EMBO/soda-nel huggingface dataset:
Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neo-substrate PLZF (2020)
Figure 3

Figure 3. Interaction regions in PLZF for binding to CRBN with thalidomide

(A) Schematic diagram of PLZF and truncated PLZFs. FL; Full-length, BTB; BTB (broad-complex, tramtrack, and bric à brac) domain.

(B) In vitro binding assay using truncated PLZF. Thalidomide-dependent interaction between bls-CRBN and FLAG-GST-PLZF-full length (FL) or truncated FLAG-GST-PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology.

(C) Immunoblot analysis of AGIA-PLZF protein levels in FLAG-CRBN and PLZF-full length (FL) or truncated FLAG-GST-PLZF expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h.

(D) Schematic diagram of swapped PLZF mutants.

(E) In vitro binding assay using swapped PLZF mutants was performed using the same procedure as in Fig 3B.

(F) Amino acid sequences of ZNF1 and ZNF3 in PLZF. The white letters indicate critical Gly residue for the interaction between PLZF and CRBN with thalidomide or its derivatives.

(G) In vitro binding assay using point mutants of PLZF was performed using the same procedure as in Fig 3B.

(H) Immunoblot analysis of AGIA-PLZF protein levels in FLAG-CRBN and PLZF-WT, PLZF-ZNF1-GA, PLZF-ZNF3-GA, or PLZF-ZNF1,3-GA expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h.

Data information: All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, ****P < 0.0001).
Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neo-substrate PLZF (2020)
Figure 4

Figure 4. 5-Hydroxythalidomide induces degradation of PLZF and SALL4 by CRBN

(A) Schematic diagram of thalidomide metabolites by CYPs.

(B) In vitro binding assay for thalidomide and 5-hydroxythalidomide. Interaction between bls-CRBN and FLAG-GST-IKZF1, -SALL4 and -PLZF in the presence of DMSO, thalidomide or 5-hydroxythalidomide (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) was analysed using the AlphaScreen technology. All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3).

(C) Immunoblot analysis of AGIA-IKZF1, AGIA-SALL4 or AGIA-PLZF in FLAG-CRBN expressing CRBN-/- HEK293T cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 16 h.

(D) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h.

(E) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP-1 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h.

(F) Immunoblot analysis of endogenous SALL4, PLZF, ZFP91 or CK1α protein levels in HuH7 cells treated with DMSO, thalidomide (Tha), lenalidomide (Len), pomalidomide (Pom) or 5-hydroxythalidomide (5-HT) for 24 h.

Data information: Data in bar graphs in (D) and (E) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100. Error bars donated ± standard deviation (n = 3) and P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01 and ***P < 0.001).
Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neo-substrate PLZF (2020)
Figure 5

Figure 5. CRBN-dependent degradation of PLZF and SALL4 from mouse and chicken by treatment with thalidomide and 5-hydroxythalidomide

(A, B) Immunoblot analysis of AGIA-MmPLZF (A) or -MmSALL4 (B) in FLAG-MmCRBN-WT, -MmCRBN-I391V or -MmCRBN-V380E/I391V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h.

(C, D) Immunoblot analysis of AGIA-GgPLZF (C) or -GgSALL4 (D) in FLAG-GgCRBN-WT or -GgCRBN-I390V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h.

(E, F) Immunoblot analysis of AGIA-MmPLZF (E) or -MmSALL4 (F) in FLAG-MmCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h.

(G, H) Immunoblot analysis of AGIA-GgPLZF (G) or -GgSALL4 (H) in FLAG-GgCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h.

Data information: Data in bar graphs in (A-H) were calculated as relative (Gg or Mm) PLZF or SALL4 band intensity with (Gg or Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neo-substrate PLZF (2020)
Figure 6

Figure 6. Downregulation of PLZF causes abnormal limb development in chicken embryo

(A) Sall4, Plzf or Crbn mRNA expression in E4 chicken embryos was analysed by whole-mount in situ hybridisation. Upper panel shows whole chicken embryo and lower panel shows right forelimb bud.

(B) Sall4, Plzf, or Crbn mRNA expression in E4 chicken limb bud was analysed by section in situ hybridisation.

(C) Immunoblot analysis of AGIA-GgPLZF in AGIA-GgPLZF expressing DF-1 cells transfected with shContol (shGFP) or shPLZF expression vector.

(D) Immunoblot analysis of PLZF from tissue of chicken forelimb or hindlimb bud. Endogenous PLZF protein expression was detected by immunoblot using E4.5 chicken embryos infected with RCAN virus packaging shControl or shPlzf (forelimb shControl (n = 4), forelimb shPLZF (n = 4), hindlimb shControl (n = 2) or hindlimb shPlzf (n = 3)).

(E) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPLZF (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. Data in bar graphs were calculated as relative bone length with bone length of shControl as 100. Error bars denote ± standard deviation (forelimb and hindlimb shControl (n = 3), forelimb shPLZF (n = 5) or hindlimb shPLZF (n = 6)). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01 and ****P < 0.0001).

(F) Teratogenic phenotypes of chicken embryos in Fig 6E.
Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neo-substrate PLZF (2020)
Figure 8

Figure 8. PLZF overexpression rescues thalidomide-induced limb phenotypes

E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS-GgPLZF was treated with HBC or 1 µg/µl (3.9 mM) thalidomide, and the chicken forelimb bud phenotypes were observed in bright field (BF) or fluorescence of enhanced GFP (EGFP). Fgf10/Fgf8 expression was analysed by section by in situ hybridisation.

(A-P) (A-D) Empty vector and HBC (n = 8), (E-H) empty vector, and thalidomide (n =4), (I-L) GgPLZF vector, and HBC (n = 5), (M-P) GgPLZF vector and thalidomide (n =7). The dashed line in (E) indicates limb bud. The yellow and pink arrowheads show the expression of Fgf10 in the mesenchyme and expression of Fgf8 at the apical ectodermal ridge (AER), respectively.

(Q) Quantitative analysis of phenotypes of chicken limb bud in Fig 8A, E, I and M. Data in bar graphs in (Q) were calculated as relative limb bud length with limb bud length of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01).

(R) Quantitative analysis of Fgf10/Fgf8 expression in Fig 8C, D, G, H, K, L, O and P. Data in bar graphs in (R) were calculated as relative Fgf10/Fgf8 mRNA expressions with Fgf10/Fgf8 mRNA expressions of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (*P < 0.05 and **P < 0.01).
Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites (2021)
Figure 1

Figure 1. RAP80 is not necessary for BRCA1 recruitment to DSB sites. (A) Schematic representation of BRCA1 and BARD1. Highlighted are RING domains (orange), PALB2-interacting coiled-coil region (CC; red), BRCA1 C-terminal domains (BRCT; green) and ankyrin repeats (ANK; pink). (B) Immunoblotting of whole cell extracts obtained from parental and RAP80-/- clones in the RPE-1 hTERT p53-/- Cas9, RPE-1 hTERT p53-/- BRCA1-/- Cas9, U2OS, U2OS-2-6-3 and U2OS Flp-In/TREx cell lines with RAP80 antibodies. Tubulin is used as loading control. Representative of at least two independent immunoblots. See Figure EV1 for further details on the gene editing strategy used to knockout RAP80. (C, D) The indicated parental and RAP80-/- cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against ABRAXAS1 and γH2AX. Shown in (C) is the percentage of cells with > 5 ABRAXAS1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs of RPE1-hTERT p53-/- Cas9 wild type (WT) and RAP90-/- cells are shown in (D). (E, F) The indicated cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against BRCA1 and γH2AX. Shown in (E) is the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates for all conditions except n=5 for U2OS WT, n=2 for U2OS 2-6-3 cells, n=7, 5, 4 for U2OS Flp-In/T-REx cells). Representative microgaphs of RPE-1 hTERT p53-/- Cas9 wild type (WT) and RAP80-/- are shown in (F). (G) RPE-1 hTERT p53-/- Cas9 parental (WT) and RAP80-/- cells were untreated (t=0 h) or irradiated with a 10 Gy dose. Samples were collected at the indicated time points, processed for immunofluorescence and stained with antibodies against BRCA1 and γH2AX. Shown is the percentage of cells with > 5 BRCA1 foci. A minimum of 100 cells per replicate were analyzed and the datapoints represent the mean ± S.D. (n=3 biological replicates). All scale bars are 5 μm.
Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites (2021)
Figure 2

Figure 2. RAP80-independent BRCA1 recruitment to DSB sites is dependent on RNF8 and RNF168. (A, B) U2OS Flp-In/T-Rex parental (WT) and RAP80-/- cells were transfected with siRNA pools targeting BRCA1, RNF8, RNF168 or BARD1 or with a non-targeting control siRNA (CTRL). 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX is shown in (A). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (B). (C, D) Parental (WT) RPE1-hTERT p53-/- Cas9, RNF8-/-, and RNF168-/- cells were treated with either a non-targeting siRNA pool (CTRL) or a pool targeting RAP80. 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 10 BRCA1 foci that colocalize with γH2AX is shown in (C). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.
Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites (2021)
Figure 3

Figure 3. The RING domain participates in BRCA1 recruitment to DNA damage sites. (A, B) U2OS Flp-In/T-REx cells with integrated transgenes encoding GFP-BRCA1BRCT or -BRCA1BRCT S1655A were transfected with non-targeting siRNA (CTRL) or siRNA targeting RAP80 or BRCA1. Following doxycycline treatment to induce transgene expression (5 μg/mL, 24 h), cells were irradiated (10 Gy) and processed for immunofluorescence 1 h post-IR for GFP and antibodies against RAP80 and γH2AX. Shown in (A) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=4). Representative micrographs are shown in (B). (C, D) U2OS Flp-In/T-REx cells stably integrated with the indicated transgenes were treated with doxycycline (5 μg/mL, 36 h) to induce protein expression and transfected with an siRNA targeting BRCA1 and also either non-targeting siRNA (CTRL) or siRNAs targeting RAP80. 1 h post-irradiation (10 Gy) cells were processed for immunofluorescence using GFP (to detect the BRCA1 fusions) and antibodies against γH2AX. Shown in (C) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.
Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites (2021)
Figure 4

Figure 4. Nucleosome- and E2-binding by the BRCA1 RING participates in promoting BRCA1 localization. (A, B) U2OS 2-6-3 parental (WT) or RAP80-/- cell lines were transfected with siRNAs targeting BRCA1 followed by nucleofection of the indicated GFP fusion proteins. 48 h post-nucleofection, mCherry-LacR-FokI expression was induced for 5 h prior to being processed for fluorescence microscopy for GFP and mCherry. Shown in (A) is the quantitation of GFP fluorescence at the mCherry focus where the bars represent the mean ± S.D (n= 50, 50, 40, 40, 40, 40, 20, 20, 30, 30 cells analyzed from 3 biological replicates). Representative micrographs for the BRCA1 and BRCA1-I26A conditions are shown in (B). Additional micrographs for the other conditions are in Figure EV2C. (C) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells (WT) or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against BRCA1 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3B. (D) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against RAD51 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3C. Scale bar is 5 μm in (B).
Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites (2021)
Figure 5

Figure 5. Loss of BRCA1 recruitment to DSB sites causes sensitization to PARP inhibition. (A, B) MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and BRCA1 antibodies. Shown in (A) is the quantitation of a minimum of 100 EdU+ cells per replicate and the bars represent the mean ± S.D (n= 3 biological replicates). Representative micrographs are shown in (B). (C, D). MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and RAD51 antibodies. Shown in (C) is the quantitation of minimum of 100 EdU+ cells per replicate and the bars represent the mean ± S.D (n= 3 biological replicates). Representative micrographs are shown in (D). (E) Clonogenic survival assays using the indicated dose of olaparib and MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions. Data is shown as the mean ± S.D (n=3 biological replicates). Representative clonogenic images are shown in Figure EV5D.

Data information: All scale bars are 5 μm.
Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction (2020)
Figure 1

Figure 1. Alpha-lipoic acid (ALA) synthesis reduces in aged Drosophila midguts, and orally administered ALA rejuvenates aged intestinal stem cells (ISCs)

A. A model illustrating the Drosophila "esg>luciferase" reporter system of chemical administration. The chemicals were fed to Drosophila with esg-GAL4-driven luciferase expression on the 26th day after eclosion. After administration for 14 days, Drosophila were dissected, and the activity of luciferase in their midguts was measured.

B. Quantification of the luciferase activity of flies with indicated ages and ALA administration. Error bars show the standard deviation (SD) of six independent experiments.

C-F. Immunofluorescence images of esg-GFP and Delta (Dl) staining with the midgut section from the R4 region of 14-day Drosophila (C), 26-day Drosophila (D), 40-day Drosophila (E), and 40-day Drosophila in response to 0.5 mM ALA administration, which started at day 26 after eclosion (F). esg-GFP (green) identifies ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs.

G. Quantification of the number of esg-GFP+ cells and Dl+ cells in experiments (C-F). n is indicated. The numbers of quantified guts from left to right are 15, 17, 16, 16, 15, 17, 16, and 16.

H. Quantification of the number of pH3+ cells in experiments (C-F). n is indicated.

I. Relative mRNA fold change of Las in sorted esg-GFP+ cells of wild-type (esg-GFP/CyO) Drosophila during aging. The Las expressions of Drosophila with different ages are plotted relative to 14-day Drosophila, which was set to 1. Error bars indicate the SD of three independent experiments.

J. Western blotting results of midguts indicate a decrease of LAS in the midguts of aged Drosophila. Loading controls, β-tubulin.

K. Quantification of LAS band intensity as seen in experiments (J). Error bars indicate the SD of three independent experiments.

L. Quantification of the content of ALA in midguts of 14-day, 26-day, and 40-day flies using LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments.

M-O. LC-ESI-MS/MS chromatograms of ALA in midguts of 14-day flies(M), 26-day flies (N), and 40-day flies (O). Arrows indicate the peak of ALA. The area of ALA peak indicates the content of ALA. cps: counts per second. MRM: Multiple Reaction Ronitoring. When the estimated peak of ALA is undetected in the chromatogram, the content of ALA is considered as 0.

Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm (C-F). Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. See also Figure EV1.
Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction (2020)
Figure 2

Figure 2. Reduced ALA synthesis in flies causes the functional decline in ISCs.

A-B. LC-ESI-MS/MS chromatograms of midguts of flies carrying Act5Cts-GAL4-driven UAS-lacZ (control, A) or Las RNAi (B).

C. Quantification of the content of ALA in midguts of flies with indicated genotypes using LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments.

D-F. Immunofluorescence images of the midgut section from the R4 region in Drosophila carrying esgts-GAL4-driven UAS-lacZ (D, control), Las RNAi (E), or Las RNAi in response to ALA administration (F). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs.

G. Quantification of the number of esg-GFP+ cells, Dl+ cells, and pH3+ cells in experiments (D-F). n is indicated. The numbers of quantified guts from left to right are 17, 19, 18, 17, 19, 18, 17, 19, and 18.

H. Quantification of the luciferase activity of midguts with indicated genotypes and manipulation. Error bars show the SD of six independent experiments.

I-J. Immunofluorescence images of control (FRT40A, I) and Las RNAi (J) MARCM clones (green, outlined by white dotted lines) 10 days after clone induction (ACI). Pdm1 staining (red) was used to visualize ECs.

K. Quantification of the ratio of Pdm1+ cells per clone of MARCM clones with indicated genotypes. n is indicated. Each dot corresponds to one clone.

L. Quantification of MARCM clone size of experiments in (I-J). n is indicated. Each dot corresponds to one clone.

Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm (D-F, I-J). Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05. See also Figure EV2.
Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction (2020)
Figure 4

Figure 4. ALA prevents ISC senescence not mainly through its antioxidative ability

A-F. Immunofluorescence images of midgut sections from the R4 region in Drosophila carrying esgts-GAL4-driven expression of CAT cDNA (A), CAT cDNA with AD4 administration (B), CAT cDNA with ALA administration (C), Keap1 RNAi (D), Keap1 RNAi with AD4 administration (E), or Keap1 RNAi with ALA administration (F). esg-GFP (green) represents ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs.

G. Quantification of the number of esg-GFP+ cells, Dl+ cells of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA, CAT cDNA, CAT cDNA with AD4 administration, CAT cDNA with ALA administration, Keap1 RNAi, Keap1 RNAi with AD4 administration, or Keap1 RNAi with ALA administration. n is indicated. The numbers of quantified guts from left to right are 18, 19, 20, 18, 18, 19, 19, 18, 19, 20, 18, 18, 19, and 19.

H. Quantification of the number of pH3+ cells and luciferase activity of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA, CAT cDNA, CAT cDNA with AD4 administration, CAT cDNA with ALA administration, Keap1 RNAi, Keap1 RNAi with AD4 administration, or Keap1 RNAi with ALA administration. The numbers of quantified guts from left to right are 18, 19, 20, 18, 18, 19, and 19.

I-L. Representative images of the midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (I), Las RNAi (J), Las RNAi and CAT cDNA (K), or Las RNAi and Keap1 RNAi (L). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs.

M. Quantification of the number of esg-GFP+ cells, Dl+ cells, and pH3+ cells in experiments I-L. n is indicated. The numbers of quantified guts from left to right are 15, 17, 18, 17, 15, 17, 18, 17, 15, 17, 18, and 17.

N. Quantification of luciferase activity of midguts with indicated genotypes. Error bars show the SD of six independent experiments.

Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm (A-F, and I-L). Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.