EMBO/soda-nel huggingface dataset:
Miro1 dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity (2016)
Figure 5

Figure 5. Miro1 is involved in homeostatic changes in presynaptic Ca2+ signals.

A, Live images of neurons co-transfected with SyGCaMP5 and MtDsRed or myc-ΔEF-Miro1-IRES-MtDsRed (ΔEF Miro) before and during 10 Hz field stimulation with and without TTX treatments. Scale bar, 10 m. B, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 2.5 0.5), n=10 neurons (62 terminals) and non-treated terminals (black trace, ΔF/F0 = 1.3 0.6), n=11 neurons (69 terminals) co-transfected with MtDsRed, *p<0.05, t-test. C, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 1.8 0.3) in n=7 neurons (33 terminals) and non-treated terminals (black trace, ΔF/F0 = 2.3 0.6) in n=7 neurons (32 terminals) co-transfected with myc-ΔEF-Miro1-IRES-MtDsRed; p=0.5, t-test. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.
A cell atlas of adult muscle precursors uncovers early events in fiber-type divergence in Drosophila (2020)
Figure 1

Figure 1. Single-cell atlas of the wing imaginal disc identifies diverse cell types

A. Origin of the flight muscles. Left panel: Trachea, air sac primordium, IFM myoblasts and DFM myoblasts in the adepithelial layer overlaying the wing disc epithelium. Right panel: Lateral view of the adult flight muscles in the thorax.

B. ﻿ Workflow for droplet-based scRNAseq. Wandering third instar larval wing discs were dissected, pouch was removed and dissociate into single-cell suspension. Droplets containing unique barcoded-beads and single cells were collected. Following library preparation, sequencing data were aligned, and a gene-cell expression matrix was generated and analyzed using Seurat for identification of variable genes and unsupervised cell clustering based on gene expression similarity.

C. Annotated cell-type, including 6,711 epithelial, 272 tracheal and 4,544 myoblast cells, in UMAP plot of the reference single cell atlas.

D. ﻿RNA expression heatmap showing the top differentially expressed gene markers for each cluster of the reference single cell atlas dataset. Cells (column) are clustered by the expression of the main marker genes (row).

E. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) used as markers to assign epithelial and myoblast cells, respectively, in the reference dataset.

F. Confocal single plane image of third instar larval wing disc and orthogonal views of the disc stained with anti-Zfh1 (red) and anti-Fas3 (green).

G. Dot plot showing the expression levels of the marker genes identified for the myoblast, epithelial and tracheal cells across the 26 clusters of the reference cell atlas. Color intensity represents the average normalized expression level. Dot diameter represents the fraction of cells expressing each gene in each cluster. Gene expression for each cell was normalized by the total expression, and then the expression of each gene was scaled.

H. Confocal single plane image of third instar larval wing disc and orthogonal view of SPARC>GFP (green) stained with anti-Ct (red) and 4´,6-diamidino-2-phenylindole (DAPI, blue). Full genotype y-, w-/w-; UAS-GFP/+; SPARC-GAL4/+.

Data information: Scale bars: 50 μm. White arrowhead: Ct expression in tracheoblasts
A cell atlas of adult muscle precursors uncovers early events in fiber-type divergence in Drosophila (2020)
Figure 2

Figure 2. Cells in epithelial clusters map to spatially distinct regions of the wing disc

A. Schematic representation of the wing imaginal disc and its relationship to the adult wing. The cell fate map of the wing disc is adapted from (Bryant, 1975).

B. Subset of 6,983 epithelial cells in UMAP plot of the reference single cell atlas.

C. Approximate map of the epithelial clusters identified in the reference single cell atlas dataset to distinct position over the disc proper.

D. Average expression level of the genes used as known markers to assign each epithelial cluster in the reference atlas dataset. ﻿Cells colored by the expression of nub, wg, zfh2, Sox15, grn, bnl, eyg, Ance, Idg4 and drm.

E. Dot plot showing the expression levels of the top marker genes identified for the epithelial cells across 17 clusters of the reference cell atlas dataset.

F. Confocal single plane image of third instar larval wing disc stained with anti-Nub, anti-Wg, anti-Svp, anti-Hairy (red), reporting expression of dpp-lacZ, Dad-lacZ (anti-β-gal, red) and counterstained with DAPI (cyan). Scale bars: 100 μm. Full genotypes: P{CaryP}attP2 (top and bottom panels), P{PZ}dpp[10638];+, and y- w-;+; P{lacW}Dad[j1E4].
A cell atlas of adult muscle precursors uncovers early events in fiber-type divergence in Drosophila (2020)
Figure 3

Figure 3. Single-cell transcriptome atlas reveals a large compendium of myoblasts

A. Subset of 4,544 myoblast cells in UMAP plot of the reference single cell atlas dataset.

B. Dot plots showing the expression levels of the top variable genes between IFM and DFM myoblasts across 7 clusters of the reference cell atlas dataset.

C-K. Confocal single plane images of third instar larval wing discs stained with: (C) anti-Ct (red) and anti-Zfh1 (green), (D) kirre[rp298]-lacZ (anti-β-gal, green) and anti-Zfh1 (red), (E) Ten-a[MI04411]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (F) anti-Nrt (red) and anti-Zfh1 (cyan), (G) Argk[CB3789]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (H) nkd[MI00209]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (I) Ama[NP1297]>GFP (green), anti-Ct (red) and anti-Zfh1 (cyan) and (J) Mef2>CD8-GFP (green), and fluorescent in situ hybridization of Ama-RNA probe (white). K. Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red), and fluorescent in situ hybridization of Ama-RNA probe (white).

L. Confocal single plane images of adult DFM of Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red) stained with DAPI (white) dorsal right, anterior up. DFM are numbered in white following (Lawrence, 1982).

Data information: Dashed yellow line points to DFM myoblast area. Yellow asterisks and arrowhead indicate Ama expression in DFM myoblasts and in a few subset of IFM myoblasts, respectively. Scale bars: 50 μm. Full genotypes: (C) 1151-GAL4, v-, w-; +; UAS-mCherry-RNAi, (D) kirre[rp298]-nlacZ; +; +, (E) y-w-; Ten-a[MI04411]::GFP, (F) y-, v-; +; P{CaryP}attP2, (G) y-, w-; +; Argk[CB03789]::GFP, (H) y w; nkd[MI00209]/TM6B, Tb, (I) w-; UAS-GFP; Ama[NP1297]-GAL4, (J) w; UAS-CD8-GFP/Mef2-GAL4 (K-L) w-; UAS-gTRACE/+; Ama[NP1297]-GAL4/+.
A cell atlas of adult muscle precursors uncovers early events in fiber-type divergence in Drosophila (2020)
Figure 4

Figure 4. IFM and DFM myoblast clusters uncover different state of differentiation

A. Dot plot showing the expression levels of the top variable genes between myoblasts across 7 clusters of the reference cell atlas dataset.

B-D. Confocal single plane images of third instar larval wing discs stained with anti-Ct (red) and (B) E(spl)m6-BFM-gapGFP (green), (C) E(spl)m7-HLH[VK00037]-GFP (green), or (D) E(spl)m3-HLH[GMR10E12]>GFP (green).

E-G. ﻿Slingshot used for inferring cell lineages and pseudotime among the IFM myoblasts. (E) Two lineages for IFM myoblast clusters visualized in subset of 3,492 cells in UMAP plot of the reference single cell atlas dataset. (F) Pseudotime inference in Lineage 1 is from IFM_1 → IFM_2 →IFM_3→IFM_4. (G) Pseudotime inference in Lineage 2 is from IFM_1 → IFM_2 →IFM_3→IFM_5.

H-I. Confocal single plane images of third instar larval wing discs stained with anti-Ct (cyan) showing the lineage of GAL4 (green) and the active GAL4 (red) in (H) E(spl)m3-HLH[GMR10E12]>gTRACE and (I) E(spl)m6-BFM-GAL4>gTRACE.

Data information: White arrowheads point to location of IFM_1-2 cells. Red asterisks indicate expression of E(spl)m3-HLH in epithelial cells. Scale bars: 50 μm. Full genotypes are (B) w-; UAS-GFP; E(spl)m3-HLH[GMR10E12]-GAL4, (C) w-; E(spl)m7-HLH-GFP[VK00037]; +, (D) E(spl)m6-BFM-gapGFP, (H) w-; UAS-gTRACE/+; E(spl)m3-HLH[GMR10E12]-GAL4/+ , (I) w-; UAS-gTRACE, E(spl)m6-BFM-GAL4.
A cell atlas of adult muscle precursors uncovers early events in fiber-type divergence in Drosophila (2020)
Figure 5

Figure 5. Exploring clusters of IFM myoblasts reveals their heterogeneity

A-E Confocal single plane images of third instar larval wing discs stained with (A) anti-LamC (green) and anti-Zfh1 (red), (B) edl[k12907]-lacZ (green) and anti-Zfh1 (red), (C) anti-H (green) and anti-Zfh1 (red), (D) Cg25C[k00405]-lacZ (green) and anti-Zfh1 (red), or (E) vgk[G00454]-GFP (green) and anti-Ct (red).

Data information: Scale bars: 50 μm. Full genotypes: (A, C) y-, v-; +; P{CaryP}attP2, (B) y-, w; edl[k12907]; +, (D) y-, w-; Col4a1[A109.1F2]-lacZ; +, (E) w-; vkg-GFP[G00454];+
A cell atlas of adult muscle precursors uncovers early events in fiber-type divergence in Drosophila (2020)
Figure 6

Figure 6. Novel markers are functionally important for the development of the flight muscles

A-D. The muscle-specific (A-B) Mef2-GAL4 and (C-D) 1151-GAL4 drivers were crossed to UAS-RNAi lines to knockdown the expression of the gene markers for the myoblast clusters. (A, C). Viability test quantified by scoring the percentage of viable animals at each developmental stage, including early pupa (black), pharate (grey) and adult (white), relative to the total number of animals. Data are expressed as stacked bars showing the median with interquartile range (n≥ 3 independent biological replicates), unless noted as viable (NQ) and lethal (NQ), which stands for animals that were viable and lethal, respectively, but no quantitative analysis were performed. (B, D). Flight ability scored by quantifying the percentage of flies landing on each section of the column (top, middle and bottom) as indicated on the diagram on the right. Data are expressed as stacked bars. The total number of flies used in each assay was pooled together and displayed next to each bar (N≥6). n/a stands for non-applicable since animals are not viable.

E. Confocal single plane images of third instar larval wing discs from Mef2>mCherry-RNAi, (left panel) and Mef2>chinmo[HM04048]-RNAi (right panel) stained with anti-Zfh1 (red) and DAPI (cyan). Scale bars: 50 μm.

F. Quantification of phenotype displayed in (E). Stacked bars showing the percentage of wing discs scored as either normal or as abnormal. Data are expressed as mean ± sem, N = 32 discs per genotype, n = 3 independent experiments.

G. Quantification of viability test. Since the UAS-Ama-RNAi[GD12733] transgene is inserted on Chromosome X the genotype of females is Mef2>Ama-RNAi whereas males are Mef2>+. Box plot showing the ratio of flies with genotype of interest (females) over control flies (males). The whiskers show 5-95 percentile, and the middle line the median. * p=0.014 (Mann-Whitney test, N≥6 independent biological replicates). Full genotypes are w, UAS-Dicer2;+; mCherry-RNAi/Mef2-GAL4 and w, UAS-Dicer2, UAS-Ama-RNAi[GD12733];+; Mef2-GAL4.
A cell atlas of adult muscle precursors uncovers early events in fiber-type divergence in Drosophila (2020)
Figure 7

Figure 7. Amalgam is required for the expansion of myoblast pool in larval wing disc.

A. Lethality of 1151>Ama-RNAi[HMS00297] animals at pharate stage was partially rescued by overexpressing the transgene Ama-cDNA. ﻿Representative images of adults and pharate pupa are shown. Full genotypes: 1151-GAL4;UAS-myrRFP;+ , 1151-GAL4;UAS-myrRFP;UAS-Ama[HMS00297]-RNAi , 1151-GAL4;UAS-AmaOE/CyO;UAS-Ama[HMS00297]-RNAi and 1151-GAL4;UAS-AmaOE;+.

B. ﻿Two-dimensional UMAP representation of single-cell RNA-seq 1151>Ama-RNAi dataset colored by cell type, including 51 tracheal, 3,107 epithelial and 180 myoblast cells.

C. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) in the 1151>Ama-RNAi dataset.

D. Confocal single plane images of 1151>mCherry-RNAi and 1151>Ama-RNAi wing discs stained with anti-Ct (red) and anti-Zfh1 (green).

E. Dot plots showing the expression levels of the top markers for myoblast clusters identified in the reference cell atlas across the cluster of myoblasts in 1151>Ama-RNAi and 7 myoblast clusters in control dataset.

F. The myoblast cells in 1151>Ama-RNAi dataset projected into the reference single cell atlas by transferring cell type labels using Seurat. The bar graph represents the total number of cells in each cluster normalized to the total number of epithelial cells per genotype.

G-H. Confocal single plane images of 1151>mCherry-RNAi and 1151>Ama-RNAi wing discs stained with anti-Zfh1 (green) and (G) anti-Nrt (white) at wandering third instar larval stage (110 - 135 h AEL) or (H) DAPI (red) at early third instar larval wing discs (72 - 110 h AEL).

Data information: Scale bars: 50 μm. Full genotypes: (D, G-H) 1151-GAL4;+; UAS-mCherry-RNAi and 1151-GAL4; +; UAS-Ama[HMS00297]-RNAi.
A cell atlas of adult muscle precursors uncovers early events in fiber-type divergence in Drosophila (2020)
Figure 8

Figure 8. The loss of Amalgam impairs the formation of both IFM and DFM in at early stages of developing pupa.

Confocal single plane images of 1151>Ama-RNAi animals and 1151>mCherry-RNAi.

A. Forming DFM at 40 h APF stained with anti-Zfh1 (green), anti-Futsch (22c10, red) and anti-Kettin (white). White arrows point to the wing hinge, wings pointing left, anterior up.

B. Forming IFM (DLM) at 16 h APF, stained with anti-Futsch (22c10, red), anti-Zfh1 (green) and DAPI (cyan). Yellow-dashed box indicates magnified area (bottom panel). Anterior up.

C. DFM at 96 h APF stained with Phalloidin (red), anti-Kettin (green) and DAPI (blue). Anterior up, dorsal right. DFM are numbered in white as in (Lawrence, 1982).

D. IFM at 96 h APF, transverse section (top) stained with anti-PS-integrin (green), Phalloidin (red) and DAPI (blue), dorsal up; sagittal section (bottom) stained with anti-Kettin (green), Phalloidin (red) and DAPI (blue), anterior up, dorsal right.

Data information: Scale bars: 50 μm (A, B), and 100 μm (C, D). Full genotypes: 1151-GAL4; +; UAS-mCherry-RNAi and 1151-GAL4; +; UAS-Ama[HMS00297]-RNAi.

Expanded View Figure Legends
Carfilzomib modulates tumor microenvironment to potentiate immune checkpoint therapy for cancer (2021)
Figure 1

Figure 1. High-throughput Screening of FDA drugs capable of inducing IL-1β expression in M2 macrophages

A. Schematic diagram of high-throughput screening.

B. Carfilzomib, Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il-1β-luciferase transgenic mice. BMDMs from IL-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours to differentiate into mature M2 macrophages, and then used for screening of FDA compounds (5 μM). The X axis stands for the code of the drug.

C. Carfilzomib, Bortezomib and MLN9708 activate Il-1β-luciferase in M2 macrophages in a dose-dependent manner. BMDMs from Il-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib, Bortezomib or MLN9708 at indicated concentrations. Luciferase activity was monitored 12 hours later. Data from three experiments are presented as the mean ± SD.

D. Carfilzomib, Bortezomib and MLN9708 activate Il-1β-luciferase in a dose-dependent manner as well as inhibit the transformation of M2 macrophages. BMDMs from Il-1β-luciferase transgenic mice were pretreated with DMSO, Carfilzomib, Bortezomib, or MLN970 at indicated concentrations for 1 hour, followed by treatment with IL-4 (20 ng/mL). Luciferase activity was monitored 12 hours later. Data from three experiments are presented as the mean ± SD.

E. Carfilzomib, Bortezomib and MLN9708 activate Il-1β-luciferase in M0 macrophages in a dose-dependent manner. BMDMs from Il-1β-luciferase transgenic mice were stimulated by DMSO, Carfilzomib, Bortezomib, or MLN970 at indicated concentrations. Luciferase activity was monitored 12 hours later. Data from three experiments are presented as the mean ± SD.

F, G. Carfilzomib, Bortezomib and MLN9708 promote the mRNA expression of Il-1β in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib (1 μM), Bortezomib (1 μM), or MLN9708 (2 μM). RNA was extracted from BMDMs or Raw264.7 cells 6 hours after stimulation and the expression of Il-1β were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test).

H, I. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory cytokine IL-1β in M2 macrophages. BMDMs (H) or Raw264.7 cells (I) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO, Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). Secretion of IL-1β were measured through ELISA 24 hours later. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. **p < 0.01, ***p < 0.001 (student's t-test).
Carfilzomib modulates tumor microenvironment to potentiate immune checkpoint therapy for cancer (2021)
Figure 3

Figure 3. Proteasome inhibitors reprogram M2 macrophages toward M1-like macrophages by inducing ER stress signaling

A, B. Carfilzomib and proteasome inhibitor MG132 activate Il-1β luciferase in macrophages. (A) BMDMs were treated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, Carfilzomib (1 μM), or MG132 (5 μM). (B) BMDMs were stimulated by DMSO, Carfilzomib (1 μM) or MG132 (5 μM)) for 1 hour, then induced with IL-4 (20 ng/mL). Luciferase assays were monitored 12 hours after stimulation. The data are means ± SD of three independent experiments. ***p < 0.001 (student's t-test).

C. MG132 promotes the expression of M1 macrophage markers and inhibits the expression of M2 macrophage markers. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO or MG132 (5 μM). RNA was extracted from BMDMs 6 hours after stimulation and the expression of M1 (Il-1β / Il-6 / Inos) or M2 (Cd206 / Arg1) macrophage markers were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, ***p < 0.001 (student's t-test).

D. Knockdown of β5 subunit of proteasome in macrophages promotes the expression of proinflammatory factors. RNA was extracted from Raw264.7 (shGFP or shPsmb5) and the expression of Il-1β and Il-6 were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, ***p < 0.001 (student's t-test).

E. Carfilzomib, Bortezomib, MLN9708 and MG132 induce ER stress response. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO or Carfilzomib (1 μM), Bortezomib (1 μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 3 hours or 6 hours after stimulation and the expression of ER stress related genes (Bip, Chop) were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).

F. ER stress agonists, TUN and TG, promote the expression of M1 macrophage markers, as well as reduce the expression of M2 macrophage markers. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, TUN (500 nM) or TG (500 nM). RNA was extracted from cells 6 hours after stimulation and indicated genes were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).

G. Inhibition of ER stress impairs the ability of Carfilzomib, Bortezomib, MLN9708 and MG132 to reprogram M2 towards M1-like macrophages. BMDMs were induced by IL-4 (20 ng/mL) for 24 hours, then pretreated with 4-PBA (5 mM) for 1 hour and stimulated by Carfilzomib (1μM), Bortezomib (1μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 6 hours after stimulation and the expression of Il-1β, Il-6 and Inos were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).
Carfilzomib modulates tumor microenvironment to potentiate immune checkpoint therapy for cancer (2021)
Figure 6

Figure 6. Carfilzomib synergizes with PD-1 inhibitors to treat lung cancer

A-C. Carfilzomib shrinks tumor through modulating tumor microenvironment. EG7 cells were inoculated in wild-type or Rag1-/- mice to allow tumor xenograft to reach a volume of around 80 mm3. Mice were randomized for treatment (n = 6) by saline or Carfilzomib for two weeks. Tumor growth was monitored (A) and the xenografts were dissected to photograph (B) or weigh (C) after treatment. Data are shown as mean ±SD and n indicates the number of biological replicates. *p < 0.05, ***p < 0.001 (student's t-test).

D. CD4+ and CD8+ T cells play a role in mediating cancer-shrinking effect of Carfilzomib. Lung-cancer-bearing TD mice were treated by saline, Carfilzomib, anti-mouse CD4 antibody (ip.), anti-mouse CD8 antibody (ip.), or Carfilzomib plus anti-mouse CD4/8 antibody. CT scanning was performed after two-week treatment.

E. Representative images of H&E staining of the treated lung tissues. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method.

F. Carfilzomib synergized with PD1 antibody in shrinking lung cancers. Lung cancer bearing TD mice were treated by saline, anti-PD1 antibody (ip), Carfilzomib, or Carfilzomib plus anti-PD1 (ip). CT scanning was performed after two-week treatment.

G. Representative images of H&E staining of the lung tissues. Expression of Ki-67 in lung tissue was detected by immunohistochemistry method.

H. Carfilzomib synergized with PD1 antibody in shrinking lung cancer through activating T cells. Treated TD mice were euthanized and the lung tissues were dissected to analyze the expression of CD69 and CD25 in CD8+ T cells by flow cytometry.

I. Statistics about the proportion of CD25 or CD69 positive cells in CD8+ T cells. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).