EMBO/soda-nel huggingface dataset:
TMEM41B is a novel regulator of autophagy and lipid mobilization (2018)
Figure 4

Figure 4 - TMEM41B is required for mobilization and utilization of endogenous fatty acids.

A H4 Cas9 TMEM41B KO and NT control cells were pulsed with the fatty acid analog Red C12, chased in complete medium or upon serum-deprivation, and stained with MitoTracker. Cells were imaged live with an automated CV7000 confocal microscope. Scale bar: 20 μm.

B Fatty acid Red C12 intensity was measured within MitoTracker signal and ratio between SD and CM conditions was calculated. Data is presented as mean ± SD (n=3 technical replicates).

C-E H4 Cas9 TMEM41B KO and NT control cells were plated in XF96 plates and analyzed using a Seahorse Bioscience XF96. (C) Basal oxygen consumption rates (OCR) and extra cellular acidification rates (ECAR) were measured in cells grown in complete medium. OCR/ECAR ratio is also reported. Data are presented as mean ± SEM (n=30-32 technical replicates) from one representative experiment. (D) OCR was measured in cells grown in substrate-limited medium and treated in the absence or presence of Etomoxir (Eto). Oligomycin (OA), FCCP (FC) and a mixture of rotenone and antimycin A (RA) was added as indicated. Data are shown as mean ± SEM (n= 69 technical replicates). (E) Endogenous FA utilization was calculating by subtracting basal normalized OCR values of Eto-treated cells from untreated cells. Data are shown as mean ± SEM (n= 69 technical replicates).
TMEM41B is a novel regulator of autophagy and lipid mobilization (2018)
Figure 5

Figure 5 - TMEM41B localizes to the ER.

A H4 Cas9 cells stably expressing N-terminally (Myc-TMEM41B) or C-terminally Myc-tagged TMEM41B (TMEM41B-Myc) were probed by calnexin (CANX) and Myc tag immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm.

B-D Endogenous TMEM41B locus was tagged with C-terminal Myc using CRISPR-directed homologous recombination in HeLa cells. HeLa knock-in (KI) cells were compared to parental HeLa (-) and HeLa cells stably transduced with a TMEM41B-Myc over-expression (OE) construct. (B) Confocal micrographs of cells stained with antibodies against CANX and Myc confirming ER localization of both KI and OE TMEM41B-Myc. Scale bar represents 20 μm. (C) Immunoblot analysis confirming expression of both KI and OE TMEM41B-Myc. (D) PCR analysis with primers binding to TMEM41B last intron and to Myc tag confirming successful targeting of the endogenous TMEM41B locus with Myc (band labeled with asterisk). M=size marker.

E H4 Cas9 cells and H4 Cas9 cells stably expressing Myc-TMEM41B or TMEM41B-Myc were lysed and subjected to anti-Myc IP. Eluates were analyzed by mass spectrometry. Enrichment of proteins in IPs from Myc-TMEM41B or TMEM41B-Myc cells versus H4 Cas9 cells is depicted as log2 fold changes. Entire data is reported in Dataset EV2.

Expanded View Figure Legends
Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-chip model (2021)
Figure 1

Fig. 1. SARS-CoV-2 infection in a human lung-on-chip model.

(A-C) (A) Schematic of the LoC model for SARS-CoV-2. Maximum intensity projections show that confluent layers of alveolar epithelial cells (ATs) and endothelial cells with strong expression of tight junction markers populate the top (B) and bottom faces (C) of the porous membrane. Actin, CD31, TJP-1 and nuclear labelling are shown in azure, spring green, bright pink, and electric indigo LUTs, respectively.

(D) ACE2 levels were characterized using RNAscope. A representative 3D view of a 232 x 232 μm2 field of view of the AT layer of an uninfected control at 3 days at air-liquid interface (3C) is shown. The nuclear labelling is indicated in electric indigo and ACE2 mRNA is labelled in spring green LUT.

(E, F) qRT-PCR characterization of the AT and endothelial layers on-chip for SARS-CoV-2 entry markers (ACE2, TMPRSS2, NRP1) for cells extracted from the apical and vascular channels from uninfected LoCs (n=3 biological replicates) (E) and infected LoCs (n=4 biological replicates) at 1 day-post infection (F). In both cases expression is normalized to GAPDH levels.

(G) Same as in (D) with infected LoC at 3 dpi (G) shown.

(H) ACE2 expression decreases between 1 and 3 dpi in the LoC model, quantified on a per-cell basis for n=4 or 5 fields of view (technical replicates) across control and infected chips (n=1 control and n=1 infected chip) at each timepoint.

(I, J) A plot of the fold change in expression of host proteins required for viral entry in the AT and endothelial layer of LoCs at 1 day-post infection (n=4 biological replicates) and 3 days-post infection (n=3 biological replicates) vs. uninfected control LoCs (n=3 biological replicates).

Data information: The bars represent the mean value, the solid line represents the median value, and the error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis one-way ANOVA test, * represents p<=0.05, ** represents p<=0.01, ** represents p<=0.001. Scale bar = 20 μm.
Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-chip model (2021)
Figure 2

Fig. 2. Viral replication but lack of release of infectious virions characterizes SARS-CoV-2 infection in the LoC model.

(A, B) The kinetics of release of SARS-CoV-2 progeny was assessed from samples obtained from a wash of the apical face of the chip (A, B), and the vascular effluent (A) collected at each day-post infection. (A) The number of viral genomes in each sample was quantified using a one-step qRT-PCR assay for the SARS-CoV-2 N gene. The starting inoculum introduced to the apical side (labelled 'Inn') corresponded to 200-300 PFU. Lines join datapoints from the same LoC, reconstituted with (green squares) and without (dark blue circles) macrophages. Viral genome numbers within the apical wash showed a declining trend over 3 days of infection. Viral genomes are not detected ('nd') in the vascular effluent for all the chips shown. (B) Measurements of plaque forming units (PFU) from the apical wash of LoCs reconstituted without macrophages (n=8 biological replicates for 1 dpi, n=2 biological replicates for 2 dpi) confirms the presence of infectious virions.

(C) Quantification of the intracellular levels of transcripts for the SARS-CoV-2 N and SARS-CoV-2 E genes in the AT and endothelial layers of infected LoCs at 1 dpi (n=3 biological replicates) relative to expression of the eukaryotic housekeeping gene GAPDH.

(D) SARS-CoV-2 N: SARS-CoV-2 E ratio in the AT and endothelial layers of the infected LoCs shown in (C), n=3 biological replicates.

Data information: In all plots, the bars represent the mean value, and the error bars represent the standard deviation, the solid black line in (A) represents the median.
Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-chip model (2021)
Figure 3

Fig. 3. SARS-CoV-2 infection rapidly disrupts confluency of the endothelial layer.

(A-D) 3D views of representative 232 x 232 μm2 (A, B) and 155 x 155 μm2 (C, D) fields of view from confocal imaging of an uninfected control (A), and infected chips without macrophages at days 2 (B) and 3 (C, D) post-infection, respectively. Actin and nuclear labelling are shown in azure and indigo, respectively. (B) At 2 dpi, adjacent areas of endothelial cell clusters identified by a stronger nuclear stain and brighter actin staining (yellow arrows) and cells with increased permeability and loss of confluence (white arrows) are indicated. (C) At 3 dpi, a significant loss of barrier integrity is observed in the endothelial layer, whereas the AT layer directly above this field of view (D) is relatively intact. A small area of missing cells in the AT layer is indicated with a white arrow in (D).

(E, F) Plots of density of nuclei (number of nuclei per 1E4 μm2) on the AT (E) and endothelial (F) faces of infected LoCs both with and without macrophages for characterization of the cellular layer integrity. The bars represent the mean value, the solid line represents the median and the error bars represent the standard deviation from at least 4 fields of view each from two independent LoCs for each timepoint. Data from uninfected controls corresponding to the 3 dpi timepoint are labelled '3C'.

(G-J) The fields of view in (C, D) are shown also in (G, H) respectively, SARS-CoV-2 spike (S) proteins labelled via immunofluorescence are false-colored in amber. Zooms in (I) and (J) show clear evidence of intracellular spike protein within both endothelial and ATs, respectively.

(K) A 3D view of the field of views in (C, D) and (G, H) respectively highlights the relative damage to the endothelial layer versus the epithelium, as well as the basolateral transmission of S protein via junctions in the pores of the membrane (identified with yellow arrows). The white arrows define the XYZ axes.

Data information: P-values are calculated using a one-way Kruskal-Wallis ANOVA test, * represents p<=0.05, ** represents p<=0.01, ** represents p<=0.001.
Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-chip model (2021)
Figure 4

Fig. 4. SARS-CoV-2 infection leads to a loss of tight junctions, generation of CD31- endothelial cell clusters and a pro-coagulatory environment.

(A-D) Maximum intensity projects of 232 x 232 μm2 fields of view from the AT (A, B) and endothelial layers of an uninfected control LoC. ATs show strong actin (A) and TJP1 expression (B, identified via antibody staining) at the cell junction boundaries. Endothelial cells also show high levels of TJP-1 expression (C) together with CD31 expression at cell junctions (D, identified via antibody labelling). Actin, TJP-1, CD31, and nuclear labelling are indicated with the azure, bright pink, spring green and electric indigo LUTs, respectively.

(E-H) 3D views of one 232 x 232 μm2 field of view of the AT layer (E) and (F-H) three 232 x 232 μm2 fields of view of the endothelial layer of an infected LoC at 1 dpi. Areas with lower TJP1 expression in the AT layer in (E) are indicated by white arrows. (F-H) Areas with loss of tight junctions between cells on the endothelial surface is indicated by white arrows. The proportion of the surface area covered by confluent cells decreases sequentially from (F) to (H). Endothelial cell clusters with low CD31 expression are clearly visible, and the proportion of the surface area covered by these clusters increases sequentially from (F) to (H). In (F-H), spike protein is identified via an anti-SARS-spike antibody and shown in amber LUT, and von Willebrand Factor identified by antibody labelling is shown in bring pink LUT.

(I-L) Zooms of two endothelial cell clusters (I, J and K, L) in the endothelial layer of another infected LoC at 1 dpi confirm that little or no CD31 expression is detected (I, K) despite high levels of actin and TJP-1 protein expression in these cells. (J, L).

(M) Plot of the fold change in expression of TJP1 (AT and endothelial layers) and CDH5, CD31, and CD146 (endothelial layers only) in infected LoCs at 1 and 3 dpi (n=3 biological replicates in each case) vs, uninfected controls (n=3 biological replicates).

(N) Plot of the fold change in gene expression of endothelial cell specific coagulation markers - Tissue Factor (F3), Tissue Factor Pathway Inhibitor (TFPI), Thrombomodulin (THBD), Plasminogen Activator Inhibitor (SERPINE1) and von Willebrand Factor (VWF) in infected LoCs at 1 and 3 dpi (n=3 biological replicates in each case) vs. uninfected controls (n=3 biological replicates).

Data information: In all plots, bars represent the mean value and error bars represent the standard deviation. Scale bar = 20 μm. P-values are calculated using a one-way Kruskal-Wallis ANOVA test, * represents p<=0.05, ** represents p<=0.01, ** represents p<=0.001.
Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-chip model (2021)
Figure 5

Fig. 5. RNAscope analysis reveals intracellular localization and slow accumulation of intracellular viral RNA in the LoC model.

(A-H) 3D views of 232 x 232 μm2 fields of view of the AT (A, G) and endothelial layer (B, H) from infected LoCs reconstituted without macrophages at 1 (A, B) and 3 dpi (G, H). Orf1ab antisense RNA, S RNA, ACE2 mRNA, and nuclear staining with DAPI are false-colored pink, amber, spring green, and electric indigo, respectively. (C, D) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (C) An example of infection and intracellular replication in a cell with no detectable ACE2 expression (white arrow) as well as nuclear localization of viral RNA (white arrowhead). (D) An example of an uninfected cell with ACE2 mRNA expression (yellow arrow). (E, F) Zooms corresponding to the regions in (B) representing increased nucleic acid staining (hyperplasic) and normal levels of nucleic acid staining highlighted with white and yellow boxes, respectively. (E) Examples of infection of hyperplasic endothelial cells both with and without ACE2 expression (F) Examples of endothelial cell infection with no ACE2 expression, nuclear localization of viral RNA in an infected endothelial cell is indicated (white arrow).

(I-L) Quantification of viral antisense RNA (I, J) and viral genomic RNA (K, L) from pairs (n=2 biological replicates) of otherwise identical LoCs reconstituted without (I, K) and with macrophages (J, L) analyzed at 1 and 3 dpi. Plots show the number of spots normalized by the total number of cells in 4-6 field of view (technical replicates) detected using RNAscope and confocal imaging using identical imaging conditions for all chips.

(M) Quantification of ACE2 expression per cell in ATs and endothelial cells from 4-6 fields of view (technical replicates) in a pair of LoCs reconstituted without macrophages (n=2 biological replicates) analyzed at 1 and 3 dpi, respectively.

Data information: Bars represent the mean value, the solid line represents the median, and error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p<=0.05, ** represents p<=0.01, ** represents p<=0.001.
Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-chip model (2021)
Figure 7

Fig. 7. Tocilizumab treatment reduces vascular permeability but does not prevent formation of endothelial cell clusters with reduced CD31 expression.

(A-F) Maximum intensity projections of representative 232 x 232 μm2 fields of view of the endothelial layer from an infected LoC reconstituted without macrophages at 2 dpi with (A) and without Tocilizumab treatment (D). Actin and nuclear staining are indicated with the azure and electric indigo LUTs respectively, the actin staining is saturated for the endothelial cell clusters to highlight cells with lower levels of actin. (B, C) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (B) A region with normal vascular profile, (C) A region of endothelial cell cluster formation with increased actin staining. (E, F) Zooms corresponding to the regions in (D) representing regions highlighted with white and yellow boxes, respectively. (E) An example of reduced tight junctions between endothelial cells; bare patches are indicated with arrows. (F) A region of endothelial cell clusters. Scale bar = 20 μm.

(G, H) 3D views from two 232 x 232 μm2 field of views of the endothelial layer of a Tocilizumab treated infected LoC at 2 dpi, immunostaining for the tight junction protein CD31 is shown in the Spring Green LUT. Endothelial cell clusters with low CD31 expression and altered actin staining are also visible and indicated by yellow arrows.

(I) A plot of the proportion of pixels (shown on a logarithmic scale) with regions of low F-actin intensity identified via cut-off thresholds defined as a percentage of the maximum intensity in regions of interest from one LoC with and without Tocilizumab administration. ROIs were chosen from between 6 and 7 fields of view (technical replicates), each field of view was 232 x 232 μm2. The ROIs were defined to exclude the occurrence of endothelial cell clusters. A significantly larger fraction of the surface area of the ROIs for the untreated chip have low F-actin intensities.

The solid bar represents the median, the boxes represent the 25-75 percentile range, and the whiskers represent the standard deviation from 6-7 regions of interest (technical replicates) from each chip. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p<=0.05, ** represents p<=0.01, ** represents p<=0.001.

Expanded View Figure Legends
Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function (2021)
Figure 1

Figure 1. Loss of Daam2 increases morphological complexity of astrocytes in the brain.

A-B. Immunostaining from Daam2LacZ/+ animals with Aldh1l1-EGFP reporter indicates that Daam2 (LacZ+, Red) is expressed in astrocytes (Green). (A) Daam2 expression is observed in multiple brain regions at postnatal day 21. (B) Expression of Daam2 in cortex persists throughout development (P7 to adult).

Scale bar: 50 μm.

C. Quantification of Daam2-expressing astrocytes (LacZ+) with Aldh1l1-EGFP labeled astrocytes in cortex during development. Data is presented as mean ± SEM. Two brain sections, N=4 for each age.

D. Schematic of the generation of astrocyte specific deletion of Daam2 in Aldh1l1-Cre and Daam2-floxed mice. Quantitative RT-PCR in FAC-sorted astrocytes confirms significant reduction of Daam2 transcript from Daam2 cKO mice. Experiments is performed as duplicates from N=3-4. Data is presented as mean ± SEM. Student's t-test is used for statistical analysis. ***p<0.001.

E-F. Quantification of astrocyte marker, Sox9+ cells in cortex (E) and OB (F) of Daam2 cHet and Daam2 cKO mice. Data is presented as mean ± SEM. 4-6 brain sections, N=4. Student's t-test is used for statistical analysis.

G. Representative images of astrocytes labeled with Aldh1l1-EGFP reporter in cortex and OB of Daam2 cHet and cKO mice at postnatal day 28 (P28). Scale bar: 50 μm.

H-K. Overall complexity of astrocytes with Aldh1l1-EGFP reporter was measured by Sholl analysis (Two-way ANOVA) and total process length (Student's t-test). Data is presented as mean ± SEM. n= 6-10 cells from N=3-5 mice per genotype, *p<0.05, p****<0.0001
Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function (2021)
Figure 2

Figure 2. Deletion of Daam2 increases intermediate filament GFAP whereas overexpression of Daam2 reduces GFAP and morphological complexity.

A. Representative images of GFAP immunostaining in Daam2 cKO mice compared to controls in cortex and OB at P28. Scale bar: 20 μm.

B. Quantification of GFAP immunostaining in Daam2 cHet and cKO mice from P3 to adult. Data is presented as mean ± SEM. n=6-17 images from N=4-6 mice per each genotype and age. Student's t-test is used for statistics. *p<0.05, ***p<0.001, p****<0.0001.

C. Quantification of GFAP immunostaining in Daam2 cHet and cKO mice in cortex and OB at P28. Data is presented as mean ± SEM. n=17-33 images from N=5 mice per each genotype and age. Student's t-test is used for statistics. p****<0.0001.

D. Western blot analysis showed a significant increase in the amount of GFAP protein in Daam2 KO primary astrocytes. N=2

E. Schematic of virus injection to overexpress Daam2 in the cortex.

F. Overexpression of Daam2 via injection of AAV-GfaABC1D-myc-Daam2 in wild type P1 pups revealed reduced GFAP intensity compared to animals injected with control virus (AAV-GfaABC1D-GFP). AAV-GfaABC1D-myc-Daam2 was injected into Aldh1l1-GFP reporter mice for morphological complexity analysis and infected cells (Myc positive) were compared with non-infected cells. Dotted white line indicates virus-infected cells. Filled triangles and empty triangles indicate astrocytic primary branches with the control and reduced level of GFAP or Aldh1l1-GFP, respectively. Scale bar: 20 μm.

G. Quantification of GFAP immunostaining in control virus-positive cells and Daam2 overexpressing cells. Data is presented as mean ± SEM. n= 22 images from N=3 mice per genotype. Relative fluorescent intensity was quantified, and Student's t-test was used, **** p<0.0001.

H-I. Sholl analysis (H) and quantification of total length (I) of filament with overexpression of Daam2. Data is presented as mean ± SEM. n=17-32 images from 3-4 mice per each. **p<0.01, ***p<0.001. Two-way ANOVA and Student's t-test was used for H and I, respectively.
Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function (2021)
Figure 3

Figure 3. Deletion of Daam2 differentially alters astrocytic calcium dynamics in the cortex and olfactory bulb.

A. Schematics of astrocytic calcium imaging in cortex from ex vivo slices and representative images of spontaneous calcium signaling (green) of Daam2 cHet and cKO mice injected with AAV-GfaABC1D-GCaMP6f virus. Approximate territory of astrocytes including soma and multiple microdomains is outlined in yellow. Representative traces of GCaMP6f signal in Daam2 cHet and cKO astrocytes are shown in black and red respectively.

B-D. Quantification of amplitude (B), frequency (C), and area under curve (AUC) (D) of ∆F/F GCaMP6f signal events in cortex. Daam2-deficient astrocytes have lower amplitude and frequency of signal events in cortex. Data is presented as mean ± SEM. Total number of cells is n=20, 17 from N=3-4 mice of each genotype. Student's t-test was used for statistics, *p<0.05.

E. Schematics of astrocytic calcium imaging and representative images of spontaneous calcium signaling (green) of Daam2 cHet and cKO mice injected with AAV-GfaABC1D-GCaMP6f virus in OB. Approximate territory of astrocytes including soma and multiple microdomains is outlined in yellow. Representative traces of GCaMP6f signal in Daam2 cHet and cKO astrocytes are shown in black and red respectively.

F-H. Quantification of amplitude (F), frequency (G), and area under curve (AUC) (H) of ∆F/F GCaMP6f signal events in OB. Amplitude and AUC of Daam2-deficient astrocytes are significantly increased in OB. Data is presented as mean ± SEM. Total number of cells is n=16, 15 (F-H) from N=3-4 mice of each genotype. Student's t-test was used for statistics, *p<0.05.
Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function (2021)
Figure 4

Figure 4. Daam2 deletion in astrocytes results in differential synaptic activity in cortex and olfactory bulb.

A-F. (A, D) Schematics of whole cell recording in ex vivo brain slices and representative traces of spontaneous EPSC and IPSC in neurons in cortex from Daam2 cHet (top, black) and cKO (bottom, red) mice. (B-C, E-F) Quantification of amplitude and frequency of sEPSC and sIPSC in each genotype. Data is presented as mean ± SEM. Total number of cells is n=19, 20 (B-C) and 17, 13 (E-F) from N=7 mice for each genotype. Student's t-test was used for comparison, *p<0.05.

G-L. (G, J) Schematics of whole cell recording in ex vivo brain slices and representative traces of spontaneous EPSC and IPSC in neurons of OB from Daam2 cHet (top, black) and cKO (bottom, red) mice. (H-I, K-L) Quantification of amplitude and frequency of sEPSC and sIPSC in each genotype. Data is presented as mean ± SEM. n=10, 13 (H-I) and 15, 19 (K-L) from N=6 mice. Student's t-test was used for comparison, *p<0.05, **p<0.01