n=8 technical replicates).
TMEM41B is a novel regulator of autophagy and lipid mobilization (2018)
Figure 2

Figure 2 - Knockout of TMEM41B impairs autophagosome biogenesis.

A H4 Cas9 cells were infected with sgRNAs targeting ATG7, TMEM41B or NT control, treated with 500 nM AZD8055 or vehicle control for 24 h, and analyzed by immunoblotting.

B Ratio of LC3-II to LC3-I band intensities are depicted as mean ± SD (n=6 independent experiments) with Wilcoxon test values.

C H4 Cas9 TMEM41B and NT control cells were probed by LC3B immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm.

D The number of LC3 puncta per cell was quantified using Yokogawa Analysis Software (YAS) and depicted as mean ± SD (n=3 technical replicates).

E LC3 and WIPI2 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at co-localized LC3 and WIPI2 puncta. Scale bar: 5 µm.

F The number of WIPI2 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=5 technical replicates).

G The total number of WIPI2 puncta per cell was quantified using the Harmony software on images acquired with an automated Operetta microscope. Data are depicted as mean ± SD (n=3 technical replicates).

H H4 Cas9 TMEM41B and NT control cells were infected with a RFP-DFCP1 expression construct for 72h, fixed, stained with LC3 antibodies and imaged with an automated CV7000 confocal microscope. Arrowheads point at co-localized LC3 and RFP-DFCP1 puncta. Scale bar: 5 µm.

I The number of RFP-DFCP1 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=10 technical replicates).

J mCherry-GFP-LC3 was expressed in H4 Cas9 TMEM41B KO and NT control cells which were treated with 50nM BafA1 or vehicle control for 24 h, fixed and imaged. Scale bar: 20 μm. The number of mCherry- and GFP-positive puncta per cell was quantified using YAS and are depicted as mean ± SD (n=3 technical replicates).
TMEM41B is a novel regulator of autophagy and lipid mobilization (2018)
Figure 3

Figure 3 - TMEM41B-deficent cells display enlarged lipid droplets

A H4 Cas9 cells stably expressing NT, TMEM41B or ATG7 sgRNAs were stained with BODIPY 493, NBD cholesterol or BODIPY FL C12 probes for 2 h at 37°C and imaged live with an automated CV7000 confocal microscope. Puncta area per cell was quantified using YAS and depicted as mean ± SD (n=4 technical replicates). Scale bar: 20 μm.

B H4 Cas9 TMEM41B KO and NT control cells were treated overnight with 400 μM BSA-conjugated oleic acid or 0.1% BSA as vehicle control, stained for 2 h with HCS LipidTox Green Neutral Lipid Stain and imaged with an automated Operetta microscope. Scale bar: 20 μm.

C Lipid droplet size, number and mean fluorescence intensity was quantified using Harmony software. Data are presented as mean ± SD (n=3 technical replicates). An average of 1500 cells was analyzed per replicate.

D H4 Cas9 TMEM41B KO and NT control cells were probed by ADRP immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm.

E Size of ADRP droplets was quantified with YAS and depicted as mean ± SD (n=3 technical replicates).

F Protein level of ADRP was probed by immunoblotting in H4 Cas9 TMEM41B KO and NT control cells.

G ADRP band intensities were quantified and depicted as mean ± SD (n=4 independent experiments) with paired t-test values.